The Scadden Lab in 2019
Scadden Lab Publications
Publications by topic
You can browse the Scadden Lab’s publications sorted by topic on the Scadden Lab website.
All Publications
2024
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2024. Cell of origin epigenetic priming determines susceptibility to Tet2 mutation. Nature communications. 15(1):4325. Pubmed: 38773071 DOI:10.1038/s41467-024-48508-6 Schiroli G, Kartha V, Duarte FM, Kristiansen TA, Mayerhofer C, Shrestha R, Earl A, Hu Y, Tay T, Rhee C, Buenrostro JD, Scadden DT. 2024. Cell of origin epigenetic priming determines susceptibility to Tet2 mutation. Nature communications. 15(1):4325. Pubmed: 38773071 DOI:10.1038/s41467-024-48508-6 Hematopoietic stem cell (HSC) mutations can result in clonal hematopoiesis (CH) with heterogeneous clinical outcomes. Here, we investigate how the cell state preceding Tet2 mutation impacts the pre-malignant phenotype. Using an inducible system for clonal analysis of myeloid progenitors, we find that the epigenetic features of clones at similar differentiation status are highly heterogeneous and functionally respond differently to Tet2 mutation. Cell differentiation stage also influences Tet2 mutation response indicating that the cell of origin's epigenome modulates clone-specific behaviors in CH. Molecular features associated with higher risk outcomes include Sox4 that sensitizes cells to Tet2 inactivation, inducing dedifferentiation, altered metabolism and increasing the in vivo clonal output of mutant cells, as confirmed in primary GMP and HSC models. Our findings validate the hypothesis that epigenetic features can predispose specific clones for dominance, explaining why identical genetic mutations can result in different phenotypes.© 2024. The Author(s). -
Mei S, Alchahin AM, Tsea I, Kfoury Y, Hirz T, Jeffries NE, Zhao T, Xu Y, Zhang H, Sarkar H, Wu S, Subtelny AO, Johnsen JI, Zhang Y, Salari K, Wu CL, Randolph MA, Scadden DT, Dahl DM, Shin J, Kharchenko PV, Saylor PJ, Sykes DB, Baryawno N. 2024. Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions. Genome medicine. 16(1):1. Pubmed: 38281962 DOI:10.1186/s13073-023-01272-6 Mei S, Alchahin AM, Tsea I, Kfoury Y, Hirz T, Jeffries NE, Zhao T, Xu Y, Zhang H, Sarkar H, Wu S, Subtelny AO, Johnsen JI, Zhang Y, Salari K, Wu CL, Randolph MA, Scadden DT, Dahl DM, Shin J, Kharchenko PV, Saylor PJ, Sykes DB, Baryawno N. 2024. Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions. Genome medicine. 16(1):1. Pubmed: 38281962 DOI:10.1186/s13073-023-01272-6 Array© 2023. The Author(s). -
Kristiansen T, Mayerhofer C, Gustafsson K, Scadden DT. 2024. Stromal Cell Isolation from Hematopoietic Organs. Journal of visualized experiments : JoVE. Pubmed: 38345255 DOI:10.3791/66231 Kristiansen T, Mayerhofer C, Gustafsson K, Scadden DT. 2024. Stromal Cell Isolation from Hematopoietic Organs. Journal of visualized experiments : JoVE. Pubmed: 38345255 DOI:10.3791/66231 Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field. -
Saha A, Palchaudhuri R, Lanieri L, Hyzy S, Riddle MJ, Panthera J, Eide CR, Tolar J, Panoskaltsis-Mortari A, Gorfinkel L, Tkachev V, Gerdemann U, Alvarez-Calderon F, Palato ER, MacMillan ML, Wagner JE, Kean LS, Osborn MJ, Kiem HP, Scadden DT, Olson LM, Blazar BR. 2024. Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate-conditioned Fanconi anemia mice. Blood. 143(21):2201-2216. Pubmed: 38447038 DOI:10.1182/blood.2023023549 Saha A, Palchaudhuri R, Lanieri L, Hyzy S, Riddle MJ, Panthera J, Eide CR, Tolar J, Panoskaltsis-Mortari A, Gorfinkel L, Tkachev V, Gerdemann U, Alvarez-Calderon F, Palato ER, MacMillan ML, Wagner JE, Kean LS, Osborn MJ, Kiem HP, Scadden DT, Olson LM, Blazar BR. 2024. Alloengraftment without significant toxicity or GVHD in CD45 antibody-drug conjugate-conditioned Fanconi anemia mice. Blood. 143(21):2201-2216. Pubmed: 38447038 DOI:10.1182/blood.2023023549 Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage-Sca1+cKit+ cells within 48 hours. Fanca-/- recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc-/- and Fancg-/- recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca-/- recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies. -
Bonilla G, Morris A, Kundu S, Ducasse A, Jeffries NE, Chetal K, Yvanovich EE, Barghout R, Scadden D, Mansour MK, Kingston RE, Sykes DB, Mercier FE, Sadreyev RI. 2024. Leukemia aggressiveness is driven by chromatin remodeling and expression changes of core regulators. bioRxiv : the preprint server for biology. Pubmed: 38496490 DOI:10.1101/2024.02.29.582846 Bonilla G, Morris A, Kundu S, Ducasse A, Jeffries NE, Chetal K, Yvanovich EE, Barghout R, Scadden D, Mansour MK, Kingston RE, Sykes DB, Mercier FE, Sadreyev RI. 2024. Leukemia aggressiveness is driven by chromatin remodeling and expression changes of core regulators. bioRxiv : the preprint server for biology. Pubmed: 38496490 DOI:10.1101/2024.02.29.582846 Molecular mechanisms driving clonal aggressiveness in leukemia are not fully understood. We tracked and analyzed two mouse MLL-rearranged leukemic clones independently evolving towards higher aggressiveness. More aggressive subclones lost their growth differential ex vivo but restored it upon secondary transplantation, suggesting molecular memory of aggressiveness. Development of aggressiveness was associated with clone-specific gradual modulation of chromatin states and expression levels across the genome, with a surprising preferential trend of reversing the earlier changes between normal and leukemic progenitors. To focus on the core aggressiveness program, we identified genes with consistent changes of expression and chromatin marks that were maintained in vivo and ex vivo in both clones. Overexpressing selected core genes (Smad1 as aggressiveness driver, Irx5 and Plag1 as suppressors) affected leukemic progenitor growth in the predicted way and had convergent downstream effects on central transcription factors and repressive epigenetic modifiers, suggesting a broader regulatory network of leukemic aggressiveness. -
Maneix L, Iakova P, Lee CG, Moree SE, Lu X, Datar GK, Hill CT, Spooner E, King JCK, Sykes DB, Saez B, Di Stefano B, Chen X, Krause DS, Sahin E, Tsai FTF, Goodell MA, Berk BC, Scadden DT, Catic A. 2024. Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing. Nature cell biology. 26(4):593-603. Pubmed: 38553595 DOI:10.1038/s41556-024-01387-x Maneix L, Iakova P, Lee CG, Moree SE, Lu X, Datar GK, Hill CT, Spooner E, King JCK, Sykes DB, Saez B, Di Stefano B, Chen X, Krause DS, Sahin E, Tsai FTF, Goodell MA, Berk BC, Scadden DT, Catic A. 2024. Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing. Nature cell biology. 26(4):593-603. Pubmed: 38553595 DOI:10.1038/s41556-024-01387-x Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.© 2024. The Author(s). -
Bonora M, Morganti C, van Gastel N, Ito K, Calura E, Zanolla I, Ferroni L, Zhang Y, Jung Y, Sales G, Martini P, Nakamura T, Lasorsa FM, Finkel T, Lin CP, Zavan B, Pinton P, Georgakoudi I, Romualdi C, Scadden DT, Ito K. 2024. A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate. Cell stem cell. 31(3):359-377.e10. Pubmed: 38458178 DOI:S1934-5909(24)00047-X Bonora M, Morganti C, van Gastel N, Ito K, Calura E, Zanolla I, Ferroni L, Zhang Y, Jung Y, Sales G, Martini P, Nakamura T, Lasorsa FM, Finkel T, Lin CP, Zavan B, Pinton P, Georgakoudi I, Romualdi C, Scadden DT, Ito K. 2024. A mitochondrial NADPH-cholesterol axis regulates extracellular vesicle biogenesis to support hematopoietic stem cell fate. Cell stem cell. 31(3):359-377.e10. Pubmed: 38458178 DOI:S1934-5909(24)00047-X Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved. 2023
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Kerr MD, McBride DA, Johnson WT, Chumber AK, Najibi AJ, Seo BR, Stafford AG, Scadden DT, Mooney DJ, Shah NJ. 2023. Immune-responsive biodegradable scaffolds for enhancing neutrophil regeneration. Bioengineering & translational medicine. 8(1):e10309. Pubmed: 36684088 DOI:10.1002/btm2.10309 Kerr MD, McBride DA, Johnson WT, Chumber AK, Najibi AJ, Seo BR, Stafford AG, Scadden DT, Mooney DJ, Shah NJ. 2023. Immune-responsive biodegradable scaffolds for enhancing neutrophil regeneration. Bioengineering & translational medicine. 8(1):e10309. Pubmed: 36684088 DOI:10.1002/btm2.10309 Neutrophils are essential effector cells for mediating rapid host defense and their insufficiency arising from therapy-induced side-effects, termed neutropenia, can lead to immunodeficiency-associated complications. In autologous hematopoietic stem cell transplantation (HSCT), neutropenia is a complication that limits therapeutic efficacy. Here, we report the development and in vivo evaluation of an injectable, biodegradable hyaluronic acid (HA)-based scaffold, termed HA cryogel, with myeloid responsive degradation behavior. In mouse models of immune deficiency, we show that the infiltration of functional myeloid-lineage cells, specifically neutrophils, is essential to mediate HA cryogel degradation. Post-HSCT neutropenia in recipient mice delayed degradation of HA cryogels by up to 3 weeks. We harnessed the neutrophil-responsive degradation to sustain the release of granulocyte colony stimulating factor (G-CSF) from HA cryogels. Sustained release of G-CSF from HA cryogels enhanced post-HSCT neutrophil recovery, comparable to pegylated G-CSF, which, in turn, accelerated cryogel degradation. HA cryogels are a potential approach for enhancing neutrophils and concurrently assessing immune recovery in neutropenic hosts.© 2022 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers. -
Hirz T, Mei S, Sarkar H, Kfoury Y, Wu S, Verhoeven BM, Subtelny AO, Zlatev DV, Wszolek MW, Salari K, Murray E, Chen F, Macosko EZ, Wu CL, Scadden DT, Dahl DM, Baryawno N, Saylor PJ, Kharchenko PV, Sykes DB. 2023. Dissecting the immune suppressive human prostate tumor microenvironment via integrated single-cell and spatial transcriptomic analyses. Nature communications. 14(1):663. Pubmed: 36750562 DOI:10.1038/s41467-023-36325-2 Hirz T, Mei S, Sarkar H, Kfoury Y, Wu S, Verhoeven BM, Subtelny AO, Zlatev DV, Wszolek MW, Salari K, Murray E, Chen F, Macosko EZ, Wu CL, Scadden DT, Dahl DM, Baryawno N, Saylor PJ, Kharchenko PV, Sykes DB. 2023. Dissecting the immune suppressive human prostate tumor microenvironment via integrated single-cell and spatial transcriptomic analyses. Nature communications. 14(1):663. Pubmed: 36750562 DOI:10.1038/s41467-023-36325-2 The treatment of low-risk primary prostate cancer entails active surveillance only, while high-risk disease requires multimodal treatment including surgery, radiation therapy, and hormonal therapy. Recurrence and development of metastatic disease remains a clinical problem, without a clear understanding of what drives immune escape and tumor progression. Here, we comprehensively describe the tumor microenvironment of localized prostate cancer in comparison with adjacent normal samples and healthy controls. Single-cell RNA sequencing and high-resolution spatial transcriptomic analyses reveal tumor context dependent changes in gene expression. Our data indicate that an immune suppressive tumor microenvironment associates with suppressive myeloid populations and exhausted T-cells, in addition to high stromal angiogenic activity. We infer cell-to-cell relationships from high throughput ligand-receptor interaction measurements within undissociated tissue sections. Our work thus provides a highly detailed and comprehensive resource of the prostate tumor microenvironment as well as tumor-stromal cell interactions.© 2023. The Author(s). -
Kawano Y, Kawano H, Ghoneim D, Fountaine TJ, Byun DK, LaMere MW, Mendler JH, Ho TC, Salama NA, Myers JR, Hussein SE, Frisch BJ, Ashton JM, Azadniv M, Liesveld JL, Kfoury Y, Scadden DT, Becker MW, Calvi LM. 2023. Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells. bioRxiv : the preprint server for biology. Pubmed: 37066307 DOI:10.1101/2023.04.09.536176 Kawano Y, Kawano H, Ghoneim D, Fountaine TJ, Byun DK, LaMere MW, Mendler JH, Ho TC, Salama NA, Myers JR, Hussein SE, Frisch BJ, Ashton JM, Azadniv M, Liesveld JL, Kfoury Y, Scadden DT, Becker MW, Calvi LM. 2023. Myelodysplastic syndromes disable human CD271+VCAM1+CD146+ niches supporting normal hematopoietic stem/progenitor cells. bioRxiv : the preprint server for biology. Pubmed: 37066307 DOI:10.1101/2023.04.09.536176 Mesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+ (NGFR+/VCAM1+/MCAM+/Lin-; NVML) cells display stem cell characteristics, are compatible with murine BM-derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression of MDS. -
Rhee C, Scadden EW, Wong LP, Schiroli G, Mazzola MC, Chea PL, Kato H, Hoyer FF, Mistry M, Lee BK, Kim J, Nahrendorf M, Mansour MK, Sykes DB, Sadreyev RI, Scadden DT. 2023. Limited plasticity of monocyte fate and function associated with epigenetic scripting at the level of progenitors. Blood. 142(7):658-674. Pubmed: 37267513 DOI:10.1182/blood.2023020257 Rhee C, Scadden EW, Wong LP, Schiroli G, Mazzola MC, Chea PL, Kato H, Hoyer FF, Mistry M, Lee BK, Kim J, Nahrendorf M, Mansour MK, Sykes DB, Sadreyev RI, Scadden DT. 2023. Limited plasticity of monocyte fate and function associated with epigenetic scripting at the level of progenitors. Blood. 142(7):658-674. Pubmed: 37267513 DOI:10.1182/blood.2023020257 Myeloid cell heterogeneity is known, but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed the definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/nonclassical categorization and had inherent, restricted characteristics that did not shift under homeostasis, after irradiation, or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another.© 2023 by The American Society of Hematology. -
Massengale M, Massengale JL, Benson CR, Baryawno N, Oki T, Steinhauser ML, Wang A, Balani D, Oh LS, Randolph MA, Gill TJ, Kronenberg HM, Scadden DT. 2023. Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo. JCI insight. 8(17). Pubmed: 37681409 DOI:10.1172/jci.insight.167858 Massengale M, Massengale JL, Benson CR, Baryawno N, Oki T, Steinhauser ML, Wang A, Balani D, Oh LS, Randolph MA, Gill TJ, Kronenberg HM, Scadden DT. 2023. Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo. JCI insight. 8(17). Pubmed: 37681409 DOI:10.1172/jci.insight.167858 The identity and origin of the stem/progenitor cells for adult joint cartilage repair remain unknown, impeding therapeutic development. Simulating the common therapeutic modality for cartilage repair in humans, i.e., full-thickness microfracture joint surgery, we combined the mouse full-thickness injury model with lineage tracing and identified a distinct skeletal progenitor cell type enabling long-term (beyond 7 days after injury) articular cartilage repair in vivo. Deriving from a population with active Prg4 expression in adulthood while lacking aggrecan expression, these progenitors proliferate, differentiate to express aggrecan and type II collagen, and predominate in long-term articular cartilage wounds, where they represent the principal repair progenitors in situ under native repair conditions without cellular transplantation. They originate outside the adult bone marrow or superficial zone articular cartilage. These findings have implications for skeletal biology and regenerative medicine for joint injury repair. -
Gustafsson K, Rhee C, Frodermann V, Scadden EW, Li D, Iwamoto Y, Palchaudhuri R, Hyzy SL, Boitano AE, Nahrendorf M, Scadden DT. 2023. Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody-drug conjugate. Blood advances. 7(22):6964-6973. Pubmed: 37748049 DOI:10.1182/bloodadvances.2023010561 Gustafsson K, Rhee C, Frodermann V, Scadden EW, Li D, Iwamoto Y, Palchaudhuri R, Hyzy SL, Boitano AE, Nahrendorf M, Scadden DT. 2023. Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody-drug conjugate. Blood advances. 7(22):6964-6973. Pubmed: 37748049 DOI:10.1182/bloodadvances.2023010561 Tissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody-drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45-ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45-ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45-ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45-ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45-ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell-driven disease.© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. -
Kfoury YS, Ji F, Jain E, Mazzola M, Schiroli G, Papazian A, Mercier F, Sykes DB, Kiem A, Randolph M, Calvi LM, Abdel-Wahab O, Sadreyev RI, Scadden DT. 2023. The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression. Blood advances. 7(21):6608-6623. Pubmed: 37450380 DOI:10.1182/bloodadvances.2022008268 Kfoury YS, Ji F, Jain E, Mazzola M, Schiroli G, Papazian A, Mercier F, Sykes DB, Kiem A, Randolph M, Calvi LM, Abdel-Wahab O, Sadreyev RI, Scadden DT. 2023. The bone marrow stroma in human myelodysplastic syndrome reveals alterations that regulate disease progression. Blood advances. 7(21):6608-6623. Pubmed: 37450380 DOI:10.1182/bloodadvances.2022008268 Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. -
Miller PG, Sperling AS, Mayerhofer C, McConkey ME, Ellegast JM, Da Silva C, Cohen DN, Wang C, Sharda A, Yan N, Saha S, Schluter C, Schechter I, Słabicki M, Sandoval B, Kahn J, Boettcher S, Gibson CJ, Scadden DT, Stegmaier K, Bhatt S, Lindsley RC, Ebert BL. 2023. PPM1D modulates hematopoietic cell fitness and response to DNA damage and is a therapeutic target in myeloid malignancy. Blood. 142(24):2079-2091. Pubmed: 37595362 DOI:10.1182/blood.2023020331 Miller PG, Sperling AS, Mayerhofer C, McConkey ME, Ellegast JM, Da Silva C, Cohen DN, Wang C, Sharda A, Yan N, Saha S, Schluter C, Schechter I, Słabicki M, Sandoval B, Kahn J, Boettcher S, Gibson CJ, Scadden DT, Stegmaier K, Bhatt S, Lindsley RC, Ebert BL. 2023. PPM1D modulates hematopoietic cell fitness and response to DNA damage and is a therapeutic target in myeloid malignancy. Blood. 142(24):2079-2091. Pubmed: 37595362 DOI:10.1182/blood.2023020331 PPM1D encodes a phosphatase that is recurrently activated across cancer, most notably in therapy-related myeloid neoplasms. However, the function of PPM1D in hematopoiesis and its contribution to tumor cell growth remain incompletely understood. Using conditional mouse models, we uncover a central role for Ppm1d in hematopoiesis and validate its potential as a therapeutic target. We find that Ppm1d regulates the competitive fitness and self-renewal of hematopoietic stem cells (HSCs) with and without exogenous genotoxic stresses. We also show that although Ppm1d activation confers cellular resistance to cytotoxic therapy, it does so to a lesser degree than p53 loss, informing the clonal competition phenotypes often observed in human studies. Notably, loss of Ppm1d sensitizes leukemias to cytotoxic therapies in vitro and in vivo, even in the absence of a Ppm1d mutation. Vulnerability to PPM1D inhibition is observed across many cancer types and dependent on p53 activity. Importantly, organism-wide loss of Ppm1d in adult mice is well tolerated, supporting the tolerability of pharmacologically targeting PPM1D. Our data link PPM1D gain-of-function mutations to the clonal expansion of HSCs, inform human genetic observations, and support the therapeutic targeting of PPM1D in cancer.© 2023 by The American Society of Hematology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). -
Gustafsson K, Rhee C, Frodermann V, Scadden EW, Li D, Iwamoto Y, Palchaudhuri R, Hyzy SL, Boitano AE, Nahrendorf M, Scadden DT. 2023. CD45-antibody-drug conjugate clears tissue resident myeloid cells from their niches enabling therapeutic adoptive cell transfer. bioRxiv : the preprint server for biology. Pubmed: 37732224 DOI:10.1101/2023.09.05.556397 Gustafsson K, Rhee C, Frodermann V, Scadden EW, Li D, Iwamoto Y, Palchaudhuri R, Hyzy SL, Boitano AE, Nahrendorf M, Scadden DT. 2023. CD45-antibody-drug conjugate clears tissue resident myeloid cells from their niches enabling therapeutic adoptive cell transfer. bioRxiv : the preprint server for biology. Pubmed: 37732224 DOI:10.1101/2023.09.05.556397 Tissue resident myeloid cells (TRM) in adults have highly variable lifespans and may be derived from early embryonic yolk sac, fetal liver or bone marrow. Some of these TRM are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplant can replace long-lived brain TRM resulting in clinical improvements in metabolic storage diseases. With the advent of antibody-drug-conjugate (ADC) targeted cell killing as a cell selective means of transplant conditioning, we assessed the impact of anti-CD45-ADC on TRM in multiple tissues. Replacement of TRM ranged from 40 to 95 percent efficiencies in liver, lung, and skin tissues, after a single anti-CD45-ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM in tissues returned to pre-treatment levels suggesting a regulated control of TRM abundance. As expected, brain, microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45-ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by mutant clonal hematopoiesis. Plaque resident myeloid cells were efficiently replaced with anti-CD45-ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that anti-CD45-ADC clears both HSC and TRM niches enabling cell replacement to achieve disease modification in a resident myeloid cell driven disease. -
Kooshesh KA, Gustafsson K, Scadden DT. 2023. Health Consequences of Thymus Removal in Adults. Reply. The New England journal of medicine. 389(18):1726-1727. Pubmed: 37913517 DOI:10.1056/NEJMc2310640#sa5 Kooshesh KA, Gustafsson K, Scadden DT. 2023. Health Consequences of Thymus Removal in Adults. Reply. The New England journal of medicine. 389(18):1726-1727. Pubmed: 37913517 DOI:10.1056/NEJMc2310640#sa5 -
Zhang S, Paccalet A, Rohde D, Cremer S, Hulsmans M, Lee IH, Mentkowski K, Grune J, Schloss MJ, Honold L, Iwamoto Y, Zheng Y, Bredella MA, Buckless C, Ghoshhajra B, Thondapu V, van der Laan AM, Piek JJ, Niessen HWM, Pallante F, Carnevale R, Perrotta S, Carnevale D, Iborra-Egea O, Muñoz-Guijosa C, Galvez-Monton C, Bayes-Genis A, Vidoudez C, Trauger SA, Scadden D, Swirski FK, Moskowitz MA, Naxerova K, Nahrendorf M. 2023. Bone marrow adipocytes fuel emergency hematopoiesis after myocardial infarction. Nature cardiovascular research. 2(12):1277-1290. Pubmed: 38344689 DOI:10.1038/s44161-023-00388-7 Zhang S, Paccalet A, Rohde D, Cremer S, Hulsmans M, Lee IH, Mentkowski K, Grune J, Schloss MJ, Honold L, Iwamoto Y, Zheng Y, Bredella MA, Buckless C, Ghoshhajra B, Thondapu V, van der Laan AM, Piek JJ, Niessen HWM, Pallante F, Carnevale R, Perrotta S, Carnevale D, Iborra-Egea O, Muñoz-Guijosa C, Galvez-Monton C, Bayes-Genis A, Vidoudez C, Trauger SA, Scadden D, Swirski FK, Moskowitz MA, Naxerova K, Nahrendorf M. 2023. Bone marrow adipocytes fuel emergency hematopoiesis after myocardial infarction. Nature cardiovascular research. 2(12):1277-1290. Pubmed: 38344689 DOI:10.1038/s44161-023-00388-7 After myocardial infarction (MI), emergency hematopoiesis produces inflammatory myeloid cells that accelerate atherosclerosis and promote heart failure. Since the balance between glycolysis and mitochondrial metabolism regulates hematopoietic stem cell homeostasis, metabolic cues may influence emergency myelopoiesis. Here, we show in humans and female mice that hematopoietic progenitor cells increase fatty acid metabolism after MI. Blockade of fatty acid oxidation by deleting carnitine palmitoyltransferase () in hematopoietic cells of mice limited hematopoietic progenitor proliferation and myeloid cell expansion after MI. We also observed reduced bone marrow adiposity in humans, pigs and mice following MI. Inhibiting lipolysis in adipocytes using mice or local depletion of bone marrow adipocytes in mice also curbed emergency hematopoiesis. Furthermore, systemic and regional sympathectomy prevented bone marrow adipocyte shrinkage after MI. These data establish a critical role for fatty acid metabolism in post-MI emergency hematopoiesis. -
Kooshesh KA, Foy BH, Sykes DB, Gustafsson K, Scadden DT. 2023. Health Consequences of Thymus Removal in Adults. The New England journal of medicine. 389(5):406-417. Pubmed: 37530823 DOI:10.1056/NEJMoa2302892 Kooshesh KA, Foy BH, Sykes DB, Gustafsson K, Scadden DT. 2023. Health Consequences of Thymus Removal in Adults. The New England journal of medicine. 389(5):406-417. Pubmed: 37530823 DOI:10.1056/NEJMoa2302892 ArrayCopyright © 2023 Massachusetts Medical Society. -
Gustafsson K, Scadden DT. 2023. Isolation of Thymus Stromal Cells from Human and Murine Tissue. Methods in molecular biology (Clifton, N.J.). 2567:191-201. Pubmed: 36255703 DOI:10.1007/978-1-0716-2679-5_13 Gustafsson K, Scadden DT. 2023. Isolation of Thymus Stromal Cells from Human and Murine Tissue. Methods in molecular biology (Clifton, N.J.). 2567:191-201. Pubmed: 36255703 DOI:10.1007/978-1-0716-2679-5_13 T cells go through most of their maturation in the thymus, and the stromal constituents of the thymus are therefore essential for T cell differentiation. The thymic stroma secretes the factors that recruit and sustain T cell progenitors, and they also partake in the shaping of a functional and tolerant T cell receptor repertoire. The damage incurred to the thymic stromal compartment by bone marrow conditioning regimens as well as by the natural aging process impairs T cell production. Yet little is known of how to prevent or reverse this damage. The development of high-throughput, single-cell analysis technologies has enabled better characterization of thymic stromal cells. This does however require tissue dissociation protocols optimized for stromal cell isolation. In this chapter, we detail the methodology of harvesting thymus stromal cells from human and murine tissue for downstream applications such as flow cytometric analysis and single-cell RNA sequencing.© 2023. Springer Science+Business Media, LLC, part of Springer Nature. 2022
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Rohde D, Vandoorne K, Lee IH, Grune J, Zhang S, McAlpine CS, Schloss MJ, Nayar R, Courties G, Frodermann V, Wojtkiewicz G, Honold L, Chen Q, Schmidt S, Iwamoto Y, Sun Y, Cremer S, Hoyer FF, Iborra-Egea O, Muñoz-Guijosa C, Ji F, Zhou B, Adams RH, Wythe JD, Hidalgo J, Watanabe H, Jung Y, van der Laan AM, Piek JJ, Kfoury Y, Désogère PA, Vinegoni C, Dutta P, Sadreyev RI, Caravan P, Bayes-Genis A, Libby P, Scadden DT, Lin CP, Naxerova K, Swirski FK, Nahrendorf M. 2022. Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease. Nature cardiovascular research. 1(1):28-44. Pubmed: 35747128 DOI:10.1038/s44161-021-00002-8 Rohde D, Vandoorne K, Lee IH, Grune J, Zhang S, McAlpine CS, Schloss MJ, Nayar R, Courties G, Frodermann V, Wojtkiewicz G, Honold L, Chen Q, Schmidt S, Iwamoto Y, Sun Y, Cremer S, Hoyer FF, Iborra-Egea O, Muñoz-Guijosa C, Ji F, Zhou B, Adams RH, Wythe JD, Hidalgo J, Watanabe H, Jung Y, van der Laan AM, Piek JJ, Kfoury Y, Désogère PA, Vinegoni C, Dutta P, Sadreyev RI, Caravan P, Bayes-Genis A, Libby P, Scadden DT, Lin CP, Naxerova K, Swirski FK, Nahrendorf M. 2022. Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease. Nature cardiovascular research. 1(1):28-44. Pubmed: 35747128 DOI:10.1038/s44161-021-00002-8 Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ's microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of or (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes. -
Haase C, Gustafsson K, Mei S, Yeh SC, Richter D, Milosevic J, Turcotte R, Kharchenko PV, Sykes DB, Scadden DT, Lin CP. 2022. Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging. Nature methods. 19(12):1622-1633. Pubmed: 36424441 DOI:10.1038/s41592-022-01673-2 Haase C, Gustafsson K, Mei S, Yeh SC, Richter D, Milosevic J, Turcotte R, Kharchenko PV, Sykes DB, Scadden DT, Lin CP. 2022. Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging. Nature methods. 19(12):1622-1633. Pubmed: 36424441 DOI:10.1038/s41592-022-01673-2 Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.© 2022. The Author(s). -
Elferich J, Schiroli G, Scadden DT, Grigorieff N. 2022. Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections. eLife. 11. Pubmed: 36382886 DOI:10.7554/eLife.80980 Elferich J, Schiroli G, Scadden DT, Grigorieff N. 2022. Defocus Corrected Large Area Cryo-EM (DeCo-LACE) for label-free detection of molecules across entire cell sections. eLife. 11. Pubmed: 36382886 DOI:10.7554/eLife.80980 A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods.© 2022, Elferich et al. -
Alchahin AM, Mei S, Tsea I, Hirz T, Kfoury Y, Dahl D, Wu CL, Subtelny AO, Wu S, Scadden DT, Shin JH, Saylor PJ, Sykes DB, Kharchenko PV, Baryawno N. 2022. A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma. Nature communications. 13(1):5747. Pubmed: 36180422 DOI:10.1038/s41467-022-33375-w Alchahin AM, Mei S, Tsea I, Hirz T, Kfoury Y, Dahl D, Wu CL, Subtelny AO, Wu S, Scadden DT, Shin JH, Saylor PJ, Sykes DB, Kharchenko PV, Baryawno N. 2022. A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma. Nature communications. 13(1):5747. Pubmed: 36180422 DOI:10.1038/s41467-022-33375-w Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults. When ccRCC is localized to the kidney, surgical resection or ablation of the tumor is often curative. However, in the metastatic setting, ccRCC remains a highly lethal disease. Here we use fresh patient samples that include treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. Single-cell transcriptomic analysis of tumor cells from the primary tumors reveals a distinct transcriptional signature that is predictive of metastatic potential and patient survival. Analysis of supporting stromal cells within the tumor environment demonstrates vascular remodeling within the endothelial cells. An in silico cell-to-cell interaction analysis highlights the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Our findings provide biological insights into the interplay between tumor cells and the ccRCC microenvironment.© 2022. The Author(s). -
McAlpine CS, Kiss MG, Zuraikat FM, Cheek D, Schiroli G, Amatullah H, Huynh P, Bhatti MZ, Wong LP, Yates AG, Poller WC, Mindur JE, Chan CT, Janssen H, Downey J, Singh S, Sadreyev RI, Nahrendorf M, Jeffrey KL, Scadden DT, Naxerova K, St-Onge MP, Swirski FK. 2022. Sleep exerts lasting effects on hematopoietic stem cell function and diversity. The Journal of experimental medicine. 219(11). Pubmed: 36129517 DOI:10.1084/jem.20220081 McAlpine CS, Kiss MG, Zuraikat FM, Cheek D, Schiroli G, Amatullah H, Huynh P, Bhatti MZ, Wong LP, Yates AG, Poller WC, Mindur JE, Chan CT, Janssen H, Downey J, Singh S, Sadreyev RI, Nahrendorf M, Jeffrey KL, Scadden DT, Naxerova K, St-Onge MP, Swirski FK. 2022. Sleep exerts lasting effects on hematopoietic stem cell function and diversity. The Journal of experimental medicine. 219(11). Pubmed: 36129517 DOI:10.1084/jem.20220081 A sleepless night may feel awful in its aftermath, but sleep's revitalizing powers are substantial, perpetuating the idea that convalescent sleep is a consequence-free physiological reset. Although recent studies have shown that catch-up sleep insufficiently neutralizes the negative effects of sleep debt, the mechanisms that control prolonged effects of sleep disruption are not understood. Here, we show that sleep interruption restructures the epigenome of hematopoietic stem and progenitor cells (HSPCs) and increases their proliferation, thus reducing hematopoietic clonal diversity through accelerated genetic drift. Sleep fragmentation exerts a lasting influence on the HSPC epigenome, skewing commitment toward a myeloid fate and priming cells for exaggerated inflammatory bursts. Combining hematopoietic clonal tracking with mathematical modeling, we infer that sleep preserves clonal diversity by limiting neutral drift. In humans, sleep restriction alters the HSPC epigenome and activates hematopoiesis. These findings show that sleep slows decay of the hematopoietic system by calibrating the hematopoietic epigenome, constraining inflammatory output, and maintaining clonal diversity.© 2022 McAlpine et al. -
Schloss MJ, Hulsmans M, Rohde D, Lee IH, Severe N, Foy BH, Pulous FE, Zhang S, Kokkaliaris KD, Frodermann V, Courties G, Yang C, Iwamoto Y, Knudsen AS, McAlpine CS, Yamazoe M, Schmidt SP, Wojtkiewicz GR, Masson GS, Gustafsson K, Capen D, Brown D, Higgins JM, Scadden DT, Libby P, Swirski FK, Naxerova K, Nahrendorf M. 2022. Author Correction: B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis. Nature immunology. 23(8):1285. Pubmed: 35705799 DOI:10.1038/s41590-022-01266-3 Schloss MJ, Hulsmans M, Rohde D, Lee IH, Severe N, Foy BH, Pulous FE, Zhang S, Kokkaliaris KD, Frodermann V, Courties G, Yang C, Iwamoto Y, Knudsen AS, McAlpine CS, Yamazoe M, Schmidt SP, Wojtkiewicz GR, Masson GS, Gustafsson K, Capen D, Brown D, Higgins JM, Scadden DT, Libby P, Swirski FK, Naxerova K, Nahrendorf M. 2022. Author Correction: B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis. Nature immunology. 23(8):1285. Pubmed: 35705799 DOI:10.1038/s41590-022-01266-3 -
Mercier FE, Shi J, Sykes DB, Oki T, Jankovic M, Man CH, Kfoury YS, Miller E, He S, Zhu A, Vasic R, Doench J, Orthwein A, Michor F, Scadden DT. 2022. In vivo genome-wide CRISPR screening in murine acute myeloid leukemia uncovers microenvironmental dependencies. Blood advances. 6(17):5072-5084. Pubmed: 35793392 DOI:10.1182/bloodadvances.2022007250 Mercier FE, Shi J, Sykes DB, Oki T, Jankovic M, Man CH, Kfoury YS, Miller E, He S, Zhu A, Vasic R, Doench J, Orthwein A, Michor F, Scadden DT. 2022. In vivo genome-wide CRISPR screening in murine acute myeloid leukemia uncovers microenvironmental dependencies. Blood advances. 6(17):5072-5084. Pubmed: 35793392 DOI:10.1182/bloodadvances.2022007250 Genome-wide CRISPR screens have been extremely useful in identifying therapeutic targets in diverse cancers by defining genes that are essential for malignant growth. However, most CRISPR screens were performed in vitro and thus cannot identify genes that are essential for interactions with the microenvironment in vivo. Here, we report genome-wide CRISPR screens in 2 in vivo murine models of acute myeloid leukemia (AML) driven by the KMT2A/MLLT3 fusion or by the constitutive coexpression of Hoxa9 and Meis1. Secondary validation using a focused library identified 72 genes specifically essential for leukemic growth in vivo, including components of the major histocompatibility complex class I complex, Cd47, complement receptor Cr1l, and the β-4-galactosylation pathway. Importantly, several of these in vivo-specific hits have a prognostic effect or are inferred to be master regulators of protein activity in human AML cases. For instance, we identified Fermt3, a master regulator of integrin signaling, as having in vivo-specific dependency with high prognostic relevance. Overall, we show an experimental and computational pipeline for genome-wide functional screens in vivo in AML and provide a genome-wide resource of essential drivers of leukemic growth in vivo.© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. -
Torres LS, Asada N, Weiss MJ, Trumpp A, Suda T, Scadden DT, Ito K. 2022. Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette. Stem cell reports. 17(7):1509-1535. Pubmed: 35830837 DOI:S2213-6711(22)00320-4 Torres LS, Asada N, Weiss MJ, Trumpp A, Suda T, Scadden DT, Ito K. 2022. Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette. Stem cell reports. 17(7):1509-1535. Pubmed: 35830837 DOI:S2213-6711(22)00320-4 In this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved. -
Man CH, Mercier FE, Liu N, Dong W, Stephanopoulos G, Jiang L, Jung Y, Lin CP, Leung AYH, Scadden DT. 2022. Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth. Blood. 139(4):502-522. Pubmed: 34610101 DOI:10.1182/blood.2021011563 Man CH, Mercier FE, Liu N, Dong W, Stephanopoulos G, Jiang L, Jung Y, Lin CP, Leung AYH, Scadden DT. 2022. Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth. Blood. 139(4):502-522. Pubmed: 34610101 DOI:10.1182/blood.2021011563 Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.© 2022 by The American Society of Hematology. -
Schloss MJ, Hulsmans M, Rohde D, Lee IH, Severe N, Foy BH, Pulous FE, Zhang S, Kokkaliaris KD, Frodermann V, Courties G, Yang C, Iwamoto Y, Knudsen AS, McAlpine CS, Yamazoe M, Schmidt SP, Wojtkiewicz GR, Masson GS, Gustafsson K, Capen D, Brown D, Higgins JM, Scadden DT, Libby P, Swirski FK, Naxerova K, Nahrendorf M. 2022. B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis. Nature immunology. 23(4):605-618. Pubmed: 35352063 DOI:10.1038/s41590-022-01165-7 Schloss MJ, Hulsmans M, Rohde D, Lee IH, Severe N, Foy BH, Pulous FE, Zhang S, Kokkaliaris KD, Frodermann V, Courties G, Yang C, Iwamoto Y, Knudsen AS, McAlpine CS, Yamazoe M, Schmidt SP, Wojtkiewicz GR, Masson GS, Gustafsson K, Capen D, Brown D, Higgins JM, Scadden DT, Libby P, Swirski FK, Naxerova K, Nahrendorf M. 2022. B lymphocyte-derived acetylcholine limits steady-state and emergency hematopoiesis. Nature immunology. 23(4):605-618. Pubmed: 35352063 DOI:10.1038/s41590-022-01165-7 Autonomic nerves control organ function through the sympathetic and parasympathetic branches, which have opposite effects. In the bone marrow, sympathetic (adrenergic) nerves promote hematopoiesis; however, how parasympathetic (cholinergic) signals modulate hematopoiesis is unclear. Here, we show that B lymphocytes are an important source of acetylcholine, a neurotransmitter of the parasympathetic nervous system, which reduced hematopoiesis. Single-cell RNA sequencing identified nine clusters of cells that expressed the cholinergic α7 nicotinic receptor (Chrna7) in the bone marrow stem cell niche, including endothelial and mesenchymal stromal cells (MSCs). Deletion of B cell-derived acetylcholine resulted in the differential expression of various genes, including Cxcl12 in leptin receptor (LepR) stromal cells. Pharmacologic inhibition of acetylcholine signaling increased the systemic supply of inflammatory myeloid cells in mice and humans with cardiovascular disease.© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc. 2021
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Castiello MC, Bosticardo M, Sacchetti N, Calzoni E, Fontana E, Yamazaki Y, Draghici E, Corsino C, Bortolomai I, Sereni L, Yu HH, Uva P, Palchaudhuri R, Scadden DT, Villa A, Notarangelo LD. 2021. Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency. The Journal of allergy and clinical immunology. 147(1):309-320.e6. Pubmed: 32387109 DOI:S0091-6749(20)30629-1 Castiello MC, Bosticardo M, Sacchetti N, Calzoni E, Fontana E, Yamazaki Y, Draghici E, Corsino C, Bortolomai I, Sereni L, Yu HH, Uva P, Palchaudhuri R, Scadden DT, Villa A, Notarangelo LD. 2021. Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency. The Journal of allergy and clinical immunology. 147(1):309-320.e6. Pubmed: 32387109 DOI:S0091-6749(20)30629-1 ArrayPublished by Elsevier Inc. -
Shen YJ, Mishima Y, Shi J, Sklavenitis-Pistofidis R, Redd RA, Moschetta M, Manier S, Roccaro AM, Sacco A, Tai YT, Mercier F, Kawano Y, Su NK, Berrios B, Doench JG, Root DE, Michor F, Scadden DT, Ghobrial IM. 2021. Progression signature underlies clonal evolution and dissemination of multiple myeloma. Blood. 137(17):2360-2372. Pubmed: 33150374 DOI:10.1182/blood.2020005885 Shen YJ, Mishima Y, Shi J, Sklavenitis-Pistofidis R, Redd RA, Moschetta M, Manier S, Roccaro AM, Sacco A, Tai YT, Mercier F, Kawano Y, Su NK, Berrios B, Doench JG, Root DE, Michor F, Scadden DT, Ghobrial IM. 2021. Progression signature underlies clonal evolution and dissemination of multiple myeloma. Blood. 137(17):2360-2372. Pubmed: 33150374 DOI:10.1182/blood.2020005885 Clonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.© 2021 by The American Society of Hematology. -
Oki T, Mercier F, Kato H, Jung Y, McDonald TO, Spencer JA, Mazzola MC, van Gastel N, Lin CP, Michor F, Kitamura T, Scadden DT. 2021. Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML. Nature communications. 12(1):245. Pubmed: 33431855 DOI:10.1038/s41467-020-20491-8 Oki T, Mercier F, Kato H, Jung Y, McDonald TO, Spencer JA, Mazzola MC, van Gastel N, Lin CP, Michor F, Kitamura T, Scadden DT. 2021. Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML. Nature communications. 12(1):245. Pubmed: 33431855 DOI:10.1038/s41467-020-20491-8 Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors. -
van Gastel N, Scadden DT. 2021. Young haematopoietic stem cells are picky eaters. Cell research. 31(4):377-378. Pubmed: 33633351 DOI:10.1038/s41422-021-00488-8 van Gastel N, Scadden DT. 2021. Young haematopoietic stem cells are picky eaters. Cell research. 31(4):377-378. Pubmed: 33633351 DOI:10.1038/s41422-021-00488-8 -
Wang YP, Sharda A, Xu SN, van Gastel N, Man CH, Choi U, Leong WZ, Li X, Scadden DT. 2021. Malic enzyme 2 connects the Krebs cycle intermediate fumarate to mitochondrial biogenesis. Cell metabolism. 33(5):1027-1041.e8. Pubmed: 33770508 DOI:S1550-4131(21)00110-8 Wang YP, Sharda A, Xu SN, van Gastel N, Man CH, Choi U, Leong WZ, Li X, Scadden DT. 2021. Malic enzyme 2 connects the Krebs cycle intermediate fumarate to mitochondrial biogenesis. Cell metabolism. 33(5):1027-1041.e8. Pubmed: 33770508 DOI:S1550-4131(21)00110-8 Mitochondria have an independent genome (mtDNA) and protein synthesis machinery that coordinately activate for mitochondrial generation. Here, we report that the Krebs cycle intermediate fumarate links metabolism to mitobiogenesis through binding to malic enzyme 2 (ME2). Mechanistically, fumarate binds ME2 with two complementary consequences. First, promoting the formation of ME2 dimers, which activate deoxyuridine 5'-triphosphate nucleotidohydrolase (DUT). DUT fosters thymidine generation and an increase of mtDNA. Second, fumarate-induced ME2 dimers abrogate ME2 monomer binding to mitochondrial ribosome protein L45, freeing it for mitoribosome assembly and mtDNA-encoded protein production. Methylation of the ME2-fumarate binding site by protein arginine methyltransferase-1 inhibits fumarate signaling to constrain mitobiogenesis. Notably, acute myeloid leukemia is highly dependent on mitochondrial function and is sensitive to targeting of the fumarate-ME2 axis. Therefore, mitobiogenesis can be manipulated in normal and malignant cells through ME2, an unanticipated governor of mitochondrial biomass production that senses nutrient availability through fumarate.Copyright © 2021 Elsevier Inc. All rights reserved. -
Daniels VW, Zoeller JJ, van Gastel N, McQueeney KE, Parvin S, Potter DS, Fell GG, Ferreira VG, Yilma B, Gupta R, Spetz J, Bhola PD, Endress JE, Harris IS, Carrilho E, Sarosiek KA, Scadden DT, Brugge JS, Letai A. 2021. Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics. Science signaling. 14(686). Pubmed: 34103421 DOI:10.1126/scisignal.abc7405 Daniels VW, Zoeller JJ, van Gastel N, McQueeney KE, Parvin S, Potter DS, Fell GG, Ferreira VG, Yilma B, Gupta R, Spetz J, Bhola PD, Endress JE, Harris IS, Carrilho E, Sarosiek KA, Scadden DT, Brugge JS, Letai A. 2021. Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics. Science signaling. 14(686). Pubmed: 34103421 DOI:10.1126/scisignal.abc7405 Cancer cells have differential metabolic dependencies compared to their nonmalignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), particularly those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize antiapoptotic proteins). We used high-throughput dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in nonmalignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific antiapoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Lévesque JP, Purton LE, Hidalgo A, Zon LI, Katayama Y, Scadden DT, Bowman TV, Stanley ER, Lucas D, Pinho S. 2021. In memory of Paul Sylvain Frenette, a pioneering explorer of the hematopoietic stem cell niche who left far too early. Experimental hematology. Pubmed: 34403758 DOI:10.1016/j.exphem.2021.08.002 Lévesque JP, Purton LE, Hidalgo A, Zon LI, Katayama Y, Scadden DT, Bowman TV, Stanley ER, Lucas D, Pinho S. 2021. In memory of Paul Sylvain Frenette, a pioneering explorer of the hematopoietic stem cell niche who left far too early. Experimental hematology. Pubmed: 34403758 DOI:10.1016/j.exphem.2021.08.002 -
Fast EM, Sporrij A, Manning M, Rocha EL, Yang S, Zhou Y, Guo J, Baryawno N, Barkas N, Scadden D, Camargo F, Zon LI. 2021. External signals regulate continuous transcriptional states in hematopoietic stem cells. eLife. 10. Pubmed: 34939923 DOI:10.7554/eLife.66512 Fast EM, Sporrij A, Manning M, Rocha EL, Yang S, Zhou Y, Guo J, Baryawno N, Barkas N, Scadden D, Camargo F, Zon LI. 2021. External signals regulate continuous transcriptional states in hematopoietic stem cells. eLife. 10. Pubmed: 34939923 DOI:10.7554/eLife.66512 Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo pharmacological perturbation of niche signals interferon, granulocyte colony-stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. External signals induced rapid transitions between HSC states but transcriptional response varied both between external stimulants and within the HSC population for a given perturbation. In contrast to LSK progenitors, HSCs were characterized by a greater link between molecular signatures at baseline and in response to external stressors. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq suggested some HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and LSK progenitor-specific chromatin and transcriptional features that represent determinants of signal receptiveness and regenerative potential during stress hematopoiesis.© 2021, Fast et al. -
Kfoury YS, Ji F, Mazzola M, Sykes DB, Scherer AK, Anselmo A, Akiyama Y, Mercier F, Severe N, Kokkaliaris KD, Zhao T, Brouse T, Saez B, Seidl J, Papazian A, Ivanov P, Mansour MK, Sadreyev RI, Scadden DT. 2021. tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche. Cell stem cell. 28(12):2090-2103.e9. Pubmed: 34551362 DOI:10.1016/j.stem.2021.08.014 Kfoury YS, Ji F, Mazzola M, Sykes DB, Scherer AK, Anselmo A, Akiyama Y, Mercier F, Severe N, Kokkaliaris KD, Zhao T, Brouse T, Saez B, Seidl J, Papazian A, Ivanov P, Mansour MK, Sadreyev RI, Scadden DT. 2021. tiRNA signaling via stress-regulated vesicle transfer in the hematopoietic niche. Cell stem cell. 28(12):2090-2103.e9. Pubmed: 34551362 DOI:10.1016/j.stem.2021.08.014 Extracellular vesicles (EVs) transfer complex biologic material between cells. However, the role of this process in vivo is poorly defined. Here, we demonstrate that osteoblastic cells in the bone marrow (BM) niche elaborate extracellular vesicles that are taken up by hematopoietic progenitor cells in vivo. Genotoxic or infectious stress rapidly increased stromal-derived extracellular vesicle transfer to granulocyte-monocyte progenitors. The extracellular vesicles contained processed tRNAs (tiRNAs) known to modulate protein translation. 5'-ti-Pro-CGG-1 was preferentially abundant in osteoblast-derived extracellular vesicles and, when transferred to granulocyte-monocyte progenitors, increased protein translation, cell proliferation, and myeloid differentiation. Upregulating EV transfer improved hematopoietic recovery from genotoxic injury and survival from fungal sepsis. Therefore, EV-mediated tiRNA transfer provides a stress-modulated signaling axis in the BM niche distinct from conventional cytokine-driven stress responses.Copyright © 2021 Elsevier Inc. All rights reserved. -
van Gastel N, Spinelli JB, Haigis MC, Scadden DT. 2021. Analysis of Leukemia Cell Metabolism through Stable Isotope Tracing in Mice. Bio-protocol. 11(19):e4171. Pubmed: 34722818 DOI:10.21769/BioProtoc.4171 van Gastel N, Spinelli JB, Haigis MC, Scadden DT. 2021. Analysis of Leukemia Cell Metabolism through Stable Isotope Tracing in Mice. Bio-protocol. 11(19):e4171. Pubmed: 34722818 DOI:10.21769/BioProtoc.4171 Once thought to be a mere consequence of the state of a cell, intermediary metabolism is now recognized as a key regulator of mammalian cell fate and function. In addition, cell metabolism is often disturbed in malignancies such as cancer, and targeting metabolic pathways can provide new therapeutic options. Cell metabolism is mostly studied in cell cultures , using techniques such as metabolomics, stable isotope tracing, and biochemical assays. Increasing evidence however shows that the metabolic profile of cells is highly dependent on the microenvironment, and metabolic vulnerabilities identified do not always translate to settings. Here, we provide a detailed protocol on how to perform stable isotope tracing in leukemia cells in mice, focusing on glutamine metabolism in acute myeloid leukemia (AML) cells. This method allows studying the metabolic profile of leukemia cells in their native bone marrow niche.Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC. -
Kfoury Y, Baryawno N, Severe N, Mei S, Gustafsson K, Hirz T, Brouse T, Scadden EW, Igolkina AA, Kokkaliaris K, Choi BD, Barkas N, Randolph MA, Shin JH, Saylor PJ, Scadden DT, Sykes DB, Kharchenko PV. 2021. Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment. Cancer cell. 39(11):1464-1478.e8. Pubmed: 34719426 DOI:S1535-6108(21)00494-3 Kfoury Y, Baryawno N, Severe N, Mei S, Gustafsson K, Hirz T, Brouse T, Scadden EW, Igolkina AA, Kokkaliaris K, Choi BD, Barkas N, Randolph MA, Shin JH, Saylor PJ, Scadden DT, Sykes DB, Kharchenko PV. 2021. Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment. Cancer cell. 39(11):1464-1478.e8. Pubmed: 34719426 DOI:S1535-6108(21)00494-3 Bone metastases are devastating complications of cancer. They are particularly common in prostate cancer (PCa), represent incurable disease, and are refractory to immunotherapy. We seek to define distinct features of the bone marrow (BM) microenvironment by analyzing single cells from bone metastatic prostate tumors, involved BM, uninvolved BM, and BM from cancer-free, orthopedic patients, and healthy individuals. Metastatic PCa is associated with multifaceted immune distortion, specifically exhaustion of distinct T cell subsets, appearance of macrophages with states specific to PCa bone metastases. The chemokine CCL20 is notably overexpressed by myeloid cells, as is its cognate CCR6 receptor on T cells. Disruption of the CCL20-CCR6 axis in mice with syngeneic PCa bone metastases restores T cell reactivity and significantly prolongs animal survival. Comparative high-resolution analysis of PCa bone metastases shows a targeted approach for relieving local immunosuppression for therapeutic effect.Copyright © 2021 Elsevier Inc. All rights reserved. -
Blanco MA, Sykes DB, Gu L, Wu M, Petroni R, Karnik R, Wawer M, Rico J, Li H, Jacobus WD, Jambhekar A, Cheloufi S, Meissner A, Hochedlinger K, Scadden DT, Shi Y. 2021. Chromatin-state barriers enforce an irreversible mammalian cell fate decision. Cell reports. 37(6):109967. Pubmed: 34758323 DOI:S2211-1247(21)01446-7 Blanco MA, Sykes DB, Gu L, Wu M, Petroni R, Karnik R, Wawer M, Rico J, Li H, Jacobus WD, Jambhekar A, Cheloufi S, Meissner A, Hochedlinger K, Scadden DT, Shi Y. 2021. Chromatin-state barriers enforce an irreversible mammalian cell fate decision. Cell reports. 37(6):109967. Pubmed: 34758323 DOI:S2211-1247(21)01446-7 Stem and progenitor cells have the capacity to balance self-renewal and differentiation. Hematopoietic myeloid progenitors replenish more than 25 billion terminally differentiated neutrophils every day under homeostatic conditions and can increase this output in response to stress or infection. At what point along the spectrum of maturation do progenitors lose capacity for self-renewal and become irreversibly committed to differentiation? Using a system of conditional myeloid development that can be toggled between self-renewal and differentiation, we interrogate determinants of this "point of no return" in differentiation commitment. Irreversible commitment is due primarily to loss of open regulatory site access and disruption of a positive feedback transcription factor activation loop. Restoration of the transcription factor feedback loop extends the window of cell plasticity and alters the point of no return. These findings demonstrate how the chromatin state enforces and perpetuates cell fate and identify potential avenues for manipulating cell identity.Copyright © 2021. Published by Elsevier Inc. -
Mulherkar N, Scadden DT. 2021. What is the role of the bone marrow microenvironment in AML?. Best practice & research. Clinical haematology. 34(4):101328. Pubmed: 34865700 DOI:S1521-6926(21)00093-1 Mulherkar N, Scadden DT. 2021. What is the role of the bone marrow microenvironment in AML?. Best practice & research. Clinical haematology. 34(4):101328. Pubmed: 34865700 DOI:S1521-6926(21)00093-1 Acute myeloid leukemia (AML) continues to be associated with relapse and resistance to chemotherapy. The bone marrow microenvironment in AML has been shown to regulate responsiveness to chemotherapy and to support disease progression. This review summarizes some recent experimental insights into the crucial role of the bone marrow microenvironment in AML and persistence after chemotherapy.Copyright © 2021. Published by Elsevier Ltd. -
Mori T, Verma R, Nakamoto-Matsubara R, Siu KT, Panaroni C, Fulzele KS, Mukaihara K, Onyewadume C, Maebius A, Kato H, Wong LP, Sadreyev RI, Scadden DT, Raje NS. 2021. Low NCOR2 levels in multiple myeloma patients drive multidrug resistance via MYC upregulation. Blood cancer journal. 11(12):194. Pubmed: 34864816 DOI:10.1038/s41408-021-00589-y Mori T, Verma R, Nakamoto-Matsubara R, Siu KT, Panaroni C, Fulzele KS, Mukaihara K, Onyewadume C, Maebius A, Kato H, Wong LP, Sadreyev RI, Scadden DT, Raje NS. 2021. Low NCOR2 levels in multiple myeloma patients drive multidrug resistance via MYC upregulation. Blood cancer journal. 11(12):194. Pubmed: 34864816 DOI:10.1038/s41408-021-00589-y MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression and discovered that NCOR2 was down-regulated in these cells. NCOR2 is a transcriptional coregulatory protein and its role in MM remains unknown. To define the role of NCOR2 in MM, we created NCOR2 knockout human myeloma cell lines and demonstrated that NCOR2 knockout led to high MYC expression. Furthermore, NCOR2 knockout conferred resistance to pomalidomide, BET and HDAC inhibitors, independent of Cereblon (CRBN), indicating high MYC expression as a cause of multidrug resistance. Moreover, NCOR2 interacted with the nucleosome remodeling and deacetylase (NuRD) complex and repressed the expression of CD180 by directly binding to its promoter and inducing MYC expression. Next, we generated lenalidomide-resistant and pomalidomide-resistant human myeloma cell lines. Whole-exome sequencing revealed that these cell lines acquired the same exonic mutations of NCOR2. These cell lines showed NCOR2 downregulation and MYC upregulation independent of CRBN and demonstrated resistance to BET and HDAC inhibitors. Our findings reveal a novel CRBN independent molecular mechanism associated with drug resistance. Low NCOR2 expression can serve as a potential biomarker for drug resistance and needs further validation in larger prospective studies.© 2021. The Author(s). -
Shah NJ, Mao AS, Shih TY, Kerr MD, Sharda A, Raimondo TM, Weaver JC, Vrbanac VD, Deruaz M, Tager AM, Mooney DJ, Scadden DT. 2021. Author Correction: An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation. Nature biotechnology. 39(11):1466. Pubmed: 34663922 DOI:10.1038/s41587-021-01081-5 Shah NJ, Mao AS, Shih TY, Kerr MD, Sharda A, Raimondo TM, Weaver JC, Vrbanac VD, Deruaz M, Tager AM, Mooney DJ, Scadden DT. 2021. Author Correction: An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation. Nature biotechnology. 39(11):1466. Pubmed: 34663922 DOI:10.1038/s41587-021-01081-5 2020
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Srikanthan MA, Humbert O, Haworth KG, Ironside C, Rajawat YS, Blazar BR, Palchaudhuri R, Boitano AE, Cooke MP, Scadden DT, Kiem HP. 2020. Effective Multi-lineage Engraftment in a Mouse Model of Fanconi Anemia Using Non-genotoxic Antibody-Based Conditioning. Molecular therapy. Methods & clinical development. 17:455-464. Pubmed: 32226796 DOI:10.1016/j.omtm.2020.02.001 Srikanthan MA, Humbert O, Haworth KG, Ironside C, Rajawat YS, Blazar BR, Palchaudhuri R, Boitano AE, Cooke MP, Scadden DT, Kiem HP. 2020. Effective Multi-lineage Engraftment in a Mouse Model of Fanconi Anemia Using Non-genotoxic Antibody-Based Conditioning. Molecular therapy. Methods & clinical development. 17:455-464. Pubmed: 32226796 DOI:10.1016/j.omtm.2020.02.001 Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow, facilitating donor cell engraftment. Although effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk for secondary malignancies. Antibody conjugates targeting hematopoietic cells are an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity. Specifically, this platform would be useful in diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita, Schwachman-Diamond syndrome, and Fanconi anemia (FA). Our approach utilizes antibody-drug conjugates (ADC) as an alternative conditioning regimen in an FA mouse model of autologous transplantation. Antibodies targeting either CD45 or CD117 were conjugated to saporin (SAP), a ribosomal toxin. knockout mice were conditioned with either CD45-SAP or CD117-SAP prior to receiving whole marrow from a heterozygous healthy donor. Bone marrow and peripheral blood analysis revealed equivalent levels of donor engraftment, with minimal toxicity in ADC-treated groups as compared with cyclophosphamide-treated controls. Our findings suggest ADCs may be an effective conditioning strategy in stem cell transplantation not only for diseases where traditional chemotherapy is not tolerated, but also more broadly for the field of blood and marrow transplantation.© 2020 The Authors. -
Yusuf RZ, Saez B, Sharda A, van Gastel N, Yu VWC, Baryawno N, Scadden EW, Acharya S, Chattophadhyay S, Huang C, Viswanathan V, S'aulis D, Cobert J, Sykes DB, Keibler MA, Das S, Hutchinson JN, Churchill M, Mukherjee S, Lee D, Mercier F, Doench J, Bullinger L, Logan DJ, Schreiber S, Stephanopoulos G, Rizzo WB, Scadden DT. 2020. Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers. Blood. 136(11):1303-1316. Pubmed: 32458004 DOI:10.1182/blood.2019001808 Yusuf RZ, Saez B, Sharda A, van Gastel N, Yu VWC, Baryawno N, Scadden EW, Acharya S, Chattophadhyay S, Huang C, Viswanathan V, S'aulis D, Cobert J, Sykes DB, Keibler MA, Das S, Hutchinson JN, Churchill M, Mukherjee S, Lee D, Mercier F, Doench J, Bullinger L, Logan DJ, Schreiber S, Stephanopoulos G, Rizzo WB, Scadden DT. 2020. Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers. Blood. 136(11):1303-1316. Pubmed: 32458004 DOI:10.1182/blood.2019001808 Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.© 2020 by The American Society of Hematology. -
van Gastel N, Stegen S, Eelen G, Schoors S, Carlier A, Daniëls VW, Baryawno N, Przybylski D, Depypere M, Stiers PJ, Lambrechts D, Van Looveren R, Torrekens S, Sharda A, Agostinis P, Lambrechts D, Maes F, Swinnen JV, Geris L, Van Oosterwyck H, Thienpont B, Carmeliet P, Scadden DT, Carmeliet G. 2020. Lipid availability determines fate of skeletal progenitor cells via SOX9. Nature. 579(7797):111-117. Pubmed: 32103177 DOI:10.1038/s41586-020-2050-1 van Gastel N, Stegen S, Eelen G, Schoors S, Carlier A, Daniëls VW, Baryawno N, Przybylski D, Depypere M, Stiers PJ, Lambrechts D, Van Looveren R, Torrekens S, Sharda A, Agostinis P, Lambrechts D, Maes F, Swinnen JV, Geris L, Van Oosterwyck H, Thienpont B, Carmeliet P, Scadden DT, Carmeliet G. 2020. Lipid availability determines fate of skeletal progenitor cells via SOX9. Nature. 579(7797):111-117. Pubmed: 32103177 DOI:10.1038/s41586-020-2050-1 The avascular nature of cartilage makes it a unique tissue, but whether and how the absence of nutrient supply regulates chondrogenesis remain unknown. Here we show that obstruction of vascular invasion during bone healing favours chondrogenic over osteogenic differentiation of skeletal progenitor cells. Unexpectedly, this process is driven by a decreased availability of extracellular lipids. When lipids are scarce, skeletal progenitors activate forkhead box O (FOXO) transcription factors, which bind to the Sox9 promoter and increase its expression. Besides initiating chondrogenesis, SOX9 acts as a regulator of cellular metabolism by suppressing oxidation of fatty acids, and thus adapts the cells to an avascular life. Our results define lipid scarcity as an important determinant of chondrogenic commitment, reveal a role for FOXO transcription factors during lipid starvation, and identify SOX9 as a critical metabolic mediator. These data highlight the importance of the nutritional microenvironment in the specification of skeletal cell fate. -
van Gastel N, Spinelli JB, Sharda A, Schajnovitz A, Baryawno N, Rhee C, Oki T, Grace E, Soled HJ, Milosevic J, Sykes DB, Hsu PP, Vander Heiden MG, Vidoudez C, Trauger SA, Haigis MC, Scadden DT. 2020. Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance. Cell metabolism. 32(3):391-403.e6. Pubmed: 32763164 DOI:S1550-4131(20)30367-3 van Gastel N, Spinelli JB, Sharda A, Schajnovitz A, Baryawno N, Rhee C, Oki T, Grace E, Soled HJ, Milosevic J, Sykes DB, Hsu PP, Vander Heiden MG, Vidoudez C, Trauger SA, Haigis MC, Scadden DT. 2020. Induction of a Timed Metabolic Collapse to Overcome Cancer Chemoresistance. Cell metabolism. 32(3):391-403.e6. Pubmed: 32763164 DOI:S1550-4131(20)30367-3 Cancer relapse begins when malignant cells pass through the extreme metabolic bottleneck of stress from chemotherapy and the byproducts of the massive cell death in the surrounding region. In acute myeloid leukemia, complete remissions are common, but few are cured. We tracked leukemia cells in vivo, defined the moment of maximal response following chemotherapy, captured persisting cells, and conducted unbiased metabolomics, revealing a metabolite profile distinct from the pre-chemo growth or post-chemo relapse phase. Persisting cells used glutamine in a distinctive manner, preferentially fueling pyrimidine and glutathione generation, but not the mitochondrial tricarboxylic acid cycle. Notably, malignant cell pyrimidine synthesis also required aspartate provided by specific bone marrow stromal cells. Blunting glutamine metabolism or pyrimidine synthesis selected against residual leukemia-initiating cells and improved survival in leukemia mouse models and patient-derived xenografts. We propose that timed cell-intrinsic or niche-focused metabolic disruption can exploit a transient vulnerability and induce metabolic collapse in cancer cells to overcome chemoresistance.Copyright © 2020. Published by Elsevier Inc. -
Kokkaliaris KD, Kunz L, Cabezas-Wallscheid N, Christodoulou C, Renders S, Camargo F, Trumpp A, Scadden DT, Schroeder T. 2020. Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations. Blood. 136(20):2296-2307. Pubmed: 32766876 DOI:10.1182/blood.2020006574 Kokkaliaris KD, Kunz L, Cabezas-Wallscheid N, Christodoulou C, Renders S, Camargo F, Trumpp A, Scadden DT, Schroeder T. 2020. Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations. Blood. 136(20):2296-2307. Pubmed: 32766876 DOI:10.1182/blood.2020006574 The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.© 2020 by The American Society of Hematology. -
Fang S, Chen S, Nurmi H, Leppänen VM, Jeltsch M, Scadden D, Silberstein L, Mikkola H, Alitalo K. 2020. VEGF-C protects the integrity of the bone marrow perivascular niche in mice. Blood. 136(16):1871-1883. Pubmed: 32842144 DOI:10.1182/blood.2020005699 Fang S, Chen S, Nurmi H, Leppänen VM, Jeltsch M, Scadden D, Silberstein L, Mikkola H, Alitalo K. 2020. VEGF-C protects the integrity of the bone marrow perivascular niche in mice. Blood. 136(16):1871-1883. Pubmed: 32842144 DOI:10.1182/blood.2020005699 Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor-positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.© 2020 by The American Society of Hematology. -
Dai C, Li Q, May HI, Li C, Zhang G, Sharma G, Sherry AD, Malloy CR, Khemtong C, Zhang Y, Deng Y, Gillette TG, Xu J, Scadden DT, Wang ZV. 2020. Lactate Dehydrogenase A Governs Cardiac Hypertrophic Growth in Response to Hemodynamic Stress. Cell reports. 32(9):108087. Pubmed: 32877669 DOI:S2211-1247(20)31076-7 Dai C, Li Q, May HI, Li C, Zhang G, Sharma G, Sherry AD, Malloy CR, Khemtong C, Zhang Y, Deng Y, Gillette TG, Xu J, Scadden DT, Wang ZV. 2020. Lactate Dehydrogenase A Governs Cardiac Hypertrophic Growth in Response to Hemodynamic Stress. Cell reports. 32(9):108087. Pubmed: 32877669 DOI:S2211-1247(20)31076-7 The heart manifests hypertrophic growth in response to high blood pressure, which may decompensate and progress to heart failure under persistent stress. Metabolic remodeling is an early event in this process. However, its role remains to be fully characterized. Here, we show that lactate dehydrogenase A (LDHA), a critical glycolytic enzyme, is elevated in the heart in response to hemodynamic stress. Cardiomyocyte-restricted deletion of LDHA leads to defective cardiac hypertrophic growth and heart failure by pressure overload. Silencing of LDHA in cultured cardiomyocytes suppresses cell growth from pro-hypertrophic stimulation in vitro, while overexpression of LDHA is sufficient to drive cardiomyocyte growth. Furthermore, we find that lactate is capable of rescuing the growth defect from LDHA knockdown. Mechanistically, lactate stabilizes NDRG3 (N-myc downregulated gene family 3) and stimulates ERK (extracellular signal-regulated kinase). Our results together suggest that the LDHA/NDRG3 axis may play a critical role in adaptive cardiomyocyte growth in response to hemodynamic stress.Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved. -
Burberry A, Wells MF, Limone F, Couto A, Smith KS, Keaney J, Gillet G, van Gastel N, Wang JY, Pietilainen O, Qian M, Eggan P, Cantrell C, Mok J, Kadiu I, Scadden DT, Eggan K. 2020. C9orf72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. 582(7810):89-94. Pubmed: 32483373 DOI:10.1038/s41586-020-2288-7 Burberry A, Wells MF, Limone F, Couto A, Smith KS, Keaney J, Gillet G, van Gastel N, Wang JY, Pietilainen O, Qian M, Eggan P, Cantrell C, Mok J, Kadiu I, Scadden DT, Eggan K. 2020. C9orf72 suppresses systemic and neural inflammation induced by gut bacteria. Nature. 582(7810):89-94. Pubmed: 32483373 DOI:10.1038/s41586-020-2288-7 A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins before its non-canonical translation into neural-toxic dipeptide proteins. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system. -
Shah NJ, Najibi AJ, Shih TY, Mao AS, Sharda A, Scadden DT, Mooney DJ. 2020. A biomaterial-based vaccine eliciting durable tumour-specific responses against acute myeloid leukaemia. Nature biomedical engineering. 4(1):40-51. Pubmed: 31937942 DOI:10.1038/s41551-019-0503-3 Shah NJ, Najibi AJ, Shih TY, Mao AS, Sharda A, Scadden DT, Mooney DJ. 2020. A biomaterial-based vaccine eliciting durable tumour-specific responses against acute myeloid leukaemia. Nature biomedical engineering. 4(1):40-51. Pubmed: 31937942 DOI:10.1038/s41551-019-0503-3 Acute myeloid leukaemia (AML) is a malignancy of haematopoietic origin that has limited therapeutic options. The standard-of-care cytoreductive chemotherapy depletes AML cells to induce remission, but is infrequently curative. An immunosuppressive AML microenvironment in the bone marrow and the paucity of suitable immunotherapy targets limit the induction of effective immune responses. Here, in mouse models of AML, we show that a macroporous-biomaterial vaccine that delivers the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), the Toll-like-receptor-9 agonist cytosine-guanosine oligodeoxynucleotide and one or multiple leukaemia antigens (in the form of a defined peptide antigen, cell lysates or antigens sourced from AML cells recruited in vivo) induces local immune-cell infiltration and activated dendritic cells, evoking a potent anti-AML response. The biomaterial-based vaccine prevented the engraftment of AML cells when administered as a prophylactic and when combined with chemotherapy, and eradicated established AML even in the absence of a defined vaccine antigen. Biomaterial-based AML vaccination can induce potent immune responses, deplete AML cells and prevent disease relapse. -
Kokkaliaris KD, Scadden DT. 2020. Cell interactions in the bone marrow microenvironment affecting myeloid malignancies. Blood advances. 4(15):3795-3803. Pubmed: 32780848 DOI:10.1182/bloodadvances.2020002127 Kokkaliaris KD, Scadden DT. 2020. Cell interactions in the bone marrow microenvironment affecting myeloid malignancies. Blood advances. 4(15):3795-3803. Pubmed: 32780848 DOI:10.1182/bloodadvances.2020002127 The bone marrow is a complex tissue in which heterogeneous populations of stromal cells interact with hematopoietic cells to dynamically respond to organismal needs in defense, hemostasis, and oxygen delivery. Physiologic challenges modify stromal/hematopoietic cell interactions to generate changes in blood cell production. When either stroma or hematopoietic cells are impaired, the system distorts. The distortions associated with myeloid malignancy are reviewed here and may provide opportunities for therapeutic intervention.© 2020 by The American Society of Hematology. 2019
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Scadden DT. 2019. Metcalf Lecture Award: Applying niche biology to engineer T-cell regenerative therapies. Experimental hematology. 80:1-10. Pubmed: 31765673 DOI:S0301-472X(19)31122-1 Scadden DT. 2019. Metcalf Lecture Award: Applying niche biology to engineer T-cell regenerative therapies. Experimental hematology. 80:1-10. Pubmed: 31765673 DOI:S0301-472X(19)31122-1 The processes generating cells of adaptive immunity render them less amenable to the single cytokine signals used so effectively to regenerate myeloid cells. T-cell neogenesis begins in the bone marrow, where specific sets of late osteolineage cells govern the specification of hematopoietic cells capable of migrating to the thymus where differentiation is completed. Osteocalcin-expressing bone marrow stromal cells producing Dll4 serve as a progenitor niche enabling this T-competent cell production. Biocompatible alginate-based cryogels containing bone morphogenetic proteins (BMP-2) and the Notch ligand Dll4 were engineered to recapitulate the endogenous niche. These cryogels are highly pliable and can be injected under the skin of animals undergoing bone marrow transplantation. The result in mice is an ectopic niche fostering T-competent progenitor generation that results in improved T-cell numbers and receptor diversity. The recipients can generate neoantigen vaccine responses while having improved tolerance manifest by reduced graft-versus-host disease upon allogeneic transplant. Through emerging details of niches in the bone marrow, therapeutics more complex than those necessary for myeloid reconstitution are possible. Niche biology-guided bioengineered design offers the possibility of regenerative therapies for T lymphoid cells.Copyright © 2019 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved. -
Baryawno N, Przybylski D, Kowalczyk MS, Kfoury Y, Severe N, Gustafsson K, Kokkaliaris KD, Mercier F, Tabaka M, Hofree M, Dionne D, Papazian A, Lee D, Ashenberg O, Subramanian A, Vaishnav ED, Rozenblatt-Rosen O, Regev A, Scadden DT. 2019. A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia. Cell. 177(7):1915-1932.e16. Pubmed: 31130381 DOI:S0092-8674(19)30459-3 Baryawno N, Przybylski D, Kowalczyk MS, Kfoury Y, Severe N, Gustafsson K, Kokkaliaris KD, Mercier F, Tabaka M, Hofree M, Dionne D, Papazian A, Lee D, Ashenberg O, Subramanian A, Vaishnav ED, Rozenblatt-Rosen O, Regev A, Scadden DT. 2019. A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia. Cell. 177(7):1915-1932.e16. Pubmed: 31130381 DOI:S0092-8674(19)30459-3 Stroma is a poorly defined non-parenchymal component of virtually every organ with key roles in organ development, homeostasis, and repair. Studies of the bone marrow stroma have defined individual populations in the stem cell niche regulating hematopoietic regeneration and capable of initiating leukemia. Here, we use single-cell RNA sequencing (scRNA-seq) to define a cellular taxonomy of the mouse bone marrow stroma and its perturbation by malignancy. We identified seventeen stromal subsets expressing distinct hematopoietic regulatory genes spanning new fibroblastic and osteoblastic subpopulations including distinct osteoblast differentiation trajectories. Emerging acute myeloid leukemia impaired mesenchymal osteogenic differentiation and reduced regulatory molecules necessary for normal hematopoiesis. These data suggest that tissue stroma responds to malignant cells by disadvantaging normal parenchymal cells. Our taxonomy of the stromal compartment provides a comprehensive bone marrow cell census and experimental support for cancer cell crosstalk with specific stromal elements to impair normal tissue function and thereby enable emergent cancer.Copyright © 2019 Elsevier Inc. All rights reserved. -
Galvin A, Weglarz M, Folz-Donahue K, Handley M, Baum M, Mazzola M, Litwa H, Scadden DT, Silberstein L. 2019. Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry. Current protocols in cytometry. 87(1):e50. Pubmed: 30335223 DOI:10.1002/cpcy.50 Galvin A, Weglarz M, Folz-Donahue K, Handley M, Baum M, Mazzola M, Litwa H, Scadden DT, Silberstein L. 2019. Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry. Current protocols in cytometry. 87(1):e50. Pubmed: 30335223 DOI:10.1002/cpcy.50 Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley & Sons, Inc.© 2018 John Wiley & Sons, Inc. -
Czechowicz A, Palchaudhuri R, Scheck A, Hu Y, Hoggatt J, Saez B, Pang WW, Mansour MK, Tate TA, Chan YY, Walck E, Wernig G, Shizuru JA, Winau F, Scadden DT, Rossi DJ. 2019. Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation. Nature communications. 10(1):617. Pubmed: 30728354 DOI:10.1038/s41467-018-08201-x Czechowicz A, Palchaudhuri R, Scheck A, Hu Y, Hoggatt J, Saez B, Pang WW, Mansour MK, Tate TA, Chan YY, Walck E, Wernig G, Shizuru JA, Winau F, Scadden DT, Rossi DJ. 2019. Selective hematopoietic stem cell ablation using CD117-antibody-drug-conjugates enables safe and effective transplantation with immunity preservation. Nature communications. 10(1):617. Pubmed: 30728354 DOI:10.1038/s41467-018-08201-x Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired. -
Shah NJ, Mao AS, Shih TY, Kerr MD, Sharda A, Raimondo TM, Weaver JC, Vrbanac VD, Deruaz M, Tager AM, Mooney DJ, Scadden DT. 2019. An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation. Nature biotechnology. 37(3):293-302. Pubmed: 30742125 DOI:10.1038/s41587-019-0017-2 Shah NJ, Mao AS, Shih TY, Kerr MD, Sharda A, Raimondo TM, Weaver JC, Vrbanac VD, Deruaz M, Tager AM, Mooney DJ, Scadden DT. 2019. An injectable bone marrow-like scaffold enhances T cell immunity after hematopoietic stem cell transplantation. Nature biotechnology. 37(3):293-302. Pubmed: 30742125 DOI:10.1038/s41587-019-0017-2 Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for multiple disorders, but deficiency and dysregulation of T cells limit its utility. Here we report a biomaterial-based scaffold that mimics features of T cell lymphopoiesis in the bone marrow. The bone marrow cryogel (BMC) releases bone morphogenetic protein-2 to recruit stromal cells and presents the Notch ligand Delta-like ligand-4 to facilitate T cell lineage specification of mouse and human hematopoietic progenitor cells. BMCs subcutaneously injected in mice at the time of HSCT enhanced T cell progenitor seeding of the thymus, T cell neogenesis and diversification of the T cell receptor repertoire. Peripheral T cell reconstitution increased ~6-fold in mouse HSCT and ~2-fold in human xenogeneic HSCT. Furthermore, BMCs promoted donor CD4 regulatory T cell generation and improved survival after allogeneic HSCT. In comparison to adoptive transfer of T cell progenitors, BMCs increased donor chimerism, T cell generation and antigen-specific T cell responses to vaccination. BMCs may provide an off-the-shelf approach for enhancing T cell regeneration and mitigating graft-versus-host disease in HSCT. -
Scadden DT. 2019. Stem Cell Transplant Approaches for Patients With Blood Cancers. Oncology (Williston Park, N.Y.). 33(3):86-8. Pubmed: 30866029 DOI:623488 Scadden DT. 2019. Stem Cell Transplant Approaches for Patients With Blood Cancers. Oncology (Williston Park, N.Y.). 33(3):86-8. Pubmed: 30866029 DOI:623488 -
Ni F, Yu WM, Wang X, Fay ME, Young KM, Qiu Y, Lam WA, Sulchek TA, Cheng T, Scadden DT, Qu CK. 2019. Ptpn21 Controls Hematopoietic Stem Cell Homeostasis and Biomechanics. Cell stem cell. 24(4):608-620.e6. Pubmed: 30880025 DOI:S1934-5909(19)30051-7 Ni F, Yu WM, Wang X, Fay ME, Young KM, Qiu Y, Lam WA, Sulchek TA, Cheng T, Scadden DT, Qu CK. 2019. Ptpn21 Controls Hematopoietic Stem Cell Homeostasis and Biomechanics. Cell stem cell. 24(4):608-620.e6. Pubmed: 30880025 DOI:S1934-5909(19)30051-7 Hematopoietic stem cell (HSC) quiescence is a tightly regulated process crucial for hematopoietic regeneration, which requires a healthy and supportive microenvironmental niche within the bone marrow (BM). Here, we show that deletion of Ptpn21, a protein tyrosine phosphatase highly expressed in HSCs, induces stem cell egress from the niche due to impaired retention within the BM. Ptpn21 HSCs exhibit enhanced mobility, decreased quiescence, increased apoptosis, and defective reconstitution capacity. Ptpn21 deletion also decreased HSC stiffness and increased physical deformability, in part by dephosphorylating Spetin1 (Tyr), a poorly described component of the cytoskeleton. Elevated phosphorylation of Spetin1 in Ptpn21 cells impaired cytoskeletal remodeling, contributed to cortical instability, and decreased cell rigidity. Collectively, these findings show that Ptpn21 maintains cellular mechanics, which is correlated with its important functions in HSC niche retention and preservation of hematopoietic regeneration capacity.Copyright © 2019 Elsevier Inc. All rights reserved. -
Christian S, Merz C, Evans L, Gradl S, Seidel H, Friberg A, Eheim A, Lejeune P, Brzezinka K, Zimmermann K, Ferrara S, Meyer H, Lesche R, Stoeckigt D, Bauser M, Haegebarth A, Sykes DB, Scadden DT, Losman JA, Janzer A. 2019. The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies. Leukemia. 33(10):2403-2415. Pubmed: 30940908 DOI:10.1038/s41375-019-0461-5 Christian S, Merz C, Evans L, Gradl S, Seidel H, Friberg A, Eheim A, Lejeune P, Brzezinka K, Zimmermann K, Ferrara S, Meyer H, Lesche R, Stoeckigt D, Bauser M, Haegebarth A, Sykes DB, Scadden DT, Losman JA, Janzer A. 2019. The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies. Leukemia. 33(10):2403-2415. Pubmed: 30940908 DOI:10.1038/s41375-019-0461-5 Acute myeloid leukemia (AML) is a devastating disease, with the majority of patients dying within a year of diagnosis. For patients with relapsed/refractory AML, the prognosis is particularly poor with currently available treatments. Although genetically heterogeneous, AML subtypes share a common differentiation arrest at hematopoietic progenitor stages. Overcoming this differentiation arrest has the potential to improve the long-term survival of patients, as is the case in acute promyelocytic leukemia (APL), which is characterized by a chromosomal translocation involving the retinoic acid receptor alpha gene. Treatment of APL with all-trans retinoic acid (ATRA) induces terminal differentiation and apoptosis of leukemic promyelocytes, resulting in cure rates of over 80%. Unfortunately, similarly efficacious differentiation therapies have, to date, been lacking outside of APL. Inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in the de novo pyrimidine synthesis pathway, was recently reported to induce differentiation of diverse AML subtypes. In this report we describe the discovery and characterization of BAY 2402234 - a novel, potent, selective and orally bioavailable DHODH inhibitor that shows monotherapy efficacy and differentiation induction across multiple AML subtypes. Herein, we present the preclinical data that led to initiation of a phase I evaluation of this inhibitor in myeloid malignancies. -
Scaramozza A, Park D, Kollu S, Beerman I, Sun X, Rossi DJ, Lin CP, Scadden DT, Crist C, Brack AS. 2019. Lineage Tracing Reveals a Subset of Reserve Muscle Stem Cells Capable of Clonal Expansion under Stress. Cell stem cell. 24(6):944-957.e5. Pubmed: 31006621 DOI:S1934-5909(19)30119-5 Scaramozza A, Park D, Kollu S, Beerman I, Sun X, Rossi DJ, Lin CP, Scadden DT, Crist C, Brack AS. 2019. Lineage Tracing Reveals a Subset of Reserve Muscle Stem Cells Capable of Clonal Expansion under Stress. Cell stem cell. 24(6):944-957.e5. Pubmed: 31006621 DOI:S1934-5909(19)30119-5 Stem cell heterogeneity is recognized as functionally relevant for tissue homeostasis and repair. The identity, context dependence, and regulation of skeletal muscle satellite cell (SC) subsets remains poorly understood. We identify a minor subset of Pax7 SCs that is indelibly marked by an inducible Mx1-Cre transgene in vivo, is enriched for Pax3 expression, and has reduced ROS (reactive oxygen species) levels. Mx1 SCs possess potent stem cell activity upon transplantation but minimally contribute to endogenous muscle repair, due to their relative low abundance. In contrast, a dramatic clonal expansion of Mx1 SCs allows extensive contribution to muscle repair and niche repopulation upon selective pressure of radiation stress, consistent with reserve stem cell (RSC) properties. Loss of Pax3 in RSCs increased ROS content and diminished survival and stress tolerance. These observations demonstrate that the Pax7 SC pool contains a discrete population of radiotolerant RSCs that undergo clonal expansion under severe stress.Copyright © 2019 Elsevier Inc. All rights reserved. -
Ortinau LC, Wang H, Lei K, Deveza L, Jeong Y, Hara Y, Grafe I, Rosenfeld SB, Lee D, Lee B, Scadden DT, Park D. 2019. Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells. Cell stem cell. 25(6):784-796.e5. Pubmed: 31809737 DOI:S1934-5909(19)30458-8 Ortinau LC, Wang H, Lei K, Deveza L, Jeong Y, Hara Y, Grafe I, Rosenfeld SB, Lee D, Lee B, Scadden DT, Park D. 2019. Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells. Cell stem cell. 25(6):784-796.e5. Pubmed: 31809737 DOI:S1934-5909(19)30458-8 The periosteum is critical for bone maintenance and healing. However, the in vivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible for lifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration in vivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.Copyright © 2019 Elsevier Inc. All rights reserved. -
Gustafsson K, Scadden DT. 2019. Growing old in the age of heterogeneity: the perils of shifting clonality. Current opinion in hematology. 26(4):222-227. Pubmed: 31045646 DOI:10.1097/MOH.0000000000000513 Gustafsson K, Scadden DT. 2019. Growing old in the age of heterogeneity: the perils of shifting clonality. Current opinion in hematology. 26(4):222-227. Pubmed: 31045646 DOI:10.1097/MOH.0000000000000513 Array -
Frodermann V, Rohde D, Courties G, Severe N, Schloss MJ, Amatullah H, McAlpine CS, Cremer S, Hoyer FF, Ji F, van Koeverden ID, Herisson F, Honold L, Masson GS, Zhang S, Grune J, Iwamoto Y, Schmidt SP, Wojtkiewicz GR, Lee IH, Gustafsson K, Pasterkamp G, de Jager SCA, Sadreyev RI, MacFadyen J, Libby P, Ridker P, Scadden DT, Naxerova K, Jeffrey KL, Swirski FK, Nahrendorf M. 2019. Exercise reduces inflammatory cell production and cardiovascular inflammation via instruction of hematopoietic progenitor cells. Nature medicine. 25(11):1761-1771. Pubmed: 31700184 DOI:10.1038/s41591-019-0633-x Frodermann V, Rohde D, Courties G, Severe N, Schloss MJ, Amatullah H, McAlpine CS, Cremer S, Hoyer FF, Ji F, van Koeverden ID, Herisson F, Honold L, Masson GS, Zhang S, Grune J, Iwamoto Y, Schmidt SP, Wojtkiewicz GR, Lee IH, Gustafsson K, Pasterkamp G, de Jager SCA, Sadreyev RI, MacFadyen J, Libby P, Ridker P, Scadden DT, Naxerova K, Jeffrey KL, Swirski FK, Nahrendorf M. 2019. Exercise reduces inflammatory cell production and cardiovascular inflammation via instruction of hematopoietic progenitor cells. Nature medicine. 25(11):1761-1771. Pubmed: 31700184 DOI:10.1038/s41591-019-0633-x A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creER; Lepr mice reveals that leptin's effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes. -
Mao AS, Özkale B, Shah NJ, Vining KH, Descombes T, Zhang L, Tringides CM, Wong SW, Shin JW, Scadden DT, Weitz DA, Mooney DJ. 2019. Programmable microencapsulation for enhanced mesenchymal stem cell persistence and immunomodulation. Proceedings of the National Academy of Sciences of the United States of America. 116(31):15392-15397. Pubmed: 31311862 DOI:10.1073/pnas.1819415116 Mao AS, Özkale B, Shah NJ, Vining KH, Descombes T, Zhang L, Tringides CM, Wong SW, Shin JW, Scadden DT, Weitz DA, Mooney DJ. 2019. Programmable microencapsulation for enhanced mesenchymal stem cell persistence and immunomodulation. Proceedings of the National Academy of Sciences of the United States of America. 116(31):15392-15397. Pubmed: 31311862 DOI:10.1073/pnas.1819415116 Mesenchymal stem cell (MSC) therapies demonstrate particular promise in ameliorating diseases of immune dysregulation but are hampered by short in vivo cell persistence and inconsistencies in phenotype. Here, we demonstrate that biomaterial encapsulation into alginate using a microfluidic device could substantially increase in vivo MSC persistence after intravenous (i.v.) injection. A combination of cell cluster formation and subsequent cross-linking with polylysine led to an increase in injected MSC half-life by more than an order of magnitude. These modifications extended persistence even in the presence of innate and adaptive immunity-mediated clearance. Licensing of encapsulated MSCs with inflammatory cytokine pretransplantation increased expression of immunomodulatory-associated genes, and licensed encapsulates promoted repopulation of recipient blood and bone marrow with allogeneic donor cells after sublethal irradiation by a ∼2-fold increase. The ability of microgel encapsulation to sustain MSC survival and increase overall immunomodulatory capacity may be applicable for improving MSC therapies in general. -
Severe N, Karabacak NM, Gustafsson K, Baryawno N, Courties G, Kfoury Y, Kokkaliaris KD, Rhee C, Lee D, Scadden EW, Garcia-Robledo JE, Brouse T, Nahrendorf M, Toner M, Scadden DT. 2019. Stress-Induced Changes in Bone Marrow Stromal Cell Populations Revealed through Single-Cell Protein Expression Mapping. Cell stem cell. 25(4):570-583.e7. Pubmed: 31279774 DOI:S1934-5909(19)30267-X Severe N, Karabacak NM, Gustafsson K, Baryawno N, Courties G, Kfoury Y, Kokkaliaris KD, Rhee C, Lee D, Scadden EW, Garcia-Robledo JE, Brouse T, Nahrendorf M, Toner M, Scadden DT. 2019. Stress-Induced Changes in Bone Marrow Stromal Cell Populations Revealed through Single-Cell Protein Expression Mapping. Cell stem cell. 25(4):570-583.e7. Pubmed: 31279774 DOI:S1934-5909(19)30267-X Stromal cell populations that maintain hematopoietic stem and progenitor cells (HSPCs) are generally characterized in steady-state conditions. Here, we report a comprehensive atlas of bone marrow stromal cell subpopulations under homeostatic and stress conditions using mass cytometry (CyTOF)-based single-cell protein analysis. We identified 28 subsets of non-hematopoietic cells during homeostasis, 14 of which expressed hematopoietic regulatory factors. Irradiation-based conditioning for HSPC transplantation led to the loss of most of these populations, including the LeptinR and Nestin subsets. In contrast, a subset expressing Ecto-5'-nucleotidase (CD73) was retained and a specific CD73NGFR population expresses high levels of cytokines during homeostasis and stress. Genetic ablation of CD73 compromised HSPC transplantation in an acute setting without long-term changes in bone marrow HSPCs. Thus, this protein-based expression mapping reveals distinct sets of stromal cells in the bone marrow and how they change in clinically relevant stress settings to contribute to early stages of hematopoietic regeneration.Copyright © 2019 Elsevier Inc. All rights reserved. -
Courties G, Frodermann V, Honold L, Zheng Y, Herisson F, Schloss MJ, Sun Y, Presumey J, Severe N, Engblom C, Hulsmans M, Cremer S, Rohde D, Pittet MJ, Scadden DT, Swirski FK, Kim DE, Moskowitz MA, Nahrendorf M. 2019. Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke. Circulation research. 124(9):1372-1385. Pubmed: 30782088 DOI:10.1161/CIRCRESAHA.118.314518 Courties G, Frodermann V, Honold L, Zheng Y, Herisson F, Schloss MJ, Sun Y, Presumey J, Severe N, Engblom C, Hulsmans M, Cremer S, Rohde D, Pittet MJ, Scadden DT, Swirski FK, Kim DE, Moskowitz MA, Nahrendorf M. 2019. Glucocorticoids Regulate Bone Marrow B Lymphopoiesis After Stroke. Circulation research. 124(9):1372-1385. Pubmed: 30782088 DOI:10.1161/CIRCRESAHA.118.314518 Array 2018
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Dietrich J, Baryawno N, Nayyar N, Valtis YK, Yang B, Ly I, Besnard A, Severe N, Gustafsson KU, Andronesi OC, Batchelor TT, Sahay A, Scadden DT. 2018. Bone marrow drives central nervous system regeneration after radiation injury. The Journal of clinical investigation. 128(1):281-293. Pubmed: 29202481 DOI:90647 Dietrich J, Baryawno N, Nayyar N, Valtis YK, Yang B, Ly I, Besnard A, Severe N, Gustafsson KU, Andronesi OC, Batchelor TT, Sahay A, Scadden DT. 2018. Bone marrow drives central nervous system regeneration after radiation injury. The Journal of clinical investigation. 128(1):281-293. Pubmed: 29202481 DOI:90647 Nervous system injury is a frequent result of cancer therapy involving cranial irradiation, leaving patients with marked memory and other neurobehavioral disabilities. Here, we report an unanticipated link between bone marrow and brain in the setting of radiation injury. Specifically, we demonstrate that bone marrow-derived monocytes and macrophages are essential for structural and functional repair mechanisms, including regeneration of cerebral white matter and improvement in neurocognitive function. Using a granulocyte-colony stimulating factor (G-CSF) receptor knockout mouse model in combination with bone marrow cell transplantation, MRI, and neurocognitive functional assessments, we demonstrate that bone marrow-derived G-CSF-responsive cells home to the injured brain and are critical for altering neural progenitor cells and brain repair. Additionally, compared with untreated animals, animals that received G-CSF following radiation injury exhibited enhanced functional brain repair. Together, these results demonstrate that, in addition to its known role in defense and debris removal, the hematopoietic system provides critical regenerative drive to the brain that can be modulated by clinically available agents. -
Hoggatt J, Singh P, Tate TA, Chou BK, Datari SR, Fukuda S, Liu L, Kharchenko PV, Schajnovitz A, Baryawno N, Mercier FE, Boyer J, Gardner J, Morrow DM, Scadden DT, Pelus LM. 2018. Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell. Cell. 172(1-2):191-204.e10. Pubmed: 29224778 DOI:S0092-8674(17)31312-0 Hoggatt J, Singh P, Tate TA, Chou BK, Datari SR, Fukuda S, Liu L, Kharchenko PV, Schajnovitz A, Baryawno N, Mercier FE, Boyer J, Gardner J, Morrow DM, Scadden DT, Pelus LM. 2018. Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell. Cell. 172(1-2):191-204.e10. Pubmed: 29224778 DOI:S0092-8674(17)31312-0 Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROβ, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.Copyright © 2017 Elsevier Inc. All rights reserved. -
Almeida FF, Tognarelli S, Marçais A, Kueh AJ, Friede ME, Liao Y, Willis SN, Luong K, Faure F, Mercier FE, Galluso J, Firth M, Narni-Mancinelli E, Rais B, Scadden DT, Spallotta F, Weil S, Giannattasio A, Kalensee F, Zöller T, Huntington ND, Schleicher U, Chiocchetti AG, Ugolini S, Herold MJ, Shi W, Koch J, Steinle A, Vivier E, Walzer T, Belz GT, Ullrich E. 2018. A point mutation in the signal peptide impairs the development of innate lymphoid cell subsets. Oncoimmunology. 7(10):e1475875. Pubmed: 30288342 DOI:10.1080/2162402X.2018.1475875 Almeida FF, Tognarelli S, Marçais A, Kueh AJ, Friede ME, Liao Y, Willis SN, Luong K, Faure F, Mercier FE, Galluso J, Firth M, Narni-Mancinelli E, Rais B, Scadden DT, Spallotta F, Weil S, Giannattasio A, Kalensee F, Zöller T, Huntington ND, Schleicher U, Chiocchetti AG, Ugolini S, Herold MJ, Shi W, Koch J, Steinle A, Vivier E, Walzer T, Belz GT, Ullrich E. 2018. A point mutation in the signal peptide impairs the development of innate lymphoid cell subsets. Oncoimmunology. 7(10):e1475875. Pubmed: 30288342 DOI:10.1080/2162402X.2018.1475875 NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCR strain. Ly5.1 NK cells expressed similar levels of mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors . Expression of the mutant NKp46 in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1 mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49aILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function. Innate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells . This C14R mutation impairs NKp46 surface expression resulting in destabilization of and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46 ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively. -
Ubellacker JM, Baryawno N, Severe N, DeCristo MJ, Sceneay J, Hutchinson JN, Haider MT, Rhee CS, Qin Y, Gregory WM, Garrido-Castro AC, Holen I, Brown JE, Coleman RE, Scadden DT, McAllister SS. 2018. Modulating Bone Marrow Hematopoietic Lineage Potential to Prevent Bone Metastasis in Breast Cancer. Cancer research. 78(18):5300-5314. Pubmed: 30065048 DOI:10.1158/0008-5472.CAN-18-0548 Ubellacker JM, Baryawno N, Severe N, DeCristo MJ, Sceneay J, Hutchinson JN, Haider MT, Rhee CS, Qin Y, Gregory WM, Garrido-Castro AC, Holen I, Brown JE, Coleman RE, Scadden DT, McAllister SS. 2018. Modulating Bone Marrow Hematopoietic Lineage Potential to Prevent Bone Metastasis in Breast Cancer. Cancer research. 78(18):5300-5314. Pubmed: 30065048 DOI:10.1158/0008-5472.CAN-18-0548 The presence of disseminated tumor cells in breast cancer patient bone marrow aspirates predicts decreased recurrence-free survival. Although it is appreciated that physiologic, pathologic, and therapeutic conditions impact hematopoiesis, it remains unclear whether targeting hematopoiesis presents opportunities for limiting bone metastasis. Using preclinical breast cancer models, we discovered that marrow from mice treated with the bisphosphonate zoledronic acid (ZA) are metastasis-suppressive. Specifically, ZA modulated hematopoietic myeloid/osteoclast progenitor cell (M/OCP) lineage potential to activate metastasis-suppressive activity. Granulocyte-colony stimulating factor (G-CSF) promoted ZA resistance by redirecting M/OCP differentiation. We identified M/OCP and bone marrow transcriptional programs associated with metastasis suppression and ZA resistance. Analysis of patient blood samples taken at randomization revealed that women with high-plasma G-CSF experienced significantly worse outcome with adjuvant ZA than those with lower G-CSF levels. Our findings support discovery of therapeutic strategies to direct M/OCP lineage potential and biomarkers that stratify responses in patients at risk of recurrence. Bone marrow myeloid/osteoclast progenitor cell lineage potential has a profound impact on breast cancer bone metastasis and can be modulated by G-CSF and bone-targeting agents. .©2018 American Association for Cancer Research. -
Petukhov V, Guo J, Baryawno N, Severe N, Scadden DT, Samsonova MG, Kharchenko PV. 2018. dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments. Genome biology. 19(1):78. Pubmed: 29921301 DOI:10.1186/s13059-018-1449-6 Petukhov V, Guo J, Baryawno N, Severe N, Scadden DT, Samsonova MG, Kharchenko PV. 2018. dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments. Genome biology. 19(1):78. Pubmed: 29921301 DOI:10.1186/s13059-018-1449-6 Recent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells. -
Shao L, Chang J, Feng W, Wang X, Williamson EA, Li Y, Schajnovitz A, Scadden D, Mortensen LJ, Lin CP, Li L, Paulson A, Downing J, Zhou D, Hromas RA. 2018. The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow. Nature communications. 9(1):2377. Pubmed: 29915352 DOI:10.1038/s41467-018-04716-5 Shao L, Chang J, Feng W, Wang X, Williamson EA, Li Y, Schajnovitz A, Scadden D, Mortensen LJ, Lin CP, Li L, Paulson A, Downing J, Zhou D, Hromas RA. 2018. The Wave2 scaffold Hem-1 is required for transition of fetal liver hematopoiesis to bone marrow. Nature communications. 9(1):2377. Pubmed: 29915352 DOI:10.1038/s41467-018-04716-5 The transition of hematopoiesis from the fetal liver (FL) to the bone marrow (BM) is incompletely characterized. We demonstrate that the Wiskott-Aldrich syndrome verprolin-homologous protein (WAVE) complex 2 is required for this transition, as complex degradation via deletion of its scaffold Hem-1 causes the premature exhaustion of neonatal BM hematopoietic stem cells (HSCs). This exhaustion of BM HSC is due to the failure of BM engraftment of Hem-1 FL HSCs, causing early death. The Hem-1 FL HSC engraftment defect is not due to the lack of the canonical function of the WAVE2 complex, the regulation of actin polymerization, because FL HSCs from Hem-1 mice exhibit no defects in chemotaxis, BM homing, or adhesion. Rather, the failure of Hem-1 FL HSC engraftment in the marrow is due to the loss of c-Abl survival signaling from degradation of the WAVE2 complex. However, c-Abl activity is dispensable for the engraftment of adult BM HSCs into the BM. These findings reveal a novel function of the WAVE2 complex and define a mechanism for FL HSC fitness in the embryonic BM niche. -
Dietrich J, Baryawno N, Nayyar N, Valtis YK, Yang B, Ly I, Besnard A, Severe N, Gustafsson KU, Andronesi OC, Batchelor TT, Sahay A, Scadden DT. 2018. Bone marrow drives central nervous system regeneration after radiation injury. The Journal of clinical investigation. 128(6):2651. Pubmed: 29856368 DOI:121592 Dietrich J, Baryawno N, Nayyar N, Valtis YK, Yang B, Ly I, Besnard A, Severe N, Gustafsson KU, Andronesi OC, Batchelor TT, Sahay A, Scadden DT. 2018. Bone marrow drives central nervous system regeneration after radiation injury. The Journal of clinical investigation. 128(6):2651. Pubmed: 29856368 DOI:121592 -
García-Peydró M, Fuentes P, Mosquera M, García-León MJ, Alcain J, Rodríguez A, García de Miguel P, Menéndez P, Weijer K, Spits H, Scadden DT, Cuesta-Mateos C, Muñoz-Calleja C, Sánchez-Madrid F, Toribio ML. 2018. The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model. The Journal of clinical investigation. 128(7):2802-2818. Pubmed: 29781813 DOI:92981 García-Peydró M, Fuentes P, Mosquera M, García-León MJ, Alcain J, Rodríguez A, García de Miguel P, Menéndez P, Weijer K, Spits H, Scadden DT, Cuesta-Mateos C, Muñoz-Calleja C, Sánchez-Madrid F, Toribio ML. 2018. The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model. The Journal of clinical investigation. 128(7):2802-2818. Pubmed: 29781813 DOI:92981 NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse. 2017
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Garrison BS, Rybak AP, Beerman I, Heesters B, Mercier FE, Scadden DT, Bryder D, Baron R, Rossi DJ. 2017. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells. Blood. 130(5):619-624. Pubmed: 28615219 DOI:10.1182/blood-2016-09-738591 Garrison BS, Rybak AP, Beerman I, Heesters B, Mercier FE, Scadden DT, Bryder D, Baron R, Rossi DJ. 2017. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells. Blood. 130(5):619-624. Pubmed: 28615219 DOI:10.1182/blood-2016-09-738591 The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify / as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using -deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that is highly and specifically upregulated in AMLs with translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of reduced proliferation in human leukemia cell lines possessing translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice. -
Schiroli G, Ferrari S, Conway A, Jacob A, Capo V, Albano L, Plati T, Castiello MC, Sanvito F, Gennery AR, Bovolenta C, Palchaudhuri R, Scadden DT, Holmes MC, Villa A, Sitia G, Lombardo A, Genovese P, Naldini L. 2017. Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1. Science translational medicine. 9(411). Pubmed: 29021165 DOI:10.1126/scitranslmed.aan0820 Schiroli G, Ferrari S, Conway A, Jacob A, Capo V, Albano L, Plati T, Castiello MC, Sanvito F, Gennery AR, Bovolenta C, Palchaudhuri R, Scadden DT, Holmes MC, Villa A, Sitia G, Lombardo A, Genovese P, Naldini L. 2017. Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1. Science translational medicine. 9(411). Pubmed: 29021165 DOI:10.1126/scitranslmed.aan0820 Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the -edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Engblom C, Pfirschke C, Zilionis R, Da Silva Martins J, Bos SA, Courties G, Rickelt S, Severe N, Baryawno N, Faget J, Savova V, Zemmour D, Kline J, Siwicki M, Garris C, Pucci F, Liao HW, Lin YJ, Newton A, Yaghi OK, Iwamoto Y, Tricot B, Wojtkiewicz GR, Nahrendorf M, Cortez-Retamozo V, Meylan E, Hynes RO, Demay M, Klein A, Bredella MA, Scadden DT, Weissleder R, Pittet MJ. 2017. Osteoblasts remotely supply lung tumors with cancer-promoting SiglecF neutrophils. Science (New York, N.Y.). 358(6367). Pubmed: 29191879 DOI:10.1126/science.aal5081 Engblom C, Pfirschke C, Zilionis R, Da Silva Martins J, Bos SA, Courties G, Rickelt S, Severe N, Baryawno N, Faget J, Savova V, Zemmour D, Kline J, Siwicki M, Garris C, Pucci F, Liao HW, Lin YJ, Newton A, Yaghi OK, Iwamoto Y, Tricot B, Wojtkiewicz GR, Nahrendorf M, Cortez-Retamozo V, Meylan E, Hynes RO, Demay M, Klein A, Bredella MA, Scadden DT, Weissleder R, Pittet MJ. 2017. Osteoblasts remotely supply lung tumors with cancer-promoting SiglecF neutrophils. Science (New York, N.Y.). 358(6367). Pubmed: 29191879 DOI:10.1126/science.aal5081 Bone marrow-derived myeloid cells can accumulate within tumors and foster cancer outgrowth. Local immune-neoplastic interactions have been intensively investigated, but the contribution of the systemic host environment to tumor growth remains poorly understood. Here, we show in mice and cancer patients ( = 70) that lung adenocarcinomas increase bone stromal activity in the absence of bone metastasis. Animal studies reveal that the cancer-induced bone phenotype involves bone-resident osteocalcin-expressing (Ocn) osteoblastic cells. These cells promote cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecF neutrophils, which exhibit cancer-promoting properties. Experimentally reducing Ocn cell numbers suppresses the neutrophil response and lung tumor outgrowth. These observations posit osteoblasts as remote regulators of lung cancer and identify SiglecF neutrophils as myeloid cell effectors of the osteoblast-driven protumoral response.Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. -
Gustafsson K, Scadden DT. 2017. Written in bone: young bone makes young blood. The EMBO journal. 36(7):831-833. Pubmed: 28289055 DOI:10.15252/embj.201796634 Gustafsson K, Scadden DT. 2017. Written in bone: young bone makes young blood. The EMBO journal. 36(7):831-833. Pubmed: 28289055 DOI:10.15252/embj.201796634 Ageing of bone marrow haematopoietic stem cells has been mostly associated with cell‐intrinsic mechanisms. New findings published in now show that reduced levels of the secreted matrix protein osteopontin in old bone marrow stroma cause ageing‐associated features in HSCs. In line, old HSCs are functionally rejuvenated by interaction with protease‐cleaved osteopontin fragment, and perform like young blood stem cells . -
Kalaitzidis D, Lee D, Efeyan A, Kfoury Y, Nayyar N, Sykes DB, Mercier FE, Papazian A, Baryawno N, Victora GD, Neuberg D, Sabatini DM, Scadden DT. 2017. Amino acid-insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells. The Journal of clinical investigation. 127(4):1405-1413. Pubmed: 28319048 DOI:89452 Kalaitzidis D, Lee D, Efeyan A, Kfoury Y, Nayyar N, Sykes DB, Mercier FE, Papazian A, Baryawno N, Victora GD, Neuberg D, Sabatini DM, Scadden DT. 2017. Amino acid-insensitive mTORC1 regulation enables nutritional stress resilience in hematopoietic stem cells. The Journal of clinical investigation. 127(4):1405-1413. Pubmed: 28319048 DOI:89452 The mTOR pathway is a critical determinant of cell persistence and growth wherein mTOR complex 1 (mTORC1) mediates a balance between growth factor stimuli and nutrient availability. Amino acids or glucose facilitates mTORC1 activation by inducing RagA GTPase recruitment of mTORC1 to the lysosomal outer surface, enabling activation of mTOR by the Ras homolog Rheb. Thereby, RagA alters mTORC1-driven growth in times of nutrient abundance or scarcity. Here, we have evaluated differential nutrient-sensing dependence through RagA and mTORC1 in hematopoietic progenitors, which dynamically drive mature cell production, and hematopoietic stem cells (HSC), which provide a quiescent cellular reserve. In nutrient-abundant conditions, RagA-deficient HSC were functionally unimpaired and upregulated mTORC1 via nutrient-insensitive mechanisms. RagA was also dispensable for HSC function under nutritional stress conditions. Similarly, hyperactivation of RagA did not affect HSC function. In contrast, RagA deficiency markedly altered progenitor population function and mature cell output. Therefore, RagA is a molecular mechanism that distinguishes the functional attributes of reactive progenitors from a reserve stem cell pool. The indifference of HSC to nutrient sensing through RagA contributes to their molecular resilience to nutritional stress, a characteristic that is relevant to organismal viability in evolution and in modern HSC transplantation approaches. -
Hahm E, Wei C, Fernandez I, Li J, Tardi NJ, Tracy M, Wadhwani S, Cao Y, Peev V, Zloza A, Lusciks J, Hayek SS, O'Connor C, Bitzer M, Gupta V, Sever S, Sykes DB, Scadden DT, Reiser J. 2017. Bone marrow-derived immature myeloid cells are a main source of circulating suPAR contributing to proteinuric kidney disease. Nature medicine. 23(1):100-106. Pubmed: 27941791 DOI:10.1038/nm.4242 Hahm E, Wei C, Fernandez I, Li J, Tardi NJ, Tracy M, Wadhwani S, Cao Y, Peev V, Zloza A, Lusciks J, Hayek SS, O'Connor C, Bitzer M, Gupta V, Sever S, Sykes DB, Scadden DT, Reiser J. 2017. Bone marrow-derived immature myeloid cells are a main source of circulating suPAR contributing to proteinuric kidney disease. Nature medicine. 23(1):100-106. Pubmed: 27941791 DOI:10.1038/nm.4242 Excess levels of protein in urine (proteinuria) is a hallmark of kidney disease that typically occurs in conjunction with diabetes, hypertension, gene mutations, toxins or infections but may also be of unknown cause (idiopathic). Systemic soluble urokinase plasminogen activator receptor (suPAR) is a circulating factor implicated in the onset and progression of chronic kidney disease (CKD), such as focal segmental glomerulosclerosis (FSGS). The cellular source(s) of elevated suPAR associated with future and progressing kidney disease is unclear, but is likely extra-renal, as the pathological uPAR is circulating and FSGS can recur even after a damaged kidney is replaced with a healthy donor organ. Here we report that bone marrow (BM) Gr-1 immature myeloid cells are responsible for the elevated, pathological levels of suPAR, as evidenced by BM chimera and BM ablation and cell transfer studies. A marked increase of Gr-1 myeloid cells was commonly found in the BM of proteinuric animals having high suPAR, and these cells efficiently transmit proteinuria when transferred to healthy mice. In accordance with the results seen in suPAR-associated proteinuric animal models, in which kidney damage is caused not by local podocyte-selective injury but more likely by systemic insults, a humanized xenograft model of FSGS resulted in an expansion of Gr-1 cells in the BM, leading to high plasma suPAR and proteinuric kidney disease. Together, these results identify suPAR as a functional connection between the BM and the kidney, and they implicate BM immature myeloid cells as a key contributor to glomerular dysfunction. -
Lee PY, Sykes DB, Ameri S, Kalaitzidis D, Charles JF, Nelson-Maney N, Wei K, Cunin P, Morris A, Cardona AE, Root DE, Scadden DT, Nigrovic PA. 2017. The metabolic regulator mTORC1 controls terminal myeloid differentiation. Science immunology. 2(11). Pubmed: 28763796 DOI:10.1126/sciimmunol.aam6641 Lee PY, Sykes DB, Ameri S, Kalaitzidis D, Charles JF, Nelson-Maney N, Wei K, Cunin P, Morris A, Cardona AE, Root DE, Scadden DT, Nigrovic PA. 2017. The metabolic regulator mTORC1 controls terminal myeloid differentiation. Science immunology. 2(11). Pubmed: 28763796 DOI:10.1126/sciimmunol.aam6641 Monocytes are derived from hematopoietic stem cells through a series of intermediate progenitor stages, but the factors that regulate this process are incompletely defined. Using a Ccr2/Cxcr1 dual-reporter system to model murine monocyte ontogeny, we conducted a small-molecule screen that identified an essential role of mechanistic target of rapamycin complex 1 (mTORC1) in the development of monocytes and other myeloid cells. Confirmatory studies using mice with inducible deletion of the mTORC1 component Raptor demonstrated absence of mature circulating monocytes, as well as disruption in neutrophil and dendritic cell development, reflecting arrest of terminal differentiation at the granulocyte-monocyte progenitor stage. Conversely, excess activation of mTORC1 through deletion of the mTORC1 inhibitor tuberous sclerosis complex 2 promoted spontaneous myeloid cell development and maturation. Inhibitor studies and stage-specific expression profiling identified failure to down-regulate the transcription factor Myc by the mTORC1 target ribosomal S6 kinase 1 (S6K1) as the mechanistic basis for disrupted myelopoiesis. Together, these findings define the mTORC1-S6K1-Myc pathway as a key checkpoint in terminal myeloid development.Copyright © 2017, American Association for the Advancement of Science. -
Papazian AE, Kfoury YS, Scadden DT. 2017. Shipping mouse bone marrow: Keep it in the bone. Experimental hematology. 49:68-72. Pubmed: 28043821 DOI:S0301-472X(16)30776-7 Papazian AE, Kfoury YS, Scadden DT. 2017. Shipping mouse bone marrow: Keep it in the bone. Experimental hematology. 49:68-72. Pubmed: 28043821 DOI:S0301-472X(16)30776-7 Sharing reagents is of self-evident value in life science research, however, primary cell populations often do not cryopreserve well or can require extensive preparation by collaborators, making shipping difficult. Here we report an evaluation of different conditions for the storage shipment of mouse bone marrow (BM) cells that would best preserve the number, viability, and frequency of different hematopoietic lineages, as well as functionality of progenitor populations. Bones were either crushed to release BM cells or stored intact in one of three media: Phosphate buffered saline (PBS) supplemented with 2% fetal bovine serum (FBS), Plasmalyte, or RPMI at 4°C. Cell numbers, viability, phenotype, and functionality were assessed 16 hours and 40 hours later and compared to freshly prepared samples. Whereas BM cells stored in suspension for 16 hours and BM cells kept in bone for 40 hours suffered major losses in cell number, hematopoietic lineages that were kept in the bone for 16 hours had only minor differences compared to fresh cells. With no significant differences among the different media used, intact long bones stored in media, Plasmalyte, or PBS 2% FBS for up to 16 hours provided a reasonable means of preserving bone marrow cell populations.Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved. -
Yu VWC, Yusuf RZ, Oki T, Wu J, Saez B, Wang X, Cook C, Baryawno N, Ziller MJ, Lee E, Gu H, Meissner A, Lin CP, Kharchenko PV, Scadden DT. 2017. Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells. Cell. 168(5):944-945. Pubmed: 28235203 DOI:S0092-8674(17)30188-5 Yu VWC, Yusuf RZ, Oki T, Wu J, Saez B, Wang X, Cook C, Baryawno N, Ziller MJ, Lee E, Gu H, Meissner A, Lin CP, Kharchenko PV, Scadden DT. 2017. Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells. Cell. 168(5):944-945. Pubmed: 28235203 DOI:S0092-8674(17)30188-5 Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of—and molecular basis for—heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted. -
Baryawno N, Severe N, Scadden DT. 2017. Hematopoiesis: Reconciling Historic Controversies about the Niche. Cell stem cell. 20(5):590-592. Pubmed: 28475884 DOI:S1934-5909(17)30124-8 Baryawno N, Severe N, Scadden DT. 2017. Hematopoiesis: Reconciling Historic Controversies about the Niche. Cell stem cell. 20(5):590-592. Pubmed: 28475884 DOI:S1934-5909(17)30124-8 The niche, as first conceptualized, was in conflict with the prevailing wisdom that stem cells have internal logic. The niche hypothesis has been indisputably confirmed. Yet, recent findings indicate little plasticity of epigenetically scripted hematopoietic stem/progenitors. Reconciling this conflict requires re-envisioning the niche as an enabler, not designer, of cell fate.Copyright © 2017 Elsevier Inc. All rights reserved. 2016
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Palchaudhuri R, Saez B, Hoggatt J, Schajnovitz A, Sykes DB, Tate TA, Czechowicz A, Kfoury Y, Ruchika F, Rossi DJ, Verdine GL, Mansour MK, Scadden DT. 2016. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nature biotechnology. 34(7):738-45. Pubmed: 27272386 DOI:10.1038/nbt.3584 Palchaudhuri R, Saez B, Hoggatt J, Schajnovitz A, Sykes DB, Tate TA, Czechowicz A, Kfoury Y, Ruchika F, Rossi DJ, Verdine GL, Mansour MK, Scadden DT. 2016. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin. Nature biotechnology. 34(7):738-45. Pubmed: 27272386 DOI:10.1038/nbt.3584 Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases. -
Silberstein L, Goncalves KA, Kharchenko PV, Turcotte R, Kfoury Y, Mercier F, Baryawno N, Severe N, Bachand J, Spencer JA, Papazian A, Lee D, Chitteti BR, Srour EF, Hoggatt J, Tate T, Lo Celso C, Ono N, Nutt S, Heino J, Sipilä K, Shioda T, Osawa M, Lin CP, Hu GF, Scadden DT. 2016. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators. Cell stem cell. 19(4):530-543. Pubmed: 27524439 DOI:S1934-5909(16)30199-0 Silberstein L, Goncalves KA, Kharchenko PV, Turcotte R, Kfoury Y, Mercier F, Baryawno N, Severe N, Bachand J, Spencer JA, Papazian A, Lee D, Chitteti BR, Srour EF, Hoggatt J, Tate T, Lo Celso C, Ono N, Nutt S, Heino J, Sipilä K, Shioda T, Osawa M, Lin CP, Hu GF, Scadden DT. 2016. Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators. Cell stem cell. 19(4):530-543. Pubmed: 27524439 DOI:S1934-5909(16)30199-0 Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function.Copyright © 2016 Elsevier Inc. All rights reserved. -
Hoggatt J, Kfoury Y, Scadden DT. 2016. Hematopoietic Stem Cell Niche in Health and Disease. Annual review of pathology. 11:555-81. Pubmed: 27193455 DOI:10.1146/annurev-pathol-012615-044414 Hoggatt J, Kfoury Y, Scadden DT. 2016. Hematopoietic Stem Cell Niche in Health and Disease. Annual review of pathology. 11:555-81. Pubmed: 27193455 DOI:10.1146/annurev-pathol-012615-044414 Regulation of stem cells in adult tissues is a key determinant of how well an organism can respond to the stresses of physiological challenge and disease. This is particularly true of the hematopoietic system, where demands on host defenses can call for an acute increase in cell production. Hematopoietic stem cells receive the regulatory signals for cell production in adult mammals in the bone marrow, a tissue with higher-order architectural and functional organization than previously appreciated. Here, we review the data defining particular structural components and heterologous cells in the bone marrow that participate in hematopoietic stem cell function. Further, we explore the case for stromal-hematopoietic cell interactions contributing to neoplastic myeloid disease. As the hematopoietic regulatory networks in the bone marrow are revealed, it is anticipated that strategies will emerge for how to enhance or inhibit production of specific blood cells. In that way, the control of hematopoiesis will enter the domain of therapies to modulate broad aspects of hematopoiesis, both normal and malignant. -
Scadden DT. 2016. Blood and Bone. The New England journal of medicine. 374(19):1891-3. Pubmed: 27168440 DOI:10.1056/NEJMcibr1601737 Scadden DT. 2016. Blood and Bone. The New England journal of medicine. 374(19):1891-3. Pubmed: 27168440 DOI:10.1056/NEJMcibr1601737 -
Yu VW, Lymperi S, Oki T, Jones A, Swiatek P, Vasic R, Ferraro F, Scadden DT. 2016. Distinctive Mesenchymal-Parenchymal Cell Pairings Govern B Cell Differentiation in the Bone Marrow. Stem cell reports. 7(2):220-35. Pubmed: 27453006 DOI:S2213-6711(16)30101-1 Yu VW, Lymperi S, Oki T, Jones A, Swiatek P, Vasic R, Ferraro F, Scadden DT. 2016. Distinctive Mesenchymal-Parenchymal Cell Pairings Govern B Cell Differentiation in the Bone Marrow. Stem cell reports. 7(2):220-35. Pubmed: 27453006 DOI:S2213-6711(16)30101-1 Bone marrow niches for hematopoietic progenitor cells are not well defined despite their critical role in blood homeostasis. We previously found that cells expressing osteocalcin, a marker of mature osteolineage cells, regulate the production of thymic-seeding T lymphoid progenitors. Here, using a selective cell deletion strategy, we demonstrate that a subset of mesenchymal cells expressing osterix, a marker of bone precursors in the adult, serve to regulate the maturation of early B lymphoid precursors by promoting pro-B to pre-B cell transition through insulin-like growth factor 1 (IGF-1) production. Loss of Osx(+) cells or Osx-specific deletion of IGF-1 led to a failure of B cell maturation and the impaired adaptive immune response. These data highlight the notion that bone marrow is a composite of specialized niches formed by pairings of specific mesenchymal cells with parenchymal stem or lineage committed progenitor cells, thereby providing distinctive functional units to regulate hematopoiesis.Copyright © 2016. Published by Elsevier Inc. -
Mercier FE, Sykes DB, Scadden DT. 2016. Single Targeted Exon Mutation Creates a True Congenic Mouse for Competitive Hematopoietic Stem Cell Transplantation: The C57BL/6-CD45.1(STEM) Mouse. Stem cell reports. 6(6):985-992. Pubmed: 27185283 DOI:S2213-6711(16)30035-2 Mercier FE, Sykes DB, Scadden DT. 2016. Single Targeted Exon Mutation Creates a True Congenic Mouse for Competitive Hematopoietic Stem Cell Transplantation: The C57BL/6-CD45.1(STEM) Mouse. Stem cell reports. 6(6):985-992. Pubmed: 27185283 DOI:S2213-6711(16)30035-2 Defining the molecular regulators of hematopoietic stem and progenitor cells (HSPCs) requires in vivo functional analyses. Competitive bone marrow transplants (BMTs) compare control and test HSPCs to demonstrate the functional role of a genetic change or chemical perturbation. Competitive BMT is enabled by antibodies that specifically recognize hematopoietic cells from congenic mouse strains due to variants of the cell surface protein CD45, designated CD45.1 and CD45.2. The current congenic competitor strain, B6.SJL-Ptprc(a) Pepc(b)/BoyJ (CD45.1), has a substantial inherent disadvantage in competition against the C57BL/6 (CD45.2) strain, confounding experimental interpretation. Despite backcrossing, the congenic interval over which the B6.SJL-Ptprc(a) Pepc(b)/BoyJ strain differs is almost 40 Mb encoding ∼300 genes. Here, we demonstrate that a single amino acid change determines the CD45.1 epitope. Further, we report on the single targeted exon mutant (STEM) mouse strain, CD45.1(STEM), which is functionally equivalent to CD45.2 cells in competitive BMT. This strain will permit the precise definition of functional roles for candidate genes using in vivo HSPC assays.Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved. -
Itkin T, Gur-Cohen S, Spencer JA, Schajnovitz A, Ramasamy SK, Kusumbe AP, Ledergor G, Jung Y, Milo I, Poulos MG, Kalinkovich A, Ludin A, Golan K, Khatib E, Kumari A, Kollet O, Shakhar G, Butler JM, Rafii S, Adams RH, Scadden DT, Lin CP, Lapidot T. 2016. Corrigendum: Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature. 538(7624):274. Pubmed: 27437586 DOI:10.1038/nature19088 Itkin T, Gur-Cohen S, Spencer JA, Schajnovitz A, Ramasamy SK, Kusumbe AP, Ledergor G, Jung Y, Milo I, Poulos MG, Kalinkovich A, Ludin A, Golan K, Khatib E, Kumari A, Kollet O, Shakhar G, Butler JM, Rafii S, Adams RH, Scadden DT, Lin CP, Lapidot T. 2016. Corrigendum: Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature. 538(7624):274. Pubmed: 27437586 DOI:10.1038/nature19088 -
Itkin T, Gur-Cohen S, Spencer JA, Schajnovitz A, Ramasamy SK, Kusumbe AP, Ledergor G, Jung Y, Milo I, Poulos MG, Kalinkovich A, Ludin A, Kollet O, Shakhar G, Butler JM, Rafii S, Adams RH, Scadden DT, Lin CP, Lapidot T. 2016. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature. 532(7599):323-8. Pubmed: 27074509 DOI:10.1038/nature17624 Itkin T, Gur-Cohen S, Spencer JA, Schajnovitz A, Ramasamy SK, Kusumbe AP, Ledergor G, Jung Y, Milo I, Poulos MG, Kalinkovich A, Ludin A, Kollet O, Shakhar G, Butler JM, Rafii S, Adams RH, Scadden DT, Lin CP, Lapidot T. 2016. Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature. 532(7599):323-8. Pubmed: 27074509 DOI:10.1038/nature17624 Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols. -
Yu VWC, Yusuf RZ, Oki T, Wu J, Saez B, Wang X, Cook C, Baryawno N, Ziller MJ, Lee E, Gu H, Meissner A, Lin CP, Kharchenko PV, Scadden DT. 2016. Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells. Cell. 167(5):1310-1322.e17. Pubmed: 27863245 DOI:S0092-8674(16)31466-0 Yu VWC, Yusuf RZ, Oki T, Wu J, Saez B, Wang X, Cook C, Baryawno N, Ziller MJ, Lee E, Gu H, Meissner A, Lin CP, Kharchenko PV, Scadden DT. 2016. Epigenetic Memory Underlies Cell-Autonomous Heterogeneous Behavior of Hematopoietic Stem Cells. Cell. 167(5):1310-1322.e17. Pubmed: 27863245 DOI:S0092-8674(16)31466-0 Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.Copyright © 2016 Elsevier Inc. All rights reserved. -
Dong L, Yu WM, Zheng H, Loh ML, Bunting ST, Pauly M, Huang G, Zhou M, Broxmeyer HE, Scadden DT, Qu CK. 2016. Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment. Nature. 539(7628):304-308. Pubmed: 27783593 DOI:10.1038/nature20131 Dong L, Yu WM, Zheng H, Loh ML, Bunting ST, Pauly M, Huang G, Zhou M, Broxmeyer HE, Scadden DT, Qu CK. 2016. Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment. Nature. 539(7628):304-308. Pubmed: 27783593 DOI:10.1038/nature20131 Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway, are found in 50% of patients with Noonan syndrome. These patients have an increased risk of developing leukaemia, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1β and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias. -
Lewis TA, Sykes DB, Law JM, Muñoz B, Rustiguel JK, Nonato MC, Scadden DT, Schreiber SL. 2016. Development of ML390: A Human DHODH Inhibitor That Induces Differentiation in Acute Myeloid Leukemia. ACS medicinal chemistry letters. 7(12):1112-1117. Pubmed: 27994748 Lewis TA, Sykes DB, Law JM, Muñoz B, Rustiguel JK, Nonato MC, Scadden DT, Schreiber SL. 2016. Development of ML390: A Human DHODH Inhibitor That Induces Differentiation in Acute Myeloid Leukemia. ACS medicinal chemistry letters. 7(12):1112-1117. Pubmed: 27994748 Homeobox transcription factor A9 (HoxA9) is overexpressed in 70% of patients diagnosed with acute myeloid leukemia (AML), whereas only a small subset of AML patients respond to current differentiation therapies. A cell line overexpressing HoxA9 was derived from the bone marrow of a lysozyme-GFP mouse. In this fashion, GFP served as an endogenous reporter of differentiation, permitting a high-throughput phenotypic screen against the MLPCN library. Two chemical scaffolds were optimized for activity yielding compound ML390, and genetic resistance and sequencing efforts identified dihydroorotate dehydrogenase (DHODH) as the target enzyme. The DHODH inhibitor brequinar works against these leukemic cells as well. The X-ray crystal structure of ML390 bound to DHODH elucidates ML390s binding interactions. -
German NJ, Yoon H, Yusuf RZ, Murphy JP, Finley LW, Laurent G, Haas W, Satterstrom FK, Guarnerio J, Zaganjor E, Santos D, Pandolfi PP, Beck AH, Gygi SP, Scadden DT, Kaelin WG, Haigis MC. 2016. PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2. Molecular cell. 63(6):1006-20. Pubmed: 27635760 DOI:S1097-2765(16)30428-2 German NJ, Yoon H, Yusuf RZ, Murphy JP, Finley LW, Laurent G, Haas W, Satterstrom FK, Guarnerio J, Zaganjor E, Santos D, Pandolfi PP, Beck AH, Gygi SP, Scadden DT, Kaelin WG, Haigis MC. 2016. PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2. Molecular cell. 63(6):1006-20. Pubmed: 27635760 DOI:S1097-2765(16)30428-2 While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.Copyright © 2016 Elsevier Inc. All rights reserved. -
Sykes DB, Kfoury YS, Mercier FE, Wawer MJ, Law JM, Haynes MK, Lewis TA, Schajnovitz A, Jain E, Lee D, Meyer H, Pierce KA, Tolliday NJ, Waller A, Ferrara SJ, Eheim AL, Stoeckigt D, Maxcy KL, Cobert JM, Bachand J, Szekely BA, Mukherjee S, Sklar LA, Kotz JD, Clish CB, Sadreyev RI, Clemons PA, Janzer A, Schreiber SL, Scadden DT. 2016. Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia. Cell. 167(1):171-186.e15. Pubmed: 27641501 DOI:S0092-8674(16)31154-0 Sykes DB, Kfoury YS, Mercier FE, Wawer MJ, Law JM, Haynes MK, Lewis TA, Schajnovitz A, Jain E, Lee D, Meyer H, Pierce KA, Tolliday NJ, Waller A, Ferrara SJ, Eheim AL, Stoeckigt D, Maxcy KL, Cobert JM, Bachand J, Szekely BA, Mukherjee S, Sklar LA, Kotz JD, Clish CB, Sadreyev RI, Clemons PA, Janzer A, Schreiber SL, Scadden DT. 2016. Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia. Cell. 167(1):171-186.e15. Pubmed: 27641501 DOI:S0092-8674(16)31154-0 While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.Copyright © 2016 Elsevier Inc. All rights reserved. -
Goncalves KA, Silberstein L, Li S, Severe N, Hu MG, Yang H, Scadden DT, Hu GF. 2016. Angiogenin Promotes Hematopoietic Regeneration by Dichotomously Regulating Quiescence of Stem and Progenitor Cells. Cell. 166(4):894-906. Pubmed: 27518564 DOI:S0092-8674(16)30850-9 Goncalves KA, Silberstein L, Li S, Severe N, Hu MG, Yang H, Scadden DT, Hu GF. 2016. Angiogenin Promotes Hematopoietic Regeneration by Dichotomously Regulating Quiescence of Stem and Progenitor Cells. Cell. 166(4):894-906. Pubmed: 27518564 DOI:S0092-8674(16)30850-9 Regulation of stem and progenitor cell populations is critical in the development, maintenance, and regeneration of tissues. Here, we define a novel mechanism by which a niche-secreted RNase, angiogenin (ANG), distinctively alters the functional characteristics of primitive hematopoietic stem/progenitor cells (HSPCs) compared with lineage-committed myeloid-restricted progenitor (MyePro) cells. Specifically, ANG reduces the proliferative capacity of HSPC while simultaneously increasing proliferation of MyePro cells. Mechanistically, ANG induces cell-type-specific RNA-processing events: tRNA-derived stress-induced small RNA (tiRNA) generation in HSPCs and rRNA induction in MyePro cells, leading to respective reduction and increase in protein synthesis. Recombinant ANG protein improves survival of irradiated animals and enhances hematopoietic regeneration of mouse and human HSPCs in transplantation. Thus, ANG plays a non-cell-autonomous role in regulation of hematopoiesis by simultaneously preserving HSPC stemness and promoting MyePro proliferation. These cell-type-specific functions of ANG suggest considerable therapeutic potential.Copyright © 2016 Elsevier Inc. All rights reserved. -
Yu VW, Scadden DT. 2016. Hematopoietic Stem Cell and Its Bone Marrow Niche. Current topics in developmental biology. 118:21-44. Pubmed: 27137653 DOI:S0070-2153(16)00010-7 Yu VW, Scadden DT. 2016. Hematopoietic Stem Cell and Its Bone Marrow Niche. Current topics in developmental biology. 118:21-44. Pubmed: 27137653 DOI:S0070-2153(16)00010-7 Stem cells do not thrive without their niche. The bone marrow microenvironment is where hematopoietic stem cells maintain their cell state while receiving physiological input to modify their activity in response to changing physiological demands. The complexity of the bone marrow microenvironment is being unraveled and indicates that multiple different cell types contribute to the regulation of stem and progenitor cells. Further, it is becoming evident that the bone marrow represents a composite of niches with different components and different functional roles in hematopoiesis. It is now evident that alterations in specific stromal cells that comprise the bone marrow microenvironment can contribute to hematologic pathology. In this chapter, we will review the history of the niche concept, evolving information about its components and how niche dysfunction may contribute to disease.© 2016 Elsevier Inc. All rights reserved. -
Lee D, Wang YH, Kalaitzidis D, Ramachandran J, Eda H, Sykes DB, Raje N, Scadden DT. 2016. Endogenous transmembrane protein UT2 inhibits pSTAT3 and suppresses hematological malignancy. The Journal of clinical investigation. 126(4):1300-10. Pubmed: 26927669 DOI:84620 Lee D, Wang YH, Kalaitzidis D, Ramachandran J, Eda H, Sykes DB, Raje N, Scadden DT. 2016. Endogenous transmembrane protein UT2 inhibits pSTAT3 and suppresses hematological malignancy. The Journal of clinical investigation. 126(4):1300-10. Pubmed: 26927669 DOI:84620 Regulation of STAT3 activation is critical for normal and malignant hematopoietic cell proliferation. Here, we have reported that the endogenous transmembrane protein upstream-of-mTORC2 (UT2) negatively regulates activation of STAT3. Specifically, we determined that UT2 interacts directly with GP130 and inhibits phosphorylation of STAT3 on tyrosine 705 (STAT3Y705). This reduces cytokine signaling including IL6 that is implicated in multiple myeloma and other hematopoietic malignancies. Modulation of UT2 resulted in inverse effects on animal survival in myeloma models. Samples from multiple myeloma patients also revealed a decreased copy number of UT2 and decreased expression of UT2 in genomic and transcriptomic analyses, respectively. Together, these studies identify a transmembrane protein that functions to negatively regulate cytokine signaling through GP130 and pSTAT3Y705 and is molecularly and mechanistically distinct from the suppressors of cytokine signaling (SOCS) family of genes. Moreover, this work provides evidence that perturbations of this activation-dampening molecule participate in hematologic malignancies and may serve as a key determinant of multiple myeloma pathophysiology. UT2 is a negative regulator shared across STAT3 and mTORC2 signaling cascades, functioning as a tumor suppressor in hematologic malignancies driven by those pathways. -
Yu VW, Scadden DT. 2016. Heterogeneity of the bone marrow niche. Current opinion in hematology. 23(4):331-8. Pubmed: 27177311 DOI:10.1097/MOH.0000000000000265 Yu VW, Scadden DT. 2016. Heterogeneity of the bone marrow niche. Current opinion in hematology. 23(4):331-8. Pubmed: 27177311 DOI:10.1097/MOH.0000000000000265 Array -
Ramasamy S, Saez B, Mukhopadhyay S, Ding D, Ahmed AM, Chen X, Pucci F, Yamin R, Wang J, Pittet MJ, Kelleher CM, Scadden DT, Sweetser DA. 2016. Tle1 tumor suppressor negatively regulates inflammation in vivo and modulates NF-κB inflammatory pathway. Proceedings of the National Academy of Sciences of the United States of America. 113(7):1871-6. Pubmed: 26831087 DOI:10.1073/pnas.1511380113 Ramasamy S, Saez B, Mukhopadhyay S, Ding D, Ahmed AM, Chen X, Pucci F, Yamin R, Wang J, Pittet MJ, Kelleher CM, Scadden DT, Sweetser DA. 2016. Tle1 tumor suppressor negatively regulates inflammation in vivo and modulates NF-κB inflammatory pathway. Proceedings of the National Academy of Sciences of the United States of America. 113(7):1871-6. Pubmed: 26831087 DOI:10.1073/pnas.1511380113 Tle1 (transducin-like enhancer of split 1) is a corepressor that interacts with a variety of DNA-binding transcription factors and has been implicated in many cellular functions; however, physiological studies are limited. Tle1-deficient (Tle1(Δ/Δ)) mice, although grossly normal at birth, exhibit skin defects, lung hypoplasia, severe runting, poor body condition, and early mortality. Tle1(Δ/Δ) mice display a chronic inflammatory phenotype with increased expression of inflammatory cytokines and chemokines in the skin, lung, and intestine and increased circulatory IL-6 and G-CSF, along with a hematopoietic shift toward granulocyte macrophage progenitor and myeloid cells. Tle1(Δ/Δ) macrophages produce increased inflammatory cytokines in response to Toll-like receptor (TLR) agonists and lipopolysaccharides (LPS), and Tle1(Δ/Δ) mice display an enhanced inflammatory response to ear skin 12-O-tetradecanoylphorbol-13-acetate treatment. Loss of Tle1 not only results in increased phosphorylation and activation of proinflammatory NF-κB but also results in decreased Hes1 (hairy and enhancer of split-1), a negative regulator of inflammation in macrophages. Furthermore, Tle1(Δ/Δ) mice exhibit accelerated growth of B6-F10 melanoma xenografts. Our work provides the first in vivo evidence, to our knowledge, that TLE1 is a major counterregulator of inflammation with potential roles in a variety of inflammatory diseases and in cancer progression. 2015
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Chattopadhyay S, Stewart AL, Mukherjee S, Huang C, Hartwell KA, Miller PG, Subramanian R, Carmody LC, Yusuf RZ, Sykes DB, Paulk J, Vetere A, Vallet S, Santo L, Cirstea DD, Hideshima T, Dančík V, Majireck MM, Hussain MM, Singh S, Quiroz R, Iaconelli J, Karmacharya R, Tolliday NJ, Clemons PA, Moore MAS, Stern AM, Shamji AF, Ebert BL, Golub TR, Raje NS, Scadden DT, Schreiber SL. 2015. Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors. Cell reports. 10(5):755-770. Pubmed: 25660025 DOI:S2211-1247(15)00030-3 Chattopadhyay S, Stewart AL, Mukherjee S, Huang C, Hartwell KA, Miller PG, Subramanian R, Carmody LC, Yusuf RZ, Sykes DB, Paulk J, Vetere A, Vallet S, Santo L, Cirstea DD, Hideshima T, Dančík V, Majireck MM, Hussain MM, Singh S, Quiroz R, Iaconelli J, Karmacharya R, Tolliday NJ, Clemons PA, Moore MAS, Stern AM, Shamji AF, Ebert BL, Golub TR, Raje NS, Scadden DT, Schreiber SL. 2015. Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors. Cell reports. 10(5):755-770. Pubmed: 25660025 DOI:S2211-1247(15)00030-3 Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. -
Yu VW, Saez B, Cook C, Lotinun S, Pardo-Saganta A, Wang YH, Lymperi S, Ferraro F, Raaijmakers MH, Wu JY, Zhou L, Rajagopal J, Kronenberg HM, Baron R, Scadden DT. 2015. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow. The Journal of experimental medicine. 212(5):759-74. Pubmed: 25918341 DOI:10.1084/jem.20141843 Yu VW, Saez B, Cook C, Lotinun S, Pardo-Saganta A, Wang YH, Lymperi S, Ferraro F, Raaijmakers MH, Wu JY, Zhou L, Rajagopal J, Kronenberg HM, Baron R, Scadden DT. 2015. Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow. The Journal of experimental medicine. 212(5):759-74. Pubmed: 25918341 DOI:10.1084/jem.20141843 Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined. Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells. Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function. The Notch ligand DLL4 is abundantly expressed on bone marrow Ocn(+) cells, and selective depletion of DLL4 from these cells recapitulated the thymopoietic abnormality. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell-based adaptive immunity.© 2015 Yu et al. -
Kfoury Y, Scadden DT. 2015. Mesenchymal cell contributions to the stem cell niche. Cell stem cell. 16(3):239-53. Pubmed: 25748931 DOI:S1934-5909(15)00075-2 Kfoury Y, Scadden DT. 2015. Mesenchymal cell contributions to the stem cell niche. Cell stem cell. 16(3):239-53. Pubmed: 25748931 DOI:S1934-5909(15)00075-2 Mesenchymal stromal cells (MSCs) are heterogeneous and primitive cells discovered first in the bone marrow (BM). They have putative roles in maintaining tissue homeostasis and are increasingly recognized as components of stem cell niches, which are best defined in the blood. The absence of in vivo MSC markers has limited our ability to track their behavior in vivo and draw comparisons with in vitro observations. Here we review the historical background of BM-MSCs, advances made in their prospective isolation, their developmental origin and contribution to maintaining subsets of hematopoietic cells, and how mesenchymal cells contribute to other stem cell niches.Copyright © 2015 Elsevier Inc. All rights reserved. -
Méndez-Ferrer S, Scadden DT, Sánchez-Aguilera A. 2015. Bone marrow stem cells: current and emerging concepts. Annals of the New York Academy of Sciences. 1335:32-44. Pubmed: 25573321 DOI:10.1111/nyas.12641 Méndez-Ferrer S, Scadden DT, Sánchez-Aguilera A. 2015. Bone marrow stem cells: current and emerging concepts. Annals of the New York Academy of Sciences. 1335:32-44. Pubmed: 25573321 DOI:10.1111/nyas.12641 The interactions of stromal cells with hematopoietic cells in the bone marrow have long been a subject of research, but only recently have technologies allowed us to dissect them at the stem cell level. On the other hand, limitations of these technical tools might explain numerous discrepancies in this field. It is becoming increasingly clear that mesenchymal stem cells (MSCs) represent an important component of the hematopoietic stem cell (HSC) niche in the bone marrow. However, there is heterogeneity among HSCs, and many putatively different mesenchymal progenitors identified in the bone marrow using Cre recombinase-driven mouse lines seem to exhibit HSC niche properties. Development of better reporter lines has demonstrated that some of these Cre lines do not always specifically mark the expected cells. Also, characterization of different cell populations has often been partial, and issues of redundancy and compensation might explain apparently contradictory results. Recognizing and overcoming these limitations, while also clearly defining the distinctions between subgroups of mesenchymal cells, will be essential to advance the field.© 2015 New York Academy of Sciences. -
Mercier FE, Scadden DT. 2015. Not All Created Equal: Lineage Hard-Wiring in the Production of Blood. Cell. 163(7):1568-70. Pubmed: 26687347 DOI:S0092-8674(15)01637-2 Mercier FE, Scadden DT. 2015. Not All Created Equal: Lineage Hard-Wiring in the Production of Blood. Cell. 163(7):1568-70. Pubmed: 26687347 DOI:S0092-8674(15)01637-2 The multiple cell types comprising blood have been thought to emerge from progenitors with progressively narrower lineage options. New data suggest that lineage fate may be determined earlier than thought and that myeloid progenitor populations are aggregates of individual lineage-restricted cells.Copyright © 2015 Elsevier Inc. All rights reserved. -
Krause DS, Scadden DT. 2015. A hostel for the hostile: the bone marrow niche in hematologic neoplasms. Haematologica. 100(11):1376-87. Pubmed: 26521296 DOI:10.3324/haematol.2014.113852 Krause DS, Scadden DT. 2015. A hostel for the hostile: the bone marrow niche in hematologic neoplasms. Haematologica. 100(11):1376-87. Pubmed: 26521296 DOI:10.3324/haematol.2014.113852 Our understanding of the biology of the normal hematopoietic stem cell niche has increased steadily due to improved murine models and sophisticated imaging tools. Less well understood, but of growing interest, is the interaction between cells in the bone marrow during the initiation, maintenance and treatment of hematologic neoplasms. This review summarizes the emerging concepts of the normal and leukemic hematopoietic bone marrow niche. Furthermore, it reviews current models of how the microenvironment of the bone marrow may contribute to or be modified by leukemogenesis. Finally, it provides the rationale for a "two-pronged" approach, directly targeting cancer cells themselves while also targeting the bone microenvironment to make it inhospitable to malignant cells and, ultimately, eradicating cancer stem-like cells.Copyright© Ferrata Storti Foundation. -
Ren X, Moser PT, Gilpin SE, Okamoto T, Wu T, Tapias LF, Mercier FE, Xiong L, Ghawi R, Scadden DT, Mathisen DJ, Ott HC. 2015. Engineering pulmonary vasculature in decellularized rat and human lungs. Nature biotechnology. 33(10):1097-102. Pubmed: 26368048 DOI:10.1038/nbt.3354 Ren X, Moser PT, Gilpin SE, Okamoto T, Wu T, Tapias LF, Mercier FE, Xiong L, Ghawi R, Scadden DT, Mathisen DJ, Ott HC. 2015. Engineering pulmonary vasculature in decellularized rat and human lungs. Nature biotechnology. 33(10):1097-102. Pubmed: 26368048 DOI:10.1038/nbt.3354 Bioengineered lungs produced from patient-derived cells may one day provide an alternative to donor lungs for transplantation therapy. Here we report the regeneration of functional pulmonary vasculature by repopulating the vascular compartment of decellularized rat and human lung scaffolds with human cells, including endothelial and perivascular cells derived from induced pluripotent stem cells. We describe improved methods for delivering cells into the lung scaffold and for maturing newly formed endothelium through co-seeding of endothelial and perivascular cells and a two-phase culture protocol. Using these methods we achieved ∼75% endothelial coverage in the rat lung scaffold relative to that of native lung. The regenerated endothelium showed reduced vascular resistance and improved barrier function over the course of in vitro culture and remained patent for 3 days after orthotopic transplantation in rats. Finally, we scaled our approach to the human lung lobe and achieved efficient cell delivery, maintenance of cell viability and establishment of perfusable vascular lumens. -
Wang YH, Scadden DT. 2015. Harnessing the apoptotic programs in cancer stem-like cells. EMBO reports. 16(9):1084-98. Pubmed: 26253117 DOI:10.15252/embr.201439675 Wang YH, Scadden DT. 2015. Harnessing the apoptotic programs in cancer stem-like cells. EMBO reports. 16(9):1084-98. Pubmed: 26253117 DOI:10.15252/embr.201439675 Elimination of malignant cells is an unmet challenge for most human cancer types even with therapies targeting specific driver mutations. Therefore, a multi-pronged strategy to alter cancer cell biology on multiple levels is increasingly recognized as essential for cancer cure. One such aspect of cancer cell biology is the relative apoptosis resistance of tumor-initiating cells. Here, we provide an overview of the mechanisms affecting the apoptotic process in tumor cells emphasizing the differences in the tumor-initiating or stem-like cells of cancer. Further, we summarize efforts to exploit these differences to design therapies targeting that important cancer cell population.© 2015 The Authors. -
Yu VW, Scadden DT. 2015. Global transcriptome analysis of T-competent progenitors in the bone marrow. Genomics data. 5:100-2. Pubmed: 26484235 DOI:10.1016/j.gdata.2015.05.024 Yu VW, Scadden DT. 2015. Global transcriptome analysis of T-competent progenitors in the bone marrow. Genomics data. 5:100-2. Pubmed: 26484235 DOI:10.1016/j.gdata.2015.05.024 T cells are known to develop in the thymus. However, molecular events that control the transition from hematopoietic progenitor cells in the bone marrow to T precursor cells seeded in the thymus remained poorly defined. Our recent report showed that osteocalcin (Ocn)-expressing bone cells in the bone marrow have major impact on T cell immunity by regulating T progenitor development in the bone marrow (Yu et al., 2015) [1]. Selective endogenous depletion of Ocn(+) cells by inducible diphtheria toxin receptor expression (OcnCre;iDTR) led to reduction of T-competent common lymphoid progenitors (Ly6D(-) CLPs) in the bone marrow and loss of T cells in the thymus. Expression of the Notch ligand DLL4 by Ocn(+) cells in the bone marrow ensures the production of Ly6D(-) CLPs, and expression of chemotactic molecules CCR7 and PSGL1 to enable subsequent thymic seeding. These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell based adaptive immunity. Here we present the transcriptome profiles of Ly6D(-) CLPs derived from Ocn(+) cells deleted mice (OcnCre(+);iDTR) compared to those derived from control littermates (OcnCre(-);iDTR). These data are publically available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE66102. -
Yu VW, Lymperi S, Ferraro F, Scadden DT. 2015. Transcriptome comparison of distinct osteolineage subsets in the hematopoietic stem cell niche using a triple fluorescent transgenic mouse model. Genomics data. 5:318-9. Pubmed: 26484277 DOI:10.1016/j.gdata.2015.06.001 Yu VW, Lymperi S, Ferraro F, Scadden DT. 2015. Transcriptome comparison of distinct osteolineage subsets in the hematopoietic stem cell niche using a triple fluorescent transgenic mouse model. Genomics data. 5:318-9. Pubmed: 26484277 DOI:10.1016/j.gdata.2015.06.001 The bone marrow niche is recognized as a central player in maintaining and regulating the behavior of hematopoietic stem and progenitor cells. Specific gain-of and loss-of function experiments perturbing a range of osteolineage cells or their secreted proteins had been shown to affect stem cell maintenance (Calvi et al, 2003 [1]; Stier et al., 2005 [2]; Zhang et al., 2003 [3]; Nilsson et al., 2005 [4]; Greenbaum et al., 2013 [5]) and engraftment (Adam et al., 2006, 2009 [6,7]). We used specific in vivo cell deletion approaches to dissect the niche cell-parenchymal cell dependency in a complex bone marrow microenvironment. Endogenous deletion of osteocalcin-expressing (Ocn(+)) cells led to a loss of T immune cells (Yu et al., 2015 [8]. Ocn(+) cells express the Notch ligand DLL4 to communicate with T-competent progenitors, and thereby ensuring T precursor production and expression of chemotactic molecules on their cell surface for subsequent thymic seeding. In contrast, depletion of osterix-expressing (Osx(+)) osteoprogenitors led to reduced B immune cells. These distinct hematopoietic phenotypes suggest specific pairing of mesenchymal niche cells and parenchymal hematopoietic cells in the bone marrow to create unique functional units to support hematopoiesis. Here, we present the global gene expression profiles of these osteolineage subtypes utilizing a triple fluorescent transgenic mouse model (OsxCre(+);Rosa-mCh(+);Ocn:Topaz(+)) that labels Osx(+) cells red, Ocn(+) cells green, and Osx(+) Ocn(+) cells yellow. This system allows isolation of distinct osteolineage subsets within the same animal by flow cytometry. Array data that have been described in our study [8] are also publically available from NCBI Gene Expression Omnibus (GEO) with the accession number GSE66042. Differences in gene expression may correlate with functional difference in supporting hematopoiesis. -
Wang W, Yu S, Zimmerman G, Wang Y, Myers J, Yu VW, Huang D, Huang X, Shim J, Huang Y, Xin W, Qiao P, Yan M, Xin W, Scadden DT, Stanley P, Lowe JB, Huang AY, Siebel CW, Zhou L. 2015. Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention. Stem cells (Dayton, Ohio). 33(7):2280-93. Pubmed: 25851125 DOI:10.1002/stem.2031 Wang W, Yu S, Zimmerman G, Wang Y, Myers J, Yu VW, Huang D, Huang X, Shim J, Huang Y, Xin W, Qiao P, Yan M, Xin W, Scadden DT, Stanley P, Lowe JB, Huang AY, Siebel CW, Zhou L. 2015. Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention. Stem cells (Dayton, Ohio). 33(7):2280-93. Pubmed: 25851125 DOI:10.1002/stem.2031 Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.© 2015 AlphaMed Press. -
Dutta P, Sager HB, Stengel KR, Naxerova K, Courties G, Saez B, Silberstein L, Heidt T, Sebas M, Sun Y, Wojtkiewicz G, Feruglio PF, King K, Baker JN, van der Laan AM, Borodovsky A, Fitzgerald K, Hulsmans M, Hoyer F, Iwamoto Y, Vinegoni C, Brown D, Di Carli M, Libby P, Hiebert SW, Scadden DT, Swirski FK, Weissleder R, Nahrendorf M. 2015. Myocardial Infarction Activates CCR2(+) Hematopoietic Stem and Progenitor Cells. Cell stem cell. 16(5):477-87. Pubmed: 25957903 DOI:S1934-5909(15)00166-6 Dutta P, Sager HB, Stengel KR, Naxerova K, Courties G, Saez B, Silberstein L, Heidt T, Sebas M, Sun Y, Wojtkiewicz G, Feruglio PF, King K, Baker JN, van der Laan AM, Borodovsky A, Fitzgerald K, Hulsmans M, Hoyer F, Iwamoto Y, Vinegoni C, Brown D, Di Carli M, Libby P, Hiebert SW, Scadden DT, Swirski FK, Weissleder R, Nahrendorf M. 2015. Myocardial Infarction Activates CCR2(+) Hematopoietic Stem and Progenitor Cells. Cell stem cell. 16(5):477-87. Pubmed: 25957903 DOI:S1934-5909(15)00166-6 Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.Copyright © 2015 Elsevier Inc. All rights reserved. -
Wang YH, Scadden DT. 2015. Targeting the Warburg effect for leukemia therapy: Magnitude matters. Molecular & cellular oncology. 2(3):e981988. Pubmed: 27308458 DOI:10.4161/23723556.2014.981988 Wang YH, Scadden DT. 2015. Targeting the Warburg effect for leukemia therapy: Magnitude matters. Molecular & cellular oncology. 2(3):e981988. Pubmed: 27308458 DOI:10.4161/23723556.2014.981988 Non-oxidative glucose metabolism represents a hallmark of cancer. It is now apparent that different cell states among normal stem or progenitor cells have distinct aerobic glycolysis (AG) dependencies. However, malignant cells are markedly more vulnerable to modifications of AG regardless of the differentiation state of their cell of origin. -
Courties G, Herisson F, Sager HB, Heidt T, Ye Y, Wei Y, Sun Y, Severe N, Dutta P, Scharff J, Scadden DT, Weissleder R, Swirski FK, Moskowitz MA, Nahrendorf M. 2015. Ischemic stroke activates hematopoietic bone marrow stem cells. Circulation research. 116(3):407-17. Pubmed: 25362208 DOI:10.1161/CIRCRESAHA.116.305207 Courties G, Herisson F, Sager HB, Heidt T, Ye Y, Wei Y, Sun Y, Severe N, Dutta P, Scharff J, Scadden DT, Weissleder R, Swirski FK, Moskowitz MA, Nahrendorf M. 2015. Ischemic stroke activates hematopoietic bone marrow stem cells. Circulation research. 116(3):407-17. Pubmed: 25362208 DOI:10.1161/CIRCRESAHA.116.305207 Array© 2014 American Heart Association, Inc. 2014
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Saez B, Ferraro F, Yusuf RZ, Cook CM, Yu VW, Pardo-Saganta A, Sykes SM, Palchaudhuri R, Schajnovitz A, Lotinun S, Lymperi S, Mendez-Ferrer S, Toro RD, Day R, Vasic R, Acharya SS, Baron R, Lin CP, Yamaguchi Y, Wagers AJ, Scadden DT. 2014. Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning. Blood. 124(19):2937-47. Pubmed: 25202142 DOI:10.1182/blood-2014-08-593426 Saez B, Ferraro F, Yusuf RZ, Cook CM, Yu VW, Pardo-Saganta A, Sykes SM, Palchaudhuri R, Schajnovitz A, Lotinun S, Lymperi S, Mendez-Ferrer S, Toro RD, Day R, Vasic R, Acharya SS, Baron R, Lin CP, Yamaguchi Y, Wagers AJ, Scadden DT. 2014. Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning. Blood. 124(19):2937-47. Pubmed: 25202142 DOI:10.1182/blood-2014-08-593426 The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions.© 2014 by The American Society of Hematology. -
Velardi E, Tsai JJ, Holland AM, Wertheimer T, Yu VW, Zakrzewski JL, Tuckett AZ, Singer NV, West ML, Smith OM, Young LF, Kreines FM, Levy ER, Boyd RL, Scadden DT, Dudakov JA, van den Brink MR. 2014. Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. The Journal of experimental medicine. 211(12):2341-9. Pubmed: 25332287 DOI:10.1084/jem.20131289 Velardi E, Tsai JJ, Holland AM, Wertheimer T, Yu VW, Zakrzewski JL, Tuckett AZ, Singer NV, West ML, Smith OM, Young LF, Kreines FM, Levy ER, Boyd RL, Scadden DT, Dudakov JA, van den Brink MR. 2014. Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. The Journal of experimental medicine. 211(12):2341-9. Pubmed: 25332287 DOI:10.1084/jem.20131289 Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.© 2014 Velardi et al. -
Spencer JA, Ferraro F, Roussakis E, Klein A, Wu J, Runnels JM, Zaher W, Mortensen LJ, Alt C, Turcotte R, Yusuf R, Côté D, Vinogradov SA, Scadden DT, Lin CP. 2014. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature. 508(7495):269-73. Pubmed: 24590072 DOI:10.1038/nature13034 Spencer JA, Ferraro F, Roussakis E, Klein A, Wu J, Runnels JM, Zaher W, Mortensen LJ, Alt C, Turcotte R, Yusuf R, Côté D, Vinogradov SA, Scadden DT, Lin CP. 2014. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature. 508(7495):269-73. Pubmed: 24590072 DOI:10.1038/nature13034 Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (∼9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment. -
Li X, Romain RD, Park D, Scadden DT, Merchant JL, Arnaout MA. 2014. Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89. Stem cells (Dayton, Ohio). 32(3):791-801. Pubmed: 24549639 DOI:10.1002/stem.1598 Li X, Romain RD, Park D, Scadden DT, Merchant JL, Arnaout MA. 2014. Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89. Stem cells (Dayton, Ohio). 32(3):791-801. Pubmed: 24549639 DOI:10.1002/stem.1598 Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.© 2013 AlphaMed Press. -
Morrison SJ, Scadden DT. 2014. The bone marrow niche for haematopoietic stem cells. Nature. 505(7483):327-34. Pubmed: 24429631 DOI:10.1038/nature12984 Morrison SJ, Scadden DT. 2014. The bone marrow niche for haematopoietic stem cells. Nature. 505(7483):327-34. Pubmed: 24429631 DOI:10.1038/nature12984 Niches are local tissue microenvironments that maintain and regulate stem cells. Haematopoiesis provides a model for understanding mammalian stem cells and their niches, but the haematopoietic stem cell (HSC) niche remains incompletely defined and beset by competing models. Recent progress has been made in elucidating the location and cellular components of the HSC niche in the bone marrow. The niche is perivascular, created partly by mesenchymal stromal cells and endothelial cells and often, but not always, located near trabecular bone. Outstanding questions concern the cellular complexity of the niche, the role of the endosteum and functional heterogeneity among perivascular microenvironments. -
Lee BC, Scadden DT. 2014. From quiescence to senescence. Cell cycle (Georgetown, Tex.). 13(22):3469-70. Pubmed: 25493411 DOI:10.4161/15384101.2014.980696 Lee BC, Scadden DT. 2014. From quiescence to senescence. Cell cycle (Georgetown, Tex.). 13(22):3469-70. Pubmed: 25493411 DOI:10.4161/15384101.2014.980696 -
Kharchenko PV, Silberstein L, Scadden DT. 2014. Bayesian approach to single-cell differential expression analysis. Nature methods. 11(7):740-2. Pubmed: 24836921 DOI:10.1038/nmeth.2967 Kharchenko PV, Silberstein L, Scadden DT. 2014. Bayesian approach to single-cell differential expression analysis. Nature methods. 11(7):740-2. Pubmed: 24836921 DOI:10.1038/nmeth.2967 Single-cell data provide a means to dissect the composition of complex tissues and specialized cellular environments. However, the analysis of such measurements is complicated by high levels of technical noise and intrinsic biological variability. We describe a probabilistic model of expression-magnitude distortions typical of single-cell RNA-sequencing measurements, which enables detection of differential expression signatures and identification of subpopulations of cells in a way that is more tolerant of noise. -
Kalaitzidis D, Scadden DT. 2014. Tic-TACs: refreshing hair growth. Cell. 157(4):769-70. Pubmed: 24813602 DOI:S0092-8674(14)00492-9 Kalaitzidis D, Scadden DT. 2014. Tic-TACs: refreshing hair growth. Cell. 157(4):769-70. Pubmed: 24813602 DOI:S0092-8674(14)00492-9 Although stem cells are subject to niche control, evidence is emerging that they also contribute to generating the niche through their offspring. Using the hair follicle as a model, Hsu at al. demonstrate that the transient-amplifying cells, downstream of stem cells and well-known cell producers, signal back to stem cells to maintain long-term regenerative capacity.Copyright © 2014 Elsevier Inc. All rights reserved. -
Scadden DT. 2014. Nice neighborhood: emerging concepts of the stem cell niche. Cell. 157(1):41-50. Pubmed: 24679525 DOI:S0092-8674(14)00205-0 Scadden DT. 2014. Nice neighborhood: emerging concepts of the stem cell niche. Cell. 157(1):41-50. Pubmed: 24679525 DOI:S0092-8674(14)00205-0 No metazoan cell survives on its own, absent the signals and support of its milieu. For multicellular life with specialized tissues to persist, organization is everything and so defining the association of position with cell state is critical to understanding how tissues function, maintain, and repair. This review focuses specifically on place for progenitor and stem cells. Especially emphasized are hematopoietic cells that balance free movement and stable position and where concepts of regulatory interrelationships have been shown with some precision. It reviews classical and emerging concepts of the niche, particularly considering how niche functions may participate in neoplastic disease.Copyright © 2014 Elsevier Inc. All rights reserved. -
Schajnovitz A, Scadden DT. 2014. Bone's dark side: mutated osteoblasts implicated in leukemia. Cell research. 24(4):383-4. Pubmed: 24589711 DOI:10.1038/cr.2014.26 Schajnovitz A, Scadden DT. 2014. Bone's dark side: mutated osteoblasts implicated in leukemia. Cell research. 24(4):383-4. Pubmed: 24589711 DOI:10.1038/cr.2014.26 Bone-lining osteolineage cells were previously implicated as contributors to hematological disorders and malignancies. A recent report in Nature now demonstrates that a specific mutation in mouse collagen-expressing osteoblastic cells leads to MDS and AML with 100% penetrance and is associated with strikingly similar findings in human patients. -
Park D, Spencer JA, Lin CP, Scadden DT. 2014. Sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair. Journal of visualized experiments : JoVE. Pubmed: 24894331 DOI:10.3791/51289 Park D, Spencer JA, Lin CP, Scadden DT. 2014. Sequential in vivo imaging of osteogenic stem/progenitor cells during fracture repair. Journal of visualized experiments : JoVE. Pubmed: 24894331 DOI:10.3791/51289 Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair. -
Wang YH, Israelsen WJ, Lee D, Yu VWC, Jeanson NT, Clish CB, Cantley LC, Vander Heiden MG, Scadden DT. 2014. Cell-state-specific metabolic dependency in hematopoiesis and leukemogenesis. Cell. 158(6):1309-1323. Pubmed: 25215489 DOI:S0092-8674(14)01038-1 Wang YH, Israelsen WJ, Lee D, Yu VWC, Jeanson NT, Clish CB, Cantley LC, Vander Heiden MG, Scadden DT. 2014. Cell-state-specific metabolic dependency in hematopoiesis and leukemogenesis. Cell. 158(6):1309-1323. Pubmed: 25215489 DOI:S0092-8674(14)01038-1 The balance between oxidative and nonoxidative glucose metabolism is essential for a number of pathophysiological processes. By deleting enzymes that affect aerobic glycolysis with different potencies, we examine how modulating glucose metabolism specifically affects hematopoietic and leukemic cell populations. We find that a deficiency in the M2 pyruvate kinase isoform (PKM2) reduces the levels of metabolic intermediates important for biosynthesis and impairs progenitor function without perturbing hematopoietic stem cells (HSCs), whereas lactate dehydrogenase A (LDHA) deletion significantly inhibits the function of both HSCs and progenitors during hematopoiesis. In contrast, leukemia initiation by transforming alleles putatively affecting either HSCs or progenitors is inhibited in the absence of either PKM2 or LDHA, indicating that the cell-state-specific responses to metabolic manipulation in hematopoiesis do not apply to the setting of leukemia. This finding suggests that fine-tuning the level of glycolysis may be explored therapeutically for treating leukemia while preserving HSC function.Copyright © 2014 Elsevier Inc. All rights reserved. -
Kfoury Y, Scadden DT. 2014. Cellular thrust and parry in the leukemic niche. Blood. 124(18):2760-1. Pubmed: 25359983 DOI:10.1182/blood-2014-09-597716 Kfoury Y, Scadden DT. 2014. Cellular thrust and parry in the leukemic niche. Blood. 124(18):2760-1. Pubmed: 25359983 DOI:10.1182/blood-2014-09-597716 -
Cho J, Yusuf R, Kook S, Attar E, Lee D, Park B, Cheng T, Scadden DT, Lee BC. 2014. Purinergic P2Y₁₄ receptor modulates stress-induced hematopoietic stem/progenitor cell senescence. The Journal of clinical investigation. 124(7):3159-71. Pubmed: 24937426 DOI:61636 Cho J, Yusuf R, Kook S, Attar E, Lee D, Park B, Cheng T, Scadden DT, Lee BC. 2014. Purinergic P2Y₁₄ receptor modulates stress-induced hematopoietic stem/progenitor cell senescence. The Journal of clinical investigation. 124(7):3159-71. Pubmed: 24937426 DOI:61636 Purinergic receptors of the P2Y family are G protein-coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that P2Y14 modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, P2Y14 deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking P2Y14. Enhanced senescence coincided with increased ROS, elevated p16(INK4a) expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the P2Y14 phenotype, suggesting that P2Y14 mediates antisenescence effects through Gi/o protein-dependent pathways. Primitive hematopoietic cells lacking P2Y14 were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that P2Y14 on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that P2Y14-mediated responses prevent the premature decline of regenerative capacity after injury. -
Lee D, Sykes SM, Kalaitzidis D, Lane AA, Kfoury Y, Raaijmakers MH, Wang YH, Armstrong SA, Scadden DT. 2014. Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors. Stem cell reports. 3(5):832-40. Pubmed: 25418727 DOI:S2213-6711(14)00265-3 Lee D, Sykes SM, Kalaitzidis D, Lane AA, Kfoury Y, Raaijmakers MH, Wang YH, Armstrong SA, Scadden DT. 2014. Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors. Stem cell reports. 3(5):832-40. Pubmed: 25418727 DOI:S2213-6711(14)00265-3 Central to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2) negatively regulates mTORC2 enzymatic activity, reducing AKT(S473), PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL) model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway. -
Baryawno N, Scadden DT. 2014. Blood loses it when nerves go bad. Cell research. 24(10):1151-2. Pubmed: 25070156 DOI:10.1038/cr.2014.98 Baryawno N, Scadden DT. 2014. Blood loses it when nerves go bad. Cell research. 24(10):1151-2. Pubmed: 25070156 DOI:10.1038/cr.2014.98 Myeloproliferative neoplasms are diseases that arise in the stem cells of the blood. In a recent paper published in Nature, Arranz et al. demonstrated that abrogation of sympathetic nerve fibers reduced bone marrow Nestin mesenchymal cells, which in turn led to an expansion of hematopoietic stem cells and progression of myeloproliferative neoplasms. -
Kfoury Y, Mercier F, Scadden DT. 2014. SnapShot: The hematopoietic stem cell niche. Cell. 158(1):228-228.e1. Pubmed: 24995988 DOI:S0092-8674(14)00805-8 Kfoury Y, Mercier F, Scadden DT. 2014. SnapShot: The hematopoietic stem cell niche. Cell. 158(1):228-228.e1. Pubmed: 24995988 DOI:S0092-8674(14)00805-8 -
Choi YJ, Saez B, Anders L, Hydbring P, Stefano J, Bacon NA, Cook C, Kalaszczynska I, Signoretti S, Young RA, Scadden DT, Sicinski P. 2014. D-cyclins repress apoptosis in hematopoietic cells by controlling death receptor Fas and its ligand FasL. Developmental cell. 30(3):255-67. Pubmed: 25087893 DOI:S1534-5807(14)00404-3 Choi YJ, Saez B, Anders L, Hydbring P, Stefano J, Bacon NA, Cook C, Kalaszczynska I, Signoretti S, Young RA, Scadden DT, Sicinski P. 2014. D-cyclins repress apoptosis in hematopoietic cells by controlling death receptor Fas and its ligand FasL. Developmental cell. 30(3):255-67. Pubmed: 25087893 DOI:S1534-5807(14)00404-3 D-type cyclins (D1, D2, and D3) are components of the mammalian core cell-cycle machinery and function to drive cell proliferation. Here, we report that D-cyclins perform a rate-limiting antiapoptotic function in vivo. We found that acute shutdown of all three D-cyclins in bone marrow of adult mice resulted in massive apoptosis of all hematopoietic cell types. We demonstrate that adult hematopoietic stem cells are particularly dependent on D-cyclins for survival and that they are especially sensitive to cyclin D loss. Surprisingly, we found that the antiapoptotic function of D-cyclins also operates in quiescent hematopoietic stem and progenitor cells. Our analyses revealed that D-cyclins repress the expression of the death receptor Fas and its ligand, FasL. Acute ablation of D-cyclins upregulated these proapoptotic genes and led to Fas- and caspase 8-dependent apoptosis. These results reveal an unexpected function of cell-cycle proteins in controlling apoptosis in normal cell homeostasis.Copyright © 2014 Elsevier Inc. All rights reserved. -
Kalaitzidis D, Scadden DT. 2014. Deep diving in the blood stem cell-ome. The EMBO journal. 33(20):2281-2. Pubmed: 25190519 DOI:10.15252/embj.201489778 Kalaitzidis D, Scadden DT. 2014. Deep diving in the blood stem cell-ome. The EMBO journal. 33(20):2281-2. Pubmed: 25190519 DOI:10.15252/embj.201489778 Defining the functional distinctions between cells comprising the bone marrow has yielded fundamental insights into lineage ordering and drivers of blood cell production. A novel, highly granular and multi-dimensional molecular characterization of functional subsets of hematopoietic stem- and progenitor cells recently published in (Cabezas-Wallscheid , 2014) will serve as a landmark and treasure trove for unanticipated insights into basic biology and the development of future targeted medicine. 2013
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Catic A, Suh CY, Hill CT, Daheron L, Henkel T, Orford KW, Dombkowski DM, Liu T, Liu XS, Scadden DT. 2013. Genome-wide map of nuclear protein degradation shows NCoR1 turnover as a key to mitochondrial gene regulation. Cell. 155(6):1380-95. Pubmed: 24315104 DOI:S0092-8674(13)01463-3 Catic A, Suh CY, Hill CT, Daheron L, Henkel T, Orford KW, Dombkowski DM, Liu T, Liu XS, Scadden DT. 2013. Genome-wide map of nuclear protein degradation shows NCoR1 turnover as a key to mitochondrial gene regulation. Cell. 155(6):1380-95. Pubmed: 24315104 DOI:S0092-8674(13)01463-3 Transcription factor activity and turnover are functionally linked, but the global patterns by which DNA-bound regulators are eliminated remain poorly understood. We established an assay to define the chromosomal location of DNA-associated proteins that are slated for degradation by the ubiquitin-proteasome system. The genome-wide map described here ties proteolysis in mammalian cells to active enhancers and to promoters of specific gene families. Nuclear-encoded mitochondrial genes in particular correlate with protein elimination, which positively affects their transcription. We show that the nuclear receptor corepressor NCoR1 is a key target of proteolysis and physically interacts with the transcription factor CREB. Proteasome inhibition stabilizes NCoR1 in a site-specific manner and restrains mitochondrial activity by repressing CREB-sensitive genes. In conclusion, this functional map of nuclear proteolysis links chromatin architecture with local protein stability and identifies proteolytic derepression as highly dynamic in regulating the transcription of genes involved in energy metabolism.Copyright © 2013 Elsevier Inc. All rights reserved. -
Zhang H, Alberich-Jorda M, Amabile G, Yang H, Staber PB, Di Ruscio A, Welner RS, Ebralidze A, Zhang J, Levantini E, Lefebvre V, Valk PJ, Delwel R, Hoogenkamp M, Nerlov C, Cammenga J, Saez B, Scadden DT, Bonifer C, Ye M, Tenen DG. 2013. Sox4 is a key oncogenic target in C/EBPα mutant acute myeloid leukemia. Cancer cell. 24(5):575-88. Pubmed: 24183681 DOI:S1535-6108(13)00425-X Zhang H, Alberich-Jorda M, Amabile G, Yang H, Staber PB, Di Ruscio A, Welner RS, Ebralidze A, Zhang J, Levantini E, Lefebvre V, Valk PJ, Delwel R, Hoogenkamp M, Nerlov C, Cammenga J, Saez B, Scadden DT, Bonifer C, Ye M, Tenen DG. 2013. Sox4 is a key oncogenic target in C/EBPα mutant acute myeloid leukemia. Cancer cell. 24(5):575-88. Pubmed: 24183681 DOI:S1535-6108(13)00425-X Mutation or epigenetic silencing of the transcription factor C/EBPα is observed in ∼10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBPα whereby its expression is inversely correlated with C/EBPα activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBPα mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPα inactivation contributes to the development of leukemia with a distinct LIC phenotype.Copyright © 2013 Elsevier Inc. All rights reserved. -
Hartwell KA, Miller PG, Mukherjee S, Kahn AR, Stewart AL, Logan DJ, Negri JM, Duvet M, Järås M, Puram R, Dancik V, Al-Shahrour F, Kindler T, Tothova Z, Chattopadhyay S, Hasaka T, Narayan R, Dai M, Huang C, Shterental S, Chu LP, Haydu JE, Shieh JH, Steensma DP, Munoz B, Bittker JA, Shamji AF, Clemons PA, Tolliday NJ, Carpenter AE, Gilliland DG, Stern AM, Moore MAS, Scadden DT, Schreiber SL, Ebert BL, Golub TR. 2013. Niche-based screening identifies small-molecule inhibitors of leukemia stem cells. Nature chemical biology. 9(12):840-848. Pubmed: 24161946 DOI:10.1038/nchembio.1367 Hartwell KA, Miller PG, Mukherjee S, Kahn AR, Stewart AL, Logan DJ, Negri JM, Duvet M, Järås M, Puram R, Dancik V, Al-Shahrour F, Kindler T, Tothova Z, Chattopadhyay S, Hasaka T, Narayan R, Dai M, Huang C, Shterental S, Chu LP, Haydu JE, Shieh JH, Steensma DP, Munoz B, Bittker JA, Shamji AF, Clemons PA, Tolliday NJ, Carpenter AE, Gilliland DG, Stern AM, Moore MAS, Scadden DT, Schreiber SL, Ebert BL, Golub TR. 2013. Niche-based screening identifies small-molecule inhibitors of leukemia stem cells. Nature chemical biology. 9(12):840-848. Pubmed: 24161946 DOI:10.1038/nchembio.1367 Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening. -
Miller PG, Al-Shahrour F, Hartwell KA, Chu LP, Järås M, Puram RV, Puissant A, Callahan KP, Ashton J, McConkey ME, Poveromo LP, Cowley GS, Kharas MG, Labelle M, Shterental S, Fujisaki J, Silberstein L, Alexe G, Al-Hajj MA, Shelton CA, Armstrong SA, Root DE, Scadden DT, Hynes RO, Mukherjee S, Stegmaier K, Jordan CT, Ebert BL. 2013. In Vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling. Cancer cell. 24(1):45-58. Pubmed: 23770013 DOI:S1535-6108(13)00197-9 Miller PG, Al-Shahrour F, Hartwell KA, Chu LP, Järås M, Puram RV, Puissant A, Callahan KP, Ashton J, McConkey ME, Poveromo LP, Cowley GS, Kharas MG, Labelle M, Shterental S, Fujisaki J, Silberstein L, Alexe G, Al-Hajj MA, Shelton CA, Armstrong SA, Root DE, Scadden DT, Hynes RO, Mukherjee S, Stegmaier K, Jordan CT, Ebert BL. 2013. In Vivo RNAi screening identifies a leukemia-specific dependence on integrin beta 3 signaling. Cancer cell. 24(1):45-58. Pubmed: 23770013 DOI:S1535-6108(13)00197-9 We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.Copyright © 2013 Elsevier Inc. All rights reserved. -
Kook SH, Cho JS, Morrison A, Wiener E, Lee SB, Scadden D, Lee BC. 2013. The purinergic P2Y14 receptor axis is a molecular determinant for organism survival under in utero radiation toxicity. Cell death & disease. 4(7):e703. Pubmed: 23828566 DOI:10.1038/cddis.2013.218 Kook SH, Cho JS, Morrison A, Wiener E, Lee SB, Scadden D, Lee BC. 2013. The purinergic P2Y14 receptor axis is a molecular determinant for organism survival under in utero radiation toxicity. Cell death & disease. 4(7):e703. Pubmed: 23828566 DOI:10.1038/cddis.2013.218 In utero exposure of the embryo and fetus to radiation has been implicated in malformations or fetal death, and often produces lifelong health consequences such as cancers and mental retardation. Here we demonstrate that deletion of a G-protein-coupled purinergic receptor, P2Y14, confers potent resistance to in utero radiation. Intriguingly, a putative P2Y14 receptor ligand, UDP-glucose, phenocopies the effect of P2Y14 deficiency. These data indicate that P2Y14 is a receptor governing in utero tolerance to genotoxic stress that may be pharmacologically targeted to mitigate radiation toxicity in pregnancy. -
Samuel R, Daheron L, Liao S, Vardam T, Kamoun WS, Batista A, Buecker C, Schäfer R, Han X, Au P, Scadden DT, Duda DG, Fukumura D, Jain RK. 2013. Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America. 110(31):12774-9. Pubmed: 23861493 DOI:10.1073/pnas.1310675110 Samuel R, Daheron L, Liao S, Vardam T, Kamoun WS, Batista A, Buecker C, Schäfer R, Han X, Au P, Scadden DT, Duda DG, Fukumura D, Jain RK. 2013. Generation of functionally competent and durable engineered blood vessels from human induced pluripotent stem cells. Proceedings of the National Academy of Sciences of the United States of America. 110(31):12774-9. Pubmed: 23861493 DOI:10.1073/pnas.1310675110 Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers--CD34, neuropilin 1, and human kinase insert domain-containing receptor--and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach. -
Carlson AL, Fujisaki J, Wu J, Runnels JM, Turcotte R, Spencer JA, Celso CL, Scadden DT, Strom TB, Lin CP. 2013. Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label. PloS one. 8(8):e69257. Pubmed: 23990881 DOI:10.1371/journal.pone.0069257 Carlson AL, Fujisaki J, Wu J, Runnels JM, Turcotte R, Spencer JA, Celso CL, Scadden DT, Strom TB, Lin CP. 2013. Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label. PloS one. 8(8):e69257. Pubmed: 23990881 DOI:10.1371/journal.pone.0069257 We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4(+) T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution. -
Krause DS, Fulzele K, Catic A, Sun CC, Dombkowski D, Hurley MP, Lezeau S, Attar E, Wu JY, Lin HY, Divieti-Pajevic P, Hasserjian RP, Schipani E, Van Etten RA, Scadden DT. 2013. Differential regulation of myeloid leukemias by the bone marrow microenvironment. Nature medicine. 19(11):1513-7. Pubmed: 24162813 DOI:10.1038/nm.3364 Krause DS, Fulzele K, Catic A, Sun CC, Dombkowski D, Hurley MP, Lezeau S, Attar E, Wu JY, Lin HY, Divieti-Pajevic P, Hasserjian RP, Schipani E, Van Etten RA, Scadden DT. 2013. Differential regulation of myeloid leukemias by the bone marrow microenvironment. Nature medicine. 19(11):1513-7. Pubmed: 24162813 DOI:10.1038/nm.3364 Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-β1 (TGF-β1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML. -
Silberstein L, Scadden DT. 2013. Blood scent. Cell. 155(5):981-2. Pubmed: 24267883 DOI:S0092-8674(13)01419-0 Silberstein L, Scadden DT. 2013. Blood scent. Cell. 155(5):981-2. Pubmed: 24267883 DOI:S0092-8674(13)01419-0 Blood cell production is tightly regulated by cell-intrinsic mechanisms and environmental factors. The study by Utpal Banerjee and colleagues and colleagues reveals that, in Drosophila, olfactory signals control hematopoietic progenitor maintenance, thus uncovering a physiological link between sensory perception and hematopoietic response to environmental stress.Copyright © 2013 Elsevier Inc. All rights reserved. -
Roccaro AM, Sacco A, Maiso P, Azab AK, Tai YT, Reagan M, Azab F, Flores LM, Campigotto F, Weller E, Anderson KC, Scadden DT, Ghobrial IM. 2013. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. The Journal of clinical investigation. 123(4):1542-55. Pubmed: 23454749 Roccaro AM, Sacco A, Maiso P, Azab AK, Tai YT, Reagan M, Azab F, Flores LM, Campigotto F, Weller E, Anderson KC, Scadden DT, Ghobrial IM. 2013. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression. The Journal of clinical investigation. 123(4):1542-55. Pubmed: 23454749 BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC-derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC-derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression. -
Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, Hu P, Poteat BA, Stilger KN, Ferraro F, Silberstein L, Wong FK, Farag SS, Czader M, Milne GL, Breyer RM, Serezani CH, Scadden DT, Guise TA, Srour EF, Pelus LM. 2013. Differential stem- and progenitor-cell trafficking by prostaglandin E2. Nature. 495(7441):365-9. Pubmed: 23485965 DOI:10.1038/nature11929 Hoggatt J, Mohammad KS, Singh P, Hoggatt AF, Chitteti BR, Speth JM, Hu P, Poteat BA, Stilger KN, Ferraro F, Silberstein L, Wong FK, Farag SS, Czader M, Milne GL, Breyer RM, Serezani CH, Scadden DT, Guise TA, Srour EF, Pelus LM. 2013. Differential stem- and progenitor-cell trafficking by prostaglandin E2. Nature. 495(7441):365-9. Pubmed: 23485965 DOI:10.1038/nature11929 To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically. -
Ruiz-Riol M, Mothe B, Gandhi RT, Bhardwaj N, Scadden DT, Sanchez-Merino V, Brander C. 2013. Influenza, but not HIV-specific CTL epitopes, elicits delayed-type hypersensitivity (DTH) reactions in HIV-infected patients. European journal of immunology. 43(6):1545-54. Pubmed: 23504637 DOI:10.1002/eji.201242732 Ruiz-Riol M, Mothe B, Gandhi RT, Bhardwaj N, Scadden DT, Sanchez-Merino V, Brander C. 2013. Influenza, but not HIV-specific CTL epitopes, elicits delayed-type hypersensitivity (DTH) reactions in HIV-infected patients. European journal of immunology. 43(6):1545-54. Pubmed: 23504637 DOI:10.1002/eji.201242732 The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA-A*02:01-restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed-type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4(+) T-cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope-specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large-scale HIV vaccine trials.© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. -
Fulzele K, Krause DS, Panaroni C, Saini V, Barry KJ, Liu X, Lotinun S, Baron R, Bonewald L, Feng JQ, Chen M, Weinstein LS, Wu JY, Kronenberg HM, Scadden DT, Divieti Pajevic P. 2013. Myelopoiesis is regulated by osteocytes through Gsα-dependent signaling. Blood. 121(6):930-9. Pubmed: 23160461 DOI:10.1182/blood-2012-06-437160 Fulzele K, Krause DS, Panaroni C, Saini V, Barry KJ, Liu X, Lotinun S, Baron R, Bonewald L, Feng JQ, Chen M, Weinstein LS, Wu JY, Kronenberg HM, Scadden DT, Divieti Pajevic P. 2013. Myelopoiesis is regulated by osteocytes through Gsα-dependent signaling. Blood. 121(6):930-9. Pubmed: 23160461 DOI:10.1182/blood-2012-06-437160 Hematopoietic progenitors are regulated in their respective niches by cells of the bone marrow microenvironment. The bone marrow microenvironment is composed of a variety of cell types, and the relative contribution of each of these cells for hematopoietic lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet least understood cells in bone, are thought to initiate adaptive bone remodeling responses via osteoblasts and osteoclasts. Here we report that these cells regulate hematopoiesis, constraining myelopoiesis through a Gsα-mediated mechanism that affects G-CSF production. Mice lacking Gsα in osteocytes showed a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic phenomenon was neither intrinsic to the hematopoietic cells nor dependent on osteoblasts but was a consequence of an altered bone marrow microenvironment imposed by Gsα deficiency in osteocytes. Conditioned media from osteocyte-enriched bone explants significantly increased myeloid colony formation in vitro, which was blocked by G-CSF–neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion. -
Yildirim E, Kirby JE, Brown DE, Mercier FE, Sadreyev RI, Scadden DT, Lee JT. 2013. Xist RNA is a potent suppressor of hematologic cancer in mice. Cell. 152(4):727-42. Pubmed: 23415223 DOI:S0092-8674(13)00086-X Yildirim E, Kirby JE, Brown DE, Mercier FE, Sadreyev RI, Scadden DT, Lee JT. 2013. Xist RNA is a potent suppressor of hematologic cancer in mice. Cell. 152(4):727-42. Pubmed: 23415223 DOI:S0092-8674(13)00086-X X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.Copyright © 2013 Elsevier Inc. All rights reserved. -
Brown K, Xie S, Qiu X, Mohrin M, Shin J, Liu Y, Zhang D, Scadden DT, Chen D. 2013. SIRT3 reverses aging-associated degeneration. Cell reports. 3(2):319-27. Pubmed: 23375372 DOI:S2211-1247(13)00012-0 Brown K, Xie S, Qiu X, Mohrin M, Shin J, Liu Y, Zhang D, Scadden DT, Chen D. 2013. SIRT3 reverses aging-associated degeneration. Cell reports. 3(2):319-27. Pubmed: 23375372 DOI:S2211-1247(13)00012-0 Despite recent controversy about their function in some organisms, sirtuins are thought to play evolutionarily conserved roles in lifespan extension. Whether sirtuins can reverse aging-associated degeneration is unknown. Tissue-specific stem cells persist throughout the entire lifespan to repair and maintain tissues, but their self-renewal and differentiation potential become dysregulated with aging. We show that SIRT3, a mammalian sirtuin that regulates the global acetylation landscape of mitochondrial proteins and reduces oxidative stress, is highly enriched in hematopoietic stem cells (HSCs) where it regulates a stress response. SIRT3 is dispensable for HSC maintenance and tissue homeostasis at a young age under homeostatic conditions but is essential under stress or at an old age. Importantly, SIRT3 is suppressed with aging, and SIRT3 upregulation in aged HSCs improves their regenerative capacity. Our study illuminates the plasticity of mitochondrial homeostasis controlling stem cell and tissue maintenance during the aging process and shows that aging-associated degeneration can be reversed by a sirtuin.Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved. -
Simic P, Zainabadi K, Bell E, Sykes DB, Saez B, Lotinun S, Baron R, Scadden D, Schipani E, Guarente L. 2013. SIRT1 regulates differentiation of mesenchymal stem cells by deacetylating β-catenin. EMBO molecular medicine. 5(3):430-40. Pubmed: 23364955 DOI:10.1002/emmm.201201606 Simic P, Zainabadi K, Bell E, Sykes DB, Saez B, Lotinun S, Baron R, Scadden D, Schipani E, Guarente L. 2013. SIRT1 regulates differentiation of mesenchymal stem cells by deacetylating β-catenin. EMBO molecular medicine. 5(3):430-40. Pubmed: 23364955 DOI:10.1002/emmm.201201606 Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. This potential declines with aging. We investigated whether the sirtuin SIRT1 had a function in MSCs by creating MSC specific SIRT1 knock-out (MSCKO) mice. Aged MSCKO mice (2.2 years old) showed defects in tissues derived from MSCs; i.e. a reduction in subcutaneous fat, cortical bone thickness and trabecular volume. Young mice showed related but less pronounced effects. MSCs isolated from MSCKO mice showed reduced differentiation towards osteoblasts and chondrocytes in vitro, but no difference in proliferation or apoptosis. Expression of β-catenin targets important for differentiation was reduced in MSCKO cells. Moreover, while β-catenin itself (T41A mutant resistant to cytosolic turnover) accumulated in the nuclei of wild-type MSCs, it was unable to do so in MSCKO cells. However, mutating K49R or K345R in β-catenin to mimic deacetylation restored nuclear localization and differentiation potential in MSCKO cells. We conclude that SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus leading to transcription of genes for MSC differentiation.Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO. -
Cutler C, Multani P, Robbins D, Kim HT, Le T, Hoggatt J, Pelus LM, Desponts C, Chen YB, Rezner B, Armand P, Koreth J, Glotzbecker B, Ho VT, Alyea E, Isom M, Kao G, Armant M, Silberstein L, Hu P, Soiffer RJ, Scadden DT, Ritz J, Goessling W, North TE, Mendlein J, Ballen K, Zon LI, Antin JH, Shoemaker DD. 2013. Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood. 122(17):3074-81. Pubmed: 23996087 DOI:10.1182/blood-2013-05-503177 Cutler C, Multani P, Robbins D, Kim HT, Le T, Hoggatt J, Pelus LM, Desponts C, Chen YB, Rezner B, Armand P, Koreth J, Glotzbecker B, Ho VT, Alyea E, Isom M, Kao G, Armant M, Silberstein L, Hu P, Soiffer RJ, Scadden DT, Ritz J, Goessling W, North TE, Mendlein J, Ballen K, Zon LI, Antin JH, Shoemaker DD. 2013. Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood. 122(17):3074-81. Pubmed: 23996087 DOI:10.1182/blood-2013-05-503177 Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants. -
Krause DS, Scadden DT, Preffer FI. 2013. The hematopoietic stem cell niche--home for friend and foe?. Cytometry. Part B, Clinical cytometry. 84(1):7-20. Pubmed: 23281119 DOI:10.1002/cyto.b.21066 Krause DS, Scadden DT, Preffer FI. 2013. The hematopoietic stem cell niche--home for friend and foe?. Cytometry. Part B, Clinical cytometry. 84(1):7-20. Pubmed: 23281119 DOI:10.1002/cyto.b.21066 The hematopoietic stem cell (HSC) niche is involved in the maintainance and regulation of quiescence, self-renewal and differentiation of hematopoietic stem cells and the fate of their progeny in mammals dealing with the daily stresses to the hematopoietic system. From the discovery that perturbations of the HSC niche can lead to hematopoietic disorders, we have now arrived at the prospect that the HSC niche may play a role in hematological malignancies and that this HSC niche may be a target for therapy. This review attempts to capture the discoveries of the last few years regarding the normal and malignant hematopoietic stem cell niche and possible ways to target this niche.Copyright © 2012 International Clinical Cytometry Society. -
Van Etten RA, Mauro M, Radich JP, Goldman JM, Saglio G, Jamieson C, Soverini S, Gambacorti-Passerini C, Hehlmann R, Martinelli G, Perrotti D, Scadden DT, Skorski T, Tefferi A, Mughal TI. 2013. Advances in the biology and therapy of chronic myeloid leukemia: proceedings from the 6th Post-ASH International Chronic Myeloid Leukemia and Myeloproliferative Neoplasms Workshop. Leukemia & lymphoma. 54(6):1151-8. Pubmed: 23121619 DOI:10.3109/10428194.2012.745524 Van Etten RA, Mauro M, Radich JP, Goldman JM, Saglio G, Jamieson C, Soverini S, Gambacorti-Passerini C, Hehlmann R, Martinelli G, Perrotti D, Scadden DT, Skorski T, Tefferi A, Mughal TI. 2013. Advances in the biology and therapy of chronic myeloid leukemia: proceedings from the 6th Post-ASH International Chronic Myeloid Leukemia and Myeloproliferative Neoplasms Workshop. Leukemia & lymphoma. 54(6):1151-8. Pubmed: 23121619 DOI:10.3109/10428194.2012.745524 Following the 53rd annual meeting of the American Society of Hematology (ASH) in San Diego in December 2011, a group of clinical and laboratory investigators convened for the 6th Post-ASH International Workshop on Chronic Myeloid Leukemia (CML) and Myeloproliferative Neoplasms (MPN). The Workshop took place on 13-14 December at the Estancia, La Jolla, California, USA. This report summarizes the most recent advances in the biology and therapy of CML that were presented at the ASH meeting and discussed at the Workshop. Preclinical studies focused on the CML stem cell and its niche, and on early results of deep sequencing of CML genomes. Clinical advances include updates on second- and third-generation tyrosine kinase inhibitors (TKIs), molecular monitoring, TKI discontinuation studies and new therapeutic agents. A report summarizing the pertinent advances in MPN has been published separately. -
Pozzi S, Fulciniti M, Yan H, Vallet S, Eda H, Patel K, Santo L, Cirstea D, Hideshima T, Schirtzinge L, Kuhstoss S, Anderson KC, Munshi N, Scadden D, Kronenberg HM, Raje N. 2013. In vivo and in vitro effects of a novel anti-Dkk1 neutralizing antibody in multiple myeloma. Bone. 53(2):487-96. Pubmed: 23333523 DOI:S8756-3282(13)00019-7 Pozzi S, Fulciniti M, Yan H, Vallet S, Eda H, Patel K, Santo L, Cirstea D, Hideshima T, Schirtzinge L, Kuhstoss S, Anderson KC, Munshi N, Scadden D, Kronenberg HM, Raje N. 2013. In vivo and in vitro effects of a novel anti-Dkk1 neutralizing antibody in multiple myeloma. Bone. 53(2):487-96. Pubmed: 23333523 DOI:S8756-3282(13)00019-7 Over-expression of the protein Dickkopf-1 (Dkk1) has been associated with multiple myeloma bone disease. Previous reports with the use of anti-Dkk1 neutralizing Ab directed strategies have demonstrated a pro-anabolic effect with associated anti-myeloma activity in 2 in vivo mouse models. However new insights on the role of the wnt pathway in osteoclasts (OC) are emerging and the potential effect of a neutralizing Ab to Dkk1 in osteoclastogenesis remains to be elucidated. In order to better define the effect of an anti-Dkk1 neutralizing Ab on osteoclastogenesis and myeloma, we studied a novel anti-Dkk1 monoclonal Ab in our preclinical models. In vivo data confirmed the pro-anabolic and anti-MM effect. In vitro data in part confirmed the in vivo observation, suggesting an indirect anti-MM effect secondary to inhibition of osteoclastogenesis and thus the interaction between MM and bone microenvironment. However, when studies on osteoclastogenesis were extended to samples derived from MM patients, we observed a variable response to anti-Dkk1 treatment without correlation to expression of surface receptors for Dkk1 in OCs suggesting potential heterogeneity in the efficacy of such a strategy. In conclusion, Dkk1 is a promising target for the treatment of both MM and bone disease, and ongoing clinical studies will help elucidate its efficacy.Copyright © 2013 Elsevier Inc. All rights reserved. -
Sykes SM, Scadden DT. 2013. Modeling human hematopoietic stem cell biology in the mouse. Seminars in hematology. 50(2):92-100. Pubmed: 24216169 DOI:S0037-1963(13)00053-X Sykes SM, Scadden DT. 2013. Modeling human hematopoietic stem cell biology in the mouse. Seminars in hematology. 50(2):92-100. Pubmed: 24216169 DOI:S0037-1963(13)00053-X Hematopoietic stem cells (HSCs) have the immense task of supplying an organism with enough blood to sustain a lifespan. Much of what is known about how this scant population of cells can meet the varying demand of producing more than 10(11) cells per day comes from studies conducted in an animal that is a fraction of our size and lives roughly 1/30th of our lifespan. The differences in longevity can be expected to impose different demands on a cell essential for existence. It is therefore unsurprising that while the mouse has proven invaluable in defining the organizing principals of how hematopoiesis is governed, mediators of cell localization as well as a range of experimental methods, the differences in cell cycling, DNA repair and specific molecular features of HSCs in humans are evident and important. Here, the utility and drawbacks of the mouse as an experimental model for human HSC biology are discussed.Copyright © 2013 Elsevier Inc. All rights reserved. 2012
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Mosam A, Shaik F, Uldrick TS, Esterhuizen T, Friedland GH, Scadden DT, Aboobaker J, Coovadia HM. 2012. A randomized controlled trial of highly active antiretroviral therapy versus highly active antiretroviral therapy and chemotherapy in therapy-naive patients with HIV-associated Kaposi sarcoma in South Africa. Journal of acquired immune deficiency syndromes (1999). 60(2):150-7. Pubmed: 22395672 DOI:10.1097/QAI.0b013e318251aedd Mosam A, Shaik F, Uldrick TS, Esterhuizen T, Friedland GH, Scadden DT, Aboobaker J, Coovadia HM. 2012. A randomized controlled trial of highly active antiretroviral therapy versus highly active antiretroviral therapy and chemotherapy in therapy-naive patients with HIV-associated Kaposi sarcoma in South Africa. Journal of acquired immune deficiency syndromes (1999). 60(2):150-7. Pubmed: 22395672 DOI:10.1097/QAI.0b013e318251aedd Array -
Krause DS, Scadden DT. 2012. Deconstructing the complexity of a microenvironmental niche. Cell. 149(1):16-7. Pubmed: 22464318 DOI:10.1016/j.cell.2012.03.005 Krause DS, Scadden DT. 2012. Deconstructing the complexity of a microenvironmental niche. Cell. 149(1):16-7. Pubmed: 22464318 DOI:10.1016/j.cell.2012.03.005 Cells acquire their fate in vivo in the context of a complex microenvironmental "niche" comprised of heterologous cell types, signaling molecules, extracellular matrix, biophysical forces, and metabolic substrates. Now Calderón and Boehm report the "refunctionalization" of a defective thymic epithelial niche, offering insights into how signaling molecules may be hierarchically organized to direct differentiation outcomes.Copyright © 2012 Elsevier Inc. All rights reserved. -
Park D, Spencer JA, Koh BI, Kobayashi T, Fujisaki J, Clemens TL, Lin CP, Kronenberg HM, Scadden DT. 2012. Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration. Cell stem cell. 10(3):259-72. Pubmed: 22385654 DOI:10.1016/j.stem.2012.02.003 Park D, Spencer JA, Koh BI, Kobayashi T, Fujisaki J, Clemens TL, Lin CP, Kronenberg HM, Scadden DT. 2012. Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration. Cell stem cell. 10(3):259-72. Pubmed: 22385654 DOI:10.1016/j.stem.2012.02.003 Mesenchymal stem cells (MSCs) commonly defined by in vitro functions have entered clinical application despite little definition of their function in residence. Here, we report genetic pulse-chase experiments that define osteoblastic cells as short-lived and nonreplicative, requiring replenishment from bone-marrow-derived, Mx1(+) stromal cells with "MSC" features. These cells respond to tissue stress and migrate to sites of injury, supplying new osteoblasts during fracture healing. Single cell transplantation yielded progeny that both preserve progenitor function and differentiate into osteoblasts, producing new bone. They are capable of local and systemic translocation and serial transplantation. While these cells meet current definitions of MSCs in vitro, they are osteolineage restricted in vivo in growing and adult animals. Therefore, bone-marrow-derived MSCs may be a heterogeneous population with the Mx1(+) population, representing a highly dynamic and stress responsive stem/progenitor cell population of fate-restricted potential that feeds the high cell replacement demands of the adult skeleton.Copyright © 2012 Elsevier Inc. All rights reserved. -
Scadden D, Srivastava A. 2012. Advancing stem cell biology toward stem cell therapeutics. Cell stem cell. 10(2):149-50. Pubmed: 22305564 DOI:10.1016/j.stem.2012.01.010 Scadden D, Srivastava A. 2012. Advancing stem cell biology toward stem cell therapeutics. Cell stem cell. 10(2):149-50. Pubmed: 22305564 DOI:10.1016/j.stem.2012.01.010 Here, the International Society for Stem Cell Research (ISSCR) Clinical Translation Committee introduces a series of disease-specific articles, outlining the challenges surrounding the clinical translation of stem cell therapeutics.Copyright © 2012 Elsevier Inc. All rights reserved. -
Park D, Sykes DB, Scadden DT. 2012. The hematopoietic stem cell niche. Frontiers in bioscience (Landmark edition). 17(1):30-9. Pubmed: 22201730 Park D, Sykes DB, Scadden DT. 2012. The hematopoietic stem cell niche. Frontiers in bioscience (Landmark edition). 17(1):30-9. Pubmed: 22201730 Hematopoietic stem cells (HSCs) possess the ability to self-renew and to differentiate to mature progeny along multiple different hematopoietic lineages. The function of HSCs depends upon the signals from surrounding cells found within the highly specialized microenvironment termed the hematopoietic stem cell niche. Understanding and exploiting the HSC niche is a goal of basic scientists and clinicians alike. Recent studies have focused on defining the cellular components and molecular factors critical to this microenvironment. Here we review recent findings, discuss unresolved questions, and examine the clinical implications of our current knowledge of the HSC niche. -
Yusuf RZ, Scadden DT. 2012. Fate through fat: lipid metabolism determines stem cell division outcome. Cell metabolism. 16(4):411-3. Pubmed: 23040066 DOI:S1550-4131(12)00371-3 Yusuf RZ, Scadden DT. 2012. Fate through fat: lipid metabolism determines stem cell division outcome. Cell metabolism. 16(4):411-3. Pubmed: 23040066 DOI:S1550-4131(12)00371-3 Distinctive metabolism associated with particular cell states is increasingly being defined in normal and malignant cells. Ito et al. (2012) now show that fatty acid oxidation is associated with hematopoietic stem cells and determines whether they undergo symmetric or asymmetric cell division, driving a fundamental property of the stem cell state.Copyright © 2012 Elsevier Inc. All rights reserved. -
Bungartz G, Land H, Scadden DT, Emerson SG. 2012. NF-Y is necessary for hematopoietic stem cell proliferation and survival. Blood. 119(6):1380-9. Pubmed: 22072554 DOI:10.1182/blood-2011-06-359406 Bungartz G, Land H, Scadden DT, Emerson SG. 2012. NF-Y is necessary for hematopoietic stem cell proliferation and survival. Blood. 119(6):1380-9. Pubmed: 22072554 DOI:10.1182/blood-2011-06-359406 HSC function depends on the tight control of proliferation and the balance between self-renewal and differentiation. Here, we report that the trimeric transcription factor NF-Y is critical for the survival of cycling, but not quiescent HSCs. With the use of a conditional knockout mouse model, we demonstrate that NF-Ya deletion creates an accumulation of HSCs in G(2)/M and prompts apoptosis, causing hematopoietic failure and death of the animal. These defects are accompanied by the dysregulation of multiple genes that influence cell cycle control (cyclin b1 and p21), apoptosis (Bcl-2), and self-renewal (HoxB4, Notch1, Bmi-1) and are independent of p53. Our results identify NF-Y as a pivotal upstream participant in a regulatory network necessary for the preservation of cycling HSCs. -
Kalaitzidis D, Sykes SM, Wang Z, Punt N, Tang Y, Ragu C, Sinha AU, Lane SW, Souza AL, Clish CB, Anastasiou D, Gilliland DG, Scadden DT, Guertin DA, Armstrong SA. 2012. mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis. Cell stem cell. 11(3):429-39. Pubmed: 22958934 DOI:10.1016/j.stem.2012.06.009 Kalaitzidis D, Sykes SM, Wang Z, Punt N, Tang Y, Ragu C, Sinha AU, Lane SW, Souza AL, Clish CB, Anastasiou D, Gilliland DG, Scadden DT, Guertin DA, Armstrong SA. 2012. mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis. Cell stem cell. 11(3):429-39. Pubmed: 22958934 DOI:10.1016/j.stem.2012.06.009 The mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition.Copyright © 2012 Elsevier Inc. All rights reserved. -
Hoggatt J, Scadden DT. 2012. The stem cell niche: tissue physiology at a single cell level. The Journal of clinical investigation. 122(9):3029-34. Pubmed: 22945635 DOI:60238 Hoggatt J, Scadden DT. 2012. The stem cell niche: tissue physiology at a single cell level. The Journal of clinical investigation. 122(9):3029-34. Pubmed: 22945635 DOI:60238 Stem cells are the critical unit affecting tissue maintenance, regeneration, and repair, with particular relevance to the tissues with high cell turnover. Stem cell regulation accommodates the conflicting needs of prompt responsiveness to injury and long-term preservation through quiescence. They are, in essence, the fundamental unit by which a tissue handles changing physiologic needs throughout the lifetime of the organism. As such, they are the focal point of dynamic tissue function, and their governance is physiology expressed at a cellular and molecular level. Here, we discuss the multiple components representing the stem cell niche in hematopoiesis and argue for a unbiased mapping of the niche constituents under different conditions as the first step in developing systems physiology. -
Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, Scadden DT, Wagner DD. 2012. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proceedings of the National Academy of Sciences of the United States of America. 109(32):13076-81. Pubmed: 22826226 DOI:10.1073/pnas.1200419109 Demers M, Krause DS, Schatzberg D, Martinod K, Voorhees JR, Fuchs TA, Scadden DT, Wagner DD. 2012. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis. Proceedings of the National Academy of Sciences of the United States of America. 109(32):13076-81. Pubmed: 22826226 DOI:10.1073/pnas.1200419109 Cancer-associated thrombosis often lacks a clear etiology. However, it is linked to a poor prognosis and represents the second-leading cause of death in cancer patients. Recent studies have shown that chromatin released into blood, through the generation of neutrophil extracellular traps (NETs), is procoagulant and prothrombotic. Using a murine model of chronic myelogenous leukemia, we show that malignant and nonmalignant neutrophils are more prone to NET formation. This increased sensitivity toward NET generation is also observed in mammary and lung carcinoma models, suggesting that cancers, through a systemic effect on the host, can induce an increase in peripheral blood neutrophils, which are predisposed to NET formation. In addition, in the late stages of the breast carcinoma model, NETosis occurs concomitant with the appearance of venous thrombi in the lung. Moreover, simulation of a minor systemic infection in tumor-bearing, but not control, mice results in the release of large quantities of chromatin and a prothrombotic state. The increase in neutrophil count and their priming is mediated by granulocyte colony-stimulating factor (G-CSF), which accumulates in the blood of tumor-bearing mice. The prothrombotic state in cancer can be reproduced by treating mice with G-CSF combined with low-dose LPS and leads to thrombocytopenia and microthrombosis. Taken together, our results identify extracellular chromatin released through NET formation as a cause for cancer-associated thrombosis and unveil a target in the effort to decrease the incidence of thrombosis in cancer patients. -
Scadden DT. 2012. Rethinking stroma: lessons from the blood. Cell stem cell. 10(6):648-649. Pubmed: 22704500 DOI:S1934-5909(12)00244-5 Scadden DT. 2012. Rethinking stroma: lessons from the blood. Cell stem cell. 10(6):648-649. Pubmed: 22704500 DOI:S1934-5909(12)00244-5 Stroma is a largely understudied component of all organs that contributes to stem cell niches. Studies to define stromal components in the bone marrow have led to some unexpected findings that prompt further research.Copyright © 2012 Elsevier Inc. All rights reserved. -
Yusuf RZ, Wang YH, Scadden DT. 2012. The secrets of the bone marrow niche: Metabolic priming for AML. Nature medicine. 18(6):865-867. Pubmed: 22673998 DOI:10.1038/nm.2831 Yusuf RZ, Wang YH, Scadden DT. 2012. The secrets of the bone marrow niche: Metabolic priming for AML. Nature medicine. 18(6):865-867. Pubmed: 22673998 DOI:10.1038/nm.2831 -
Guo S, Bai H, Megyola CM, Halene S, Krause DS, Scadden DT, Lu J. 2012. Complex oncogene dependence in microRNA-125a-induced myeloproliferative neoplasms. Proceedings of the National Academy of Sciences of the United States of America. 109(41):16636-41. Pubmed: 23012470 DOI:10.1073/pnas.1213196109 Guo S, Bai H, Megyola CM, Halene S, Krause DS, Scadden DT, Lu J. 2012. Complex oncogene dependence in microRNA-125a-induced myeloproliferative neoplasms. Proceedings of the National Academy of Sciences of the United States of America. 109(41):16636-41. Pubmed: 23012470 DOI:10.1073/pnas.1213196109 Deregulation of microRNA (miRNA) expression can lead to cancer initiation and progression. However, limited information exists on the function of miRNAs in cancer maintenance. We examined these issues in the case of myeloproliferative diseases and neoplasms (MPN), a collection of hematopoietic neoplasms regarded as preleukemic, thereby representing early neoplastic states. We report here that microRNA-125a (miR-125a)-induced MPN display a complex manner of oncogene dependence. Following a gain-of-function genomics screen, we overexpressed candidate miR-125a in vivo, which led to phenotypes consistent with an atypical MPN characterized by leukocytosis, monocytosis, splenomegaly, and progressive anemia. The diseased MPN state could be recapitulated in a doxycycline-inducible mouse model. Upon doxycycline withdrawal, the primary MPN phenotypes rapidly resolved after the discontinuation of miR-125a overexpression. However, reinduction of miR-125a led to complex phenotypes, with some animals rapidly developing lethal anemia with extensive damages in the spleen. Forced expression of miR-125a resulted in elevated cellular tyrosine phosphorylation and hypersensitivity toward hematopoietic cytokines. Furthermore, we demonstrate that miR-125a targets multiple protein phosphatases. Our data demonstrate that miR-125a-induced MPN is addicted to its sustained overexpression, and highlight the complex nature of oncogenic miRNA dependence in an early neoplastic state. 2011
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Mercier FE, Ragu C, Scadden DT. 2011. The bone marrow at the crossroads of blood and immunity. Nature reviews. Immunology. 12(1):49-60. Pubmed: 22193770 DOI:10.1038/nri3132 Mercier FE, Ragu C, Scadden DT. 2011. The bone marrow at the crossroads of blood and immunity. Nature reviews. Immunology. 12(1):49-60. Pubmed: 22193770 DOI:10.1038/nri3132 Progenitor cells that are the basis for all blood cell production share the bone marrow with more mature elements of the adaptive immune system. Specialized niches within the bone marrow guide and, at times, constrain the development of haematopoietic stem and progenitor cells (HSPCs) and lineage-restricted immune progenitor cells. Specific niche components are organized into distinct domains to create a diversified landscape in which specialized cell differentiation or population expansion programmes proceed. Local cues that reflect the tissue and organismal state affect cellular interactions to alter the production of a range of cell types. Here, we review the organization of regulatory elements in the bone marrow and discuss how these elements provide a dynamic means for the host to modulate stem cell and adaptive immune cell responses to physiological challenges. -
Sykes SM, Lane SW, Bullinger L, Kalaitzidis D, Yusuf R, Saez B, Ferraro F, Mercier F, Singh H, Brumme KM, Acharya SS, Scholl C, Tothova Z, Attar EC, Fröhling S, DePinho RA, Armstrong SA, Gilliland DG, Scadden DT. 2011. AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell. 146(5):697-708. Pubmed: 21884932 DOI:10.1016/j.cell.2011.07.032 Sykes SM, Lane SW, Bullinger L, Kalaitzidis D, Yusuf R, Saez B, Ferraro F, Mercier F, Singh H, Brumme KM, Acharya SS, Scholl C, Tothova Z, Attar EC, Fröhling S, DePinho RA, Armstrong SA, Gilliland DG, Scadden DT. 2011. AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell. 146(5):697-708. Pubmed: 21884932 DOI:10.1016/j.cell.2011.07.032 AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ∼40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.Copyright © 2011 Elsevier Inc. All rights reserved. -
Sanchez-Aguilera A, Lee YJ, Lo Celso C, Ferraro F, Brumme K, Mondal S, Kim C, Dorrance A, Luo HR, Scadden DT, Williams DA. 2011. Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells. Proceedings of the National Academy of Sciences of the United States of America. 108(23):9607-12. Pubmed: 21606370 DOI:10.1073/pnas.1102018108 Sanchez-Aguilera A, Lee YJ, Lo Celso C, Ferraro F, Brumme K, Mondal S, Kim C, Dorrance A, Luo HR, Scadden DT, Williams DA. 2011. Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells. Proceedings of the National Academy of Sciences of the United States of America. 108(23):9607-12. Pubmed: 21606370 DOI:10.1073/pnas.1102018108 Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1(-/-) HSPC showed impaired early localization near nestin(+) perivascular mesenchymal stem cells; only 6.25% of Vav1(-/-) HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a nestin(+) cell. Abnormal perivascular localization correlated with decreased retention of Vav1(-/-) HSPC in the bone marrow (44-60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1(-/-) cells vs. 53-63% for wild-type cells). The engraftment defect of Vav1(-/-) HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1(-/-) HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment. -
Cornejo MG, Mabialah V, Sykes SM, Khandan T, Lo Celso C, Lopez CK, Rivera-Muñoz P, Rameau P, Tothova Z, Aster JC, DePinho RA, Scadden DT, Gilliland DG, Mercher T. 2011. Crosstalk between NOTCH and AKT signaling during murine megakaryocyte lineage specification. Blood. 118(5):1264-73. Pubmed: 21653327 DOI:10.1182/blood-2011-01-328567 Cornejo MG, Mabialah V, Sykes SM, Khandan T, Lo Celso C, Lopez CK, Rivera-Muñoz P, Rameau P, Tothova Z, Aster JC, DePinho RA, Scadden DT, Gilliland DG, Mercher T. 2011. Crosstalk between NOTCH and AKT signaling during murine megakaryocyte lineage specification. Blood. 118(5):1264-73. Pubmed: 21653327 DOI:10.1182/blood-2011-01-328567 The NOTCH signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. However, the molecular basis for its role at the different steps of stem cell lineage commitment is unclear. We recently identified the NOTCH signaling pathway as a positive regulator of megakaryocyte lineage specification during hematopoiesis, but the developmental pathways that allow hematopoietic stem cell differentiation into the erythro-megakaryocytic lineages remain controversial. Here, we investigated the role of downstream mediators of NOTCH during megakaryopoiesis and report crosstalk between the NOTCH and PI3K/AKT pathways. We demonstrate the inhibitory role of phosphatase with tensin homolog and Forkhead Box class O factors on megakaryopoiesis in vivo. Finally, our data annotate developmental mechanisms in the hematopoietic system that enable a decision to be made either at the hematopoietic stem cell or the committed progenitor level to commit to the megakaryocyte lineage, supporting the existence of 2 distinct developmental pathways. -
Fujisaki J, Wu J, Carlson AL, Silberstein L, Putheti P, Larocca R, Gao W, Saito TI, Lo Celso C, Tsuyuzaki H, Sato T, Côté D, Sykes M, Strom TB, Scadden DT, Lin CP. 2011. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature. 474(7350):216-9. Pubmed: 21654805 DOI:10.1038/nature10160 Fujisaki J, Wu J, Carlson AL, Silberstein L, Putheti P, Larocca R, Gao W, Saito TI, Lo Celso C, Tsuyuzaki H, Sato T, Côté D, Sykes M, Strom TB, Scadden DT, Lin CP. 2011. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature. 474(7350):216-9. Pubmed: 21654805 DOI:10.1038/nature10160 Stem cells reside in a specialized regulatory microenvironment or niche, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity. The niche may also protect stem cells from environmental insults including cytotoxic chemotherapy and perhaps pathogenic immunity. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche in the bone marrow, a site where immune reactivity exists. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30 days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (T(reg)) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with T(reg) cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. T(reg) cells seem to participate in creating a localized zone where HSPCs reside and where T(reg) cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack. -
Lane SW, Wang YJ, Lo Celso C, Ragu C, Bullinger L, Sykes SM, Ferraro F, Shterental S, Lin CP, Gilliland DG, Scadden DT, Armstrong SA, Williams DA. 2011. Differential niche and Wnt requirements during acute myeloid leukemia progression. Blood. 118(10):2849-56. Pubmed: 21765021 DOI:10.1182/blood-2011-03-345165 Lane SW, Wang YJ, Lo Celso C, Ragu C, Bullinger L, Sykes SM, Ferraro F, Shterental S, Lin CP, Gilliland DG, Scadden DT, Armstrong SA, Williams DA. 2011. Differential niche and Wnt requirements during acute myeloid leukemia progression. Blood. 118(10):2849-56. Pubmed: 21765021 DOI:10.1182/blood-2011-03-345165 Hematopoietic stem cells (HSCs) engage in complex bidirectional signals with the hematopoietic microenvironment (HM), and there is emerging evidence that leukemia stem cells (LSCs) may use similar interactions. Using a syngeneic retroviral model of MLL-AF9 induced acute myeloid leukemia (AML), we have identified 2 different stages of leukemia progression, propagated by "pre-LSCs" and established leukemia (LSCs) and compared the homing properties of these distinctive entities to that of normal HSCs. The homing and microlocalization of pre-LSCs was most similar to long-term HSCs and was dependent on cell-intrinsic Wnt signaling. In contrast, the homing of established LSCs was most similar to that of committed myeloid progenitors and distinct from HSCs. Although osteoblast-derived Dickkopf-1, a potent Wnt inhibitor known to impair HSC function, dramatically impaired normal HSC localization within the bone marrow, it did not affect pre-LSCs, LSC homing, or AML development. Mechanistically, cell-intrinsic Wnt activation was observed in human and murine AML samples, explaining the independence of MLL-AF9 LSCs from niche-derived Wnt signals. These data identify differential engagement of HM associated with leukemic progression and identify an LSC niche that is physically distinct and independent of the constraints of Wnt signaling that apply to normal HSCs. -
Lo Celso C, Scadden DT. 2011. The haematopoietic stem cell niche at a glance. Journal of cell science. 124(Pt 21):3529-35. Pubmed: 22083139 DOI:10.1242/jcs.074112 Lo Celso C, Scadden DT. 2011. The haematopoietic stem cell niche at a glance. Journal of cell science. 124(Pt 21):3529-35. Pubmed: 22083139 DOI:10.1242/jcs.074112 -
Ferraro F, Lymperi S, Méndez-Ferrer S, Saez B, Spencer JA, Yeap BY, Masselli E, Graiani G, Prezioso L, Rizzini EL, Mangoni M, Rizzoli V, Sykes SM, Lin CP, Frenette PS, Quaini F, Scadden DT. 2011. Diabetes impairs hematopoietic stem cell mobilization by altering niche function. Science translational medicine. 3(104):104ra101. Pubmed: 21998408 DOI:10.1126/scitranslmed.3002191 Ferraro F, Lymperi S, Méndez-Ferrer S, Saez B, Spencer JA, Yeap BY, Masselli E, Graiani G, Prezioso L, Rizzini EL, Mangoni M, Rizzoli V, Sykes SM, Lin CP, Frenette PS, Quaini F, Scadden DT. 2011. Diabetes impairs hematopoietic stem cell mobilization by altering niche function. Science translational medicine. 3(104):104ra101. Pubmed: 21998408 DOI:10.1126/scitranslmed.3002191 Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients. -
Lo Celso C, Lin CP, Scadden DT. 2011. In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Nature protocols. 6(1):1-14. Pubmed: 21212779 DOI:10.1038/nprot.2010.168 Lo Celso C, Lin CP, Scadden DT. 2011. In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow. Nature protocols. 6(1):1-14. Pubmed: 21212779 DOI:10.1038/nprot.2010.168 In vivo imaging of transplanted hematopoietic stem and progenitor cells (HSPCs) was developed to investigate the relationship between HSPCs and components of their microenvironment in the bone marrow. In particular, it allows a direct observation of the behavior of hematopoietic cells during the first few days after transplantation, when the critical events in homing and early engraftment are occurring. By directly imaging these events in living animals, this method permits a detailed assessment of functions previously evaluated by crude assessments of cell counts (homing) or after prolonged periods (engraftment). This protocol offers a new means of investigating the role of cell-intrinsic and cell-extrinsic molecular regulators of hematopoiesis during the early stages of transplantation, and it is the first to allow the study of cell-cell interactions within the bone marrow in three dimensions and in real time. In this paper, we describe how to isolate, label and inject HSPCs, as well as how to perform calvarium intravital microscopy and analyze the resulting images. A typical experiment can be performed and analyzed in ∼1 week. -
Vunjak-Novakovic G, Scadden DT. 2011. Biomimetic platforms for human stem cell research. Cell stem cell. 8(3):252-61. Pubmed: 21362565 DOI:10.1016/j.stem.2011.02.014 Vunjak-Novakovic G, Scadden DT. 2011. Biomimetic platforms for human stem cell research. Cell stem cell. 8(3):252-61. Pubmed: 21362565 DOI:10.1016/j.stem.2011.02.014 Stem cells are central to developing new treatment options for tissue regeneration and constructing controllable models for biological research. Bioengineered cell culture environments that combine microenvironmental control with tissue-specific transport and signaling are critical tools in our efforts to study tissue development, regeneration, and disease under conditions that predict the human in vivo context. We propose that experimentation at the interfaces of biology, engineering, and medical sciences is critical for unlocking the full potential of stem cells. Here, we focus on the design and utilization of in vitro platforms that recapitulate the environments associated with tissue development, disease, and regeneration.Copyright © 2011 Elsevier Inc. All rights reserved. -
Steinbicker AU, Sachidanandan C, Vonner AJ, Yusuf RZ, Deng DY, Lai CS, Rauwerdink KM, Winn JC, Saez B, Cook CM, Szekely BA, Roy CN, Seehra JS, Cuny GD, Scadden DT, Peterson RT, Bloch KD, Yu PB. 2011. Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation. Blood. 117(18):4915-23. Pubmed: 21393479 DOI:10.1182/blood-2010-10-313064 Steinbicker AU, Sachidanandan C, Vonner AJ, Yusuf RZ, Deng DY, Lai CS, Rauwerdink KM, Winn JC, Saez B, Cook CM, Szekely BA, Roy CN, Seehra JS, Cuny GD, Scadden DT, Peterson RT, Bloch KD, Yu PB. 2011. Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation. Blood. 117(18):4915-23. Pubmed: 21393479 DOI:10.1182/blood-2010-10-313064 Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation. -
Vallet S, Pozzi S, Patel K, Vaghela N, Fulciniti MT, Veiby P, Hideshima T, Santo L, Cirstea D, Scadden DT, Anderson KC, Raje N. 2011. A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function. Leukemia. 25(7):1174-81. Pubmed: 21403648 DOI:10.1038/leu.2011.43 Vallet S, Pozzi S, Patel K, Vaghela N, Fulciniti MT, Veiby P, Hideshima T, Santo L, Cirstea D, Scadden DT, Anderson KC, Raje N. 2011. A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function. Leukemia. 25(7):1174-81. Pubmed: 21403648 DOI:10.1038/leu.2011.43 Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine, levels of which in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. In this study, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function, and therefore contributes to OB/OC uncoupling in MM. 2010
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Jeanson NT, Scadden DT. 2010. Vitamin D receptor deletion leads to increased hematopoietic stem and progenitor cells residing in the spleen. Blood. 116(20):4126-9. Pubmed: 20664059 DOI:10.1182/blood-2010-04-280552 Jeanson NT, Scadden DT. 2010. Vitamin D receptor deletion leads to increased hematopoietic stem and progenitor cells residing in the spleen. Blood. 116(20):4126-9. Pubmed: 20664059 DOI:10.1182/blood-2010-04-280552 Bone components participate in the regulation of hematopoietic stem cells in the adult mammal. Vitamin D regulates bone mineralization and is associated with pleiotropic effects in many cell types, including putative roles in hematopoietic differentiation. We report that deletion of the vitamin D receptor (VDR) in hematopoietic cells did not result in cell autonomous perturbation of hematopoietic stem cell or progenitor function. However, deletion of VDR in the microenvironment resulted in a marked accumulation of hematopoietic stem cells in the spleen that could be reversed by calcium dietary supplementation. These data suggest that VDR participates in restricting splenic hematopoiesis through maintenance of bone calcium homeostasis and are consistent with the concept that calcium regulation through VDR is a central participant in localizing adult hematopoiesis preferentially to bone marrow. -
Guo S, Lu J, Schlanger R, Zhang H, Wang JY, Fox MC, Purton LE, Fleming HH, Cobb B, Merkenschlager M, Golub TR, Scadden DT. 2010. MicroRNA miR-125a controls hematopoietic stem cell number. Proceedings of the National Academy of Sciences of the United States of America. 107(32):14229-34. Pubmed: 20616003 DOI:10.1073/pnas.0913574107 Guo S, Lu J, Schlanger R, Zhang H, Wang JY, Fox MC, Purton LE, Fleming HH, Cobb B, Merkenschlager M, Golub TR, Scadden DT. 2010. MicroRNA miR-125a controls hematopoietic stem cell number. Proceedings of the National Academy of Sciences of the United States of America. 107(32):14229-34. Pubmed: 20616003 DOI:10.1073/pnas.0913574107 MicroRNAs influence hematopoietic differentiation, but little is known about their effects on the stem cell state. Here, we report that the microRNA processing enzyme Dicer is essential for stem cell persistence in vivo and a specific microRNA, miR-125a, controls the size of the stem cell population by regulating hematopoietic stem/progenitor cell (HSPC) apoptosis. Conditional deletion of Dicer revealed an absolute dependence for the multipotent HSPC population in a cell-autonomous manner, with increased HSPC apoptosis in mutant animals. An evolutionarily conserved microRNA cluster containing miR-99b, let-7e, and miR-125a was preferentially expressed in long-term hematopoietic stem cells. MicroRNA miR-125a alone was capable of increasing the number of hematopoietic stem cells in vivo by more than 8-fold. This result was accomplished through a differentiation stage-specific reduction of apoptosis in immature hematopoietic progenitors, possibly through targeting multiple proapoptotic genes. Bak1 was directly down-regulated by miR-125a and expression of a 3'UTR-less Bak1 blocked miR-125a-induced hematopoietic expansion in vivo. These data demonstrate cell-state-specific regulation by microRNA and identify a unique microRNA functioning to regulate the stem cell pool size. -
Lymperi S, Ferraro F, Scadden DT. 2010. The HSC niche concept has turned 31. Has our knowledge matured?. Annals of the New York Academy of Sciences. 1192:12-8. Pubmed: 20392212 DOI:10.1111/j.1749-6632.2009.05223.x Lymperi S, Ferraro F, Scadden DT. 2010. The HSC niche concept has turned 31. Has our knowledge matured?. Annals of the New York Academy of Sciences. 1192:12-8. Pubmed: 20392212 DOI:10.1111/j.1749-6632.2009.05223.x The hematopoietic stem cell (HSC) niche is currently defined as the specific microenvironment in the bone marrow (BM) which anatomically harbors HSCs and governs their fate. It plays a pivotal role in regulating the survival and self-renewal ability of HSCs, protecting them from exhaustion while preventing their excessive proliferation. Many different stromal cell types have been proposed as putative constituents of the niche, but their integrated function is still unrevealed. Mechanisms by which stem/progenitor cell behavior is regulated in the niche include cell-to-cell interaction and the production of growth factors, cytokines, and extracellular matrix proteins. The HSC niche is a dynamic entity reflecting and responding to the needs of the organism. An understanding of how the niche participates in the maintenance of tissue homeostasis and repair offers new opportunities for the development of novel therapeutic tools. -
Vallet S, Mukherjee S, Vaghela N, Hideshima T, Fulciniti M, Pozzi S, Santo L, Cirstea D, Patel K, Sohani AR, Guimaraes A, Xie W, Chauhan D, Schoonmaker JA, Attar E, Churchill M, Weller E, Munshi N, Seehra JS, Weissleder R, Anderson KC, Scadden DT, Raje N. 2010. Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease. Proceedings of the National Academy of Sciences of the United States of America. 107(11):5124-9. Pubmed: 20194748 DOI:10.1073/pnas.0911929107 Vallet S, Mukherjee S, Vaghela N, Hideshima T, Fulciniti M, Pozzi S, Santo L, Cirstea D, Patel K, Sohani AR, Guimaraes A, Xie W, Chauhan D, Schoonmaker JA, Attar E, Churchill M, Weller E, Munshi N, Seehra JS, Weissleder R, Anderson KC, Scadden DT, Raje N. 2010. Activin A promotes multiple myeloma-induced osteolysis and is a promising target for myeloma bone disease. Proceedings of the National Academy of Sciences of the United States of America. 107(11):5124-9. Pubmed: 20194748 DOI:10.1073/pnas.0911929107 Understanding the pathogenesis of cancer-related bone disease is crucial to the discovery of new therapies. Here we identify activin A, a TGF-beta family member, as a therapeutically amenable target exploited by multiple myeloma (MM) to alter its microenvironmental niche favoring osteolysis. Increased bone marrow plasma activin A levels were found in MM patients with osteolytic disease. MM cell engagement of marrow stromal cells enhanced activin A secretion via adhesion-mediated JNK activation. Activin A, in turn, inhibited osteoblast differentiation via SMAD2-dependent distal-less homeobox-5 down-regulation. Targeting activin A by a soluble decoy receptor reversed osteoblast inhibition, ameliorated MM bone disease, and inhibited tumor growth in an in vivo humanized MM model, setting the stage for testing in human clinical trials. -
Ferraro F, Celso CL, Scadden D. 2010. Adult stem cels and their niches. Advances in experimental medicine and biology. 695:155-68. Pubmed: 21222205 DOI:10.1007/978-1-4419-7037-4_11 Ferraro F, Celso CL, Scadden D. 2010. Adult stem cels and their niches. Advances in experimental medicine and biology. 695:155-68. Pubmed: 21222205 DOI:10.1007/978-1-4419-7037-4_11 Stem cells participate in dynamic physiologic systems that dictate the outcome of developmental events and organismal stress, Since these cells are fundamental to tissue maintenance and repair, the signals they receive play a critical role in the integrity of the organism. Much work has focused on stem cell identification and the molecular pathways involved in their regulation. Yet, we understand little about how these pathways achieve physiologically responsive stem cell functions. This chapter will review the state of our understanding of stem cells in the context of their microenvironment regarding the relation between stem cell niche dysfunction, carcinogenesis and aging. -
Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA, Ebert BL, Al-Shahrour F, Hasserjian RP, Scadden EO, Aung Z, Matza M, Merkenschlager M, Lin C, Rommens JM, Scadden DT. 2010. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature. 464(7290):852-7. Pubmed: 20305640 DOI:10.1038/nature08851 Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA, Ebert BL, Al-Shahrour F, Hasserjian RP, Scadden EO, Aung Z, Matza M, Merkenschlager M, Lin C, Rommens JM, Scadden DT. 2010. Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature. 464(7290):852-7. Pubmed: 20305640 DOI:10.1038/nature08851 Mesenchymal cells contribute to the 'stroma' of most normal and malignant tissues, with specific mesenchymal cells participating in the regulatory niches of stem cells. By examining how mesenchymal osteolineage cells modulate haematopoiesis, here we show that deletion of Dicer1 specifically in mouse osteoprogenitors, but not in mature osteoblasts, disrupts the integrity of haematopoiesis. Myelodysplasia resulted and acute myelogenous leukaemia emerged that had acquired several genetic abnormalities while having intact Dicer1. Examining gene expression altered in osteoprogenitors as a result of Dicer1 deletion showed reduced expression of Sbds, the gene mutated in Schwachman-Bodian-Diamond syndrome-a human bone marrow failure and leukaemia pre-disposition condition. Deletion of Sbds in mouse osteoprogenitors induced bone marrow dysfunction with myelodysplasia. Therefore, perturbation of specific mesenchymal subsets of stromal cells can disorder differentiation, proliferation and apoptosis of heterologous cells, and disrupt tissue homeostasis. Furthermore, primary stromal dysfunction can result in secondary neoplastic disease, supporting the concept of niche-induced oncogenesis. -
Gurumurthy S, Xie SZ, Alagesan B, Kim J, Yusuf RZ, Saez B, Tzatsos A, Ozsolak F, Milos P, Ferrari F, Park PJ, Shirihai OS, Scadden DT, Bardeesy N. 2010. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature. 468(7324):659-63. Pubmed: 21124451 DOI:10.1038/nature09572 Gurumurthy S, Xie SZ, Alagesan B, Kim J, Yusuf RZ, Saez B, Tzatsos A, Ozsolak F, Milos P, Ferrari F, Park PJ, Shirihai OS, Scadden DT, Bardeesy N. 2010. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature. 468(7324):659-63. Pubmed: 21124451 DOI:10.1038/nature09572 Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint. -
Guo S, Scadden DT. 2010. A microRNA regulating adult hematopoietic stem cells. Cell cycle (Georgetown, Tex.). 9(18):3637-8. Pubmed: 20855952 Guo S, Scadden DT. 2010. A microRNA regulating adult hematopoietic stem cells. Cell cycle (Georgetown, Tex.). 9(18):3637-8. Pubmed: 20855952 -
Méndez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma'ayan A, Enikolopov GN, Frenette PS. 2010. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature. 466(7308):829-34. Pubmed: 20703299 DOI:10.1038/nature09262 Méndez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA, Scadden DT, Ma'ayan A, Enikolopov GN, Frenette PS. 2010. Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature. 466(7308):829-34. Pubmed: 20703299 DOI:10.1038/nature09262 The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs. -
Lane AA, Scadden DT. 2010. Stem cells and DNA damage: persist or perish?. Cell. 142(3):360-2. Pubmed: 20691895 DOI:10.1016/j.cell.2010.07.030 Lane AA, Scadden DT. 2010. Stem cells and DNA damage: persist or perish?. Cell. 142(3):360-2. Pubmed: 20691895 DOI:10.1016/j.cell.2010.07.030 Stem cells repopulate tissues after injury while also renewing themselves, but this makes them vulnerable to genotoxic damage. Mohrin et al. (2010) and Milyavsky et al. (2010) now show that mouse and human hematopoietic stem cells make opposing decisions about whether to die or to persist in response to DNA damage.Copyright 2010 Elsevier Inc. All rights reserved. 2009
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Jones R, Capen DE, Jacobson M, Cohen KS, Scadden DT, Duda DG. 2009. VEGFR2+PDGFRbeta+ circulating precursor cells participate in capillary restoration after hyperoxia acute lung injury (HALI). Journal of cellular and molecular medicine. 13(9B):3720-9. Pubmed: 19426150 DOI:10.1111/j.1582-4934.2009.00785.x Jones R, Capen DE, Jacobson M, Cohen KS, Scadden DT, Duda DG. 2009. VEGFR2+PDGFRbeta+ circulating precursor cells participate in capillary restoration after hyperoxia acute lung injury (HALI). Journal of cellular and molecular medicine. 13(9B):3720-9. Pubmed: 19426150 DOI:10.1111/j.1582-4934.2009.00785.x The in vivo morphology and phenotype of circulating cells that spontaneously contribute to new vessel formation in adults remain unclear. Here, we use high-resolution imaging and flow cytometry to characterize the morphology and phenotype of a distinct population of circulating mononuclear cells contributing to spontaneous new vessel formation after hyperoxia acute lung injury (HALI). We identify a subpopulation of myeloid (CD11b/Mac1(+)) haematopoietic cells co-expressing vascular endothelial growth factor receptor 2 (VEGFR2) and platelet derived growth factor receptor beta (PDGFRbeta). Moreover, we show that these CD11b(+)VEGFR2(+)PDGFRbeta(+) circulating precursor cells (CPCs) contribute structurally to the luminal surface of capillaries re-forming 2 weeks post-HALI. This indicates that these myeloid CPCs may function, at least transiently, as putative vascular precursors, and has important implications for capillary growth and repair in injury and in pathologies of the lung and other organs. -
Wu JY, Scadden DT, Kronenberg HM. 2009. Role of the osteoblast lineage in the bone marrow hematopoietic niches. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 24(5):759-64. Pubmed: 19257832 DOI:10.1359/jbmr.090225 Wu JY, Scadden DT, Kronenberg HM. 2009. Role of the osteoblast lineage in the bone marrow hematopoietic niches. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 24(5):759-64. Pubmed: 19257832 DOI:10.1359/jbmr.090225 -
Lane SW, Scadden DT, Gilliland DG. 2009. The leukemic stem cell niche: current concepts and therapeutic opportunities. Blood. 114(6):1150-7. Pubmed: 19401558 DOI:10.1182/blood-2009-01-202606 Lane SW, Scadden DT, Gilliland DG. 2009. The leukemic stem cell niche: current concepts and therapeutic opportunities. Blood. 114(6):1150-7. Pubmed: 19401558 DOI:10.1182/blood-2009-01-202606 The genetic events that contribute to the pathogenesis of acute myeloid leukemia are among the best characterized of all human malignancies. However, with notable exceptions such as acute promyelocytic leukemia, significant improvements in outcome based on these insights have not been forthcoming. Acute myeloid leukemia is a paradigm of cancer stem (or leukemia initiating) cells with hierarchy analogous to that seen in hematopoiesis. Normal hematopoiesis requires complex bidirectional interactions between the bone marrow microenvironment (or niche) and hematopoietic stem cells (HSCs). These interactions are critical for the maintenance of normal HSC quiescence and perturbations can influence HSC self-renewal. Leukemia stem cells (LSCs), which also possess limitless self-renewal, may hijack these homeostatic mechanisms, take refuge within the sanctuary of the niche during chemotherapy, and consequently contribute to eventual disease relapse. We will discuss the emerging evidence supporting the importance of the bone marrow microenvironment in LSC survival and consider the physiologic interactions of HSCs and the niche that inform our understanding of microenvironment support of LSCs. Finally, we will discuss approaches for the rational development of therapies that target the microenvironment. -
Qiu J, Takagi Y, Harada J, Topalkara K, Wang Y, Sims JR, Zheng G, Huang P, Ling Y, Scadden DT, Moskowitz MA, Cheng T. 2009. p27Kip1 constrains proliferation of neural progenitor cells in adult brain under homeostatic and ischemic conditions. Stem cells (Dayton, Ohio). 27(4):920-7. Pubmed: 19353520 DOI:10.1002/stem.1 Qiu J, Takagi Y, Harada J, Topalkara K, Wang Y, Sims JR, Zheng G, Huang P, Ling Y, Scadden DT, Moskowitz MA, Cheng T. 2009. p27Kip1 constrains proliferation of neural progenitor cells in adult brain under homeostatic and ischemic conditions. Stem cells (Dayton, Ohio). 27(4):920-7. Pubmed: 19353520 DOI:10.1002/stem.1 Cell cycle inhibition of neural stem and progenitor cells is critical for maintaining the stability of central nervous system in adults, but it may represent a significant hurdle for neural regeneration after injury. We have previously demonstrated that the cyclin-dependent kinase inhibitor (CKI) p21(cip1/waf1) (p21) maintains the quiescence of neural stem-like cells under cerebral ischemia, as similarly shown for the hematopoietic stem cells. Here, we report the distinct role of another CKI member, p27(kip1) (p27) in neural progenitor cells (NPCs) from adult brain (subventricular zone and hippocampal subgranular zone) under both homeostatic and ischemic conditions. The basal level of NPC proliferation in the p27-/- mice was higher than that in p27+/+ mice. Upon ischemia, the overall proliferation of NPCs continued to be higher in p27-/- mice than that in p27+/+ mice. Moreover, the increase of NPC proliferation in p27-/- mice remained until 2 weeks after ischemia, whereas it resumed back to the basal level in p27+/+ mice. As a result, newly generated neuronal cells in the granular layer of p27-/- brain were more abundant compared with p27+/+ controls. These new data demonstrate that p27 functions as a distinct inhibitor for NPC proliferation under homeostatic as well as ischemic conditions. -
Curley MD, Therrien VA, Cummings CL, Sergent PA, Koulouris CR, Friel AM, Roberts DJ, Seiden MV, Scadden DT, Rueda BR, Foster R. 2009. CD133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem cells (Dayton, Ohio). 27(12):2875-83. Pubmed: 19816957 DOI:10.1002/stem.236 Curley MD, Therrien VA, Cummings CL, Sergent PA, Koulouris CR, Friel AM, Roberts DJ, Seiden MV, Scadden DT, Rueda BR, Foster R. 2009. CD133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem cells (Dayton, Ohio). 27(12):2875-83. Pubmed: 19816957 DOI:10.1002/stem.236 Evidence is accumulating that solid tumors contain a rare phenotypically distinct population of cells, termed cancer stem cells (CSC), which give rise to and maintain the bulk of the tumor. These CSC are thought to be resistant to current chemotherapeutic strategies due to their intrinsic stem-like properties and thus may provide the principal driving force behind recurrent tumor growth. Given the high frequency of recurrent metastasis associated with human ovarian cancer, we sought to determine whether primary human ovarian tumors contain populations of cells with enhanced tumor-initiating capacity, a characteristic of CSC. Using an in vivo serial transplantation model, we show that primary uncultured human ovarian tumors can be reliably propagated in NOD/SCID mice, generating heterogeneous tumors that maintain the histological integrity of the parental tumor. The observed frequency of tumor engraftment suggests only certain subpopulations of ovarian tumor cells have the capacity to recapitulate tumor growth. Further profiling of human ovarian tumors for expression of candidate CSC surface markers indicated consistent expression of CD133. To determine whether CD133 expression could define a tumor-initiating cell population in primary human ovarian tumors, fluorescence-activated cell sorting (FACS) methods were employed. Injection of sorted CD133(+) and CD133(-) cell populations into NOD/SCID mice established that tumor-derived CD133(+) cells have an increased tumorigenic capacity and are capable of recapitulating the original heterogeneous tumor. Our data indicate that CD133 expression defines a NOD/SCID tumor initiating subpopulation of cells in human ovarian cancer that may be an important target for new chemotherapeutic strategies aimed at eliminating ovarian cancer. -
Adams GB, Alley IR, Chung UI, Chabner KT, Jeanson NT, Lo Celso C, Marsters ES, Chen M, Weinstein LS, Lin CP, Kronenberg HM, Scadden DT. 2009. Haematopoietic stem cells depend on Galpha(s)-mediated signalling to engraft bone marrow. Nature. 459(7243):103-7. Pubmed: 19322176 DOI:10.1038/nature07859 Adams GB, Alley IR, Chung UI, Chabner KT, Jeanson NT, Lo Celso C, Marsters ES, Chen M, Weinstein LS, Lin CP, Kronenberg HM, Scadden DT. 2009. Haematopoietic stem cells depend on Galpha(s)-mediated signalling to engraft bone marrow. Nature. 459(7243):103-7. Pubmed: 19322176 DOI:10.1038/nature07859 Haematopoietic stem and progenitor cells (HSPCs) change location during development and circulate in mammals throughout life, moving into and out of the bloodstream to engage bone marrow niches in sequential steps of homing, engraftment and retention. Here we show that HSPC engraftment of bone marrow in fetal development is dependent on the guanine-nucleotide-binding protein stimulatory alpha subunit (Galpha(s)). HSPCs from adult mice deficient in Galpha(s) (Galpha(s)(-/-)) differentiate and undergo chemotaxis, but also do not home to or engraft in the bone marrow in adult mice and demonstrate a marked inability to engage the marrow microvasculature. If deleted after engraftment, Galpha(s) deficiency did not lead to lack of retention in the marrow, rather cytokine-induced mobilization into the blood was impaired. Testing whether activation of Galpha(s) affects HSPCs, pharmacological activators enhanced homing and engraftment in vivo. Galpha(s) governs specific aspects of HSPC localization under physiological conditions in vivo and may be pharmacologically targeted to improve transplantation efficiency. -
Ali N, Karlsson C, Aspling M, Hu G, Hacohen N, Scadden DT, Larsson J. 2009. Forward RNAi screens in primary human hematopoietic stem/progenitor cells. Blood. 113(16):3690-5. Pubmed: 19188664 DOI:10.1182/blood-2008-10-176396 Ali N, Karlsson C, Aspling M, Hu G, Hacohen N, Scadden DT, Larsson J. 2009. Forward RNAi screens in primary human hematopoietic stem/progenitor cells. Blood. 113(16):3690-5. Pubmed: 19188664 DOI:10.1182/blood-2008-10-176396 The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations. -
Raje N, Hideshima T, Mukherjee S, Raab M, Vallet S, Chhetri S, Cirstea D, Pozzi S, Mitsiades C, Rooney M, Kiziltepe T, Podar K, Okawa Y, Ikeda H, Carrasco R, Richardson PG, Chauhan D, Munshi NC, Sharma S, Parikh H, Chabner B, Scadden D, Anderson KC. 2009. Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma. Leukemia. 23(5):961-70. Pubmed: 19151776 DOI:10.1038/leu.2008.378 Raje N, Hideshima T, Mukherjee S, Raab M, Vallet S, Chhetri S, Cirstea D, Pozzi S, Mitsiades C, Rooney M, Kiziltepe T, Podar K, Okawa Y, Ikeda H, Carrasco R, Richardson PG, Chauhan D, Munshi NC, Sharma S, Parikh H, Chabner B, Scadden D, Anderson KC. 2009. Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma. Leukemia. 23(5):961-70. Pubmed: 19151776 DOI:10.1038/leu.2008.378 Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM. -
Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J, Côté D, Rowe DW, Lin CP, Scadden DT. 2009. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature. 457(7225):92-6. Pubmed: 19052546 DOI:10.1038/nature07434 Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J, Côté D, Rowe DW, Lin CP, Scadden DT. 2009. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature. 457(7225):92-6. Pubmed: 19052546 DOI:10.1038/nature07434 Stem cells reside in a specialized, regulatory environment termed the niche that dictates how they generate, maintain and repair tissues. We have previously documented that transplanted haematopoietic stem and progenitor cell populations localize to subdomains of bone-marrow microvessels where the chemokine CXCL12 is particularly abundant. Using a combination of high-resolution confocal microscopy and two-photon video imaging of individual haematopoietic cells in the calvarium bone marrow of living mice over time, we examine the relationship of haematopoietic stem and progenitor cells to blood vessels, osteoblasts and endosteal surface as they home and engraft in irradiated and c-Kit-receptor-deficient recipient mice. Osteoblasts were enmeshed in microvessels and relative positioning of stem/progenitor cells within this complex tissue was nonrandom and dynamic. Both cell autonomous and non-autonomous factors influenced primitive cell localization. Different haematopoietic cell subsets localized to distinct locations according to the stage of differentiation. When physiological challenges drove either engraftment or expansion, bone-marrow stem/progenitor cells assumed positions in close proximity to bone and osteoblasts. Our analysis permits observing in real time, at a single cell level, processes that previously have been studied only by their long-term outcome at the organismal level. -
Yusuf RZ, Scadden DT. 2009. Homing of hematopoietic cells to the bone marrow. Journal of visualized experiments : JoVE. Pubmed: 19295497 DOI:10.3791/1104 Yusuf RZ, Scadden DT. 2009. Homing of hematopoietic cells to the bone marrow. Journal of visualized experiments : JoVE. Pubmed: 19295497 DOI:10.3791/1104 Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment. 2008
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Raaijmakers MH, Scadden DT. 2008. Evolving concepts on the microenvironmental niche for hematopoietic stem cells. Current opinion in hematology. 15(4):301-6. Pubmed: 18536566 DOI:10.1097/MOH.0b013e328303e14c Raaijmakers MH, Scadden DT. 2008. Evolving concepts on the microenvironmental niche for hematopoietic stem cells. Current opinion in hematology. 15(4):301-6. Pubmed: 18536566 DOI:10.1097/MOH.0b013e328303e14c Array -
Larsson J, Ohishi M, Garrison B, Aspling M, Janzen V, Adams GB, Curto M, McClatchey AI, Schipani E, Scadden DT. 2008. Nf2/merlin regulates hematopoietic stem cell behavior by altering microenvironmental architecture. Cell stem cell. 3(2):221-7. Pubmed: 18682243 DOI:10.1016/j.stem.2008.06.005 Larsson J, Ohishi M, Garrison B, Aspling M, Janzen V, Adams GB, Curto M, McClatchey AI, Schipani E, Scadden DT. 2008. Nf2/merlin regulates hematopoietic stem cell behavior by altering microenvironmental architecture. Cell stem cell. 3(2):221-7. Pubmed: 18682243 DOI:10.1016/j.stem.2008.06.005 Stem cell population size is highly regulated across species and tissue types, and alterations are associated with premature tissue failure or cancer. We assessed whether the tumor suppressor and mediator of cell contact inhibition Nf2/merlin plays a role in governing the hematopoietic stem cell pool by stem cell-autonomous or niche-determined processes. Hematopoietic stem cells in Nf2-deficient mice were increased in number and demonstrated a marked shift in location to the circulation. These changes were entirely dependent on changes in the microenvironment, with a marked increase in trabecular bone and marrow vascularity associated with increased VEGF, but without cell-autonomous alterations in stem cell characteristics. Nf2/merlin is critical for maintaining normal structure and function of the hematopoietic stem cell niche. It limits both bone and vascular components, and our model suggests that it thereby constrains stem cell number and position. -
Rodrigues NP, Boyd AS, Fugazza C, May GE, Guo Y, Tipping AJ, Scadden DT, Vyas P, Enver T. 2008. GATA-2 regulates granulocyte-macrophage progenitor cell function. Blood. 112(13):4862-73. Pubmed: 18840712 DOI:10.1182/blood-2008-01-136564 Rodrigues NP, Boyd AS, Fugazza C, May GE, Guo Y, Tipping AJ, Scadden DT, Vyas P, Enver T. 2008. GATA-2 regulates granulocyte-macrophage progenitor cell function. Blood. 112(13):4862-73. Pubmed: 18840712 DOI:10.1182/blood-2008-01-136564 The zinc finger transcription factor GATA-2 has been implicated in the regulation of hematopoietic stem cells. Herein, we explored the role of GATA-2 as a candidate regulator of the hematopoietic progenitor cell compartment. We showed that bone marrow from GATA-2 heterozygote (GATA-2(+/-)) mice displayed attenuated granulocyte-macrophage progenitor function in colony-forming cell (CFC) and serial replating CFC assays. This defect was mapped to the Lin(-)CD117(+)Sca-1(-)CD34(+)CD16/32(high) granulocyte-macrophage progenitor (GMP) compartment of GATA-2(+/-) marrow, which was reduced in size and functionally impaired in CFC assays and competitive transplantation. Similar functional impairments were obtained using a RNA interference approach to stably knockdown GATA-2 in wild-type GMP. Although apoptosis and cell-cycle distribution remained unperturbed in GATA-2(+/-) GMP, quiescent cells from GATA-2(+/-) GMP exhibited altered functionality. Gene expression analysis showed attenuated expression of HES-1 mRNA in GATA-2-deficient GMP. Binding of GATA-2 to the HES-1 locus was detected in the myeloid progenitor cell line 32Dcl3, and enforced expression of HES-1 expression in GATA-2(+/-) GMP rectified the functional defect, suggesting that GATA-2 regulates myeloid progenitor function through HES-1. These data collectively point to GATA-2 as a novel, pivotal determinant of GMP cell fate. -
Wu JY, Purton LE, Rodda SJ, Chen M, Weinstein LS, McMahon AP, Scadden DT, Kronenberg HM. 2008. Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}-dependent signaling pathways. Proceedings of the National Academy of Sciences of the United States of America. 105(44):16976-81. Pubmed: 18957542 DOI:10.1073/pnas.0802898105 Wu JY, Purton LE, Rodda SJ, Chen M, Weinstein LS, McMahon AP, Scadden DT, Kronenberg HM. 2008. Osteoblastic regulation of B lymphopoiesis is mediated by Gs{alpha}-dependent signaling pathways. Proceedings of the National Academy of Sciences of the United States of America. 105(44):16976-81. Pubmed: 18957542 DOI:10.1073/pnas.0802898105 Osteoblasts play an increasingly recognized role in supporting hematopoietic development and recently have been implicated in the regulation of B lymphopoiesis. Here we demonstrate that the heterotrimeric G protein alpha subunit G(s)alpha is required in cells of the osteoblast lineage for normal postnatal B lymphocyte production. Deletion of G(s)alpha early in the osteoblast lineage results in a 59% decrease in the percentage of B cell precursors in the bone marrow. Analysis of peripheral blood from mutant mice revealed a 67% decrease in the number of circulating B lymphocytes by 10 days of age. Strikingly, other mature hematopoietic lineages are not decreased significantly. Mice lacking G(s)alpha in cells of the osteoblast lineage exhibit a reduction in pro-B and pre-B cells. Furthermore, interleukin (IL)-7 expression is attenuated in G(s)alpha-deficient osteoblasts, and exogenous IL-7 is able to restore B cell precursor populations in the bone marrow of mutant mice. Finally, the defect in B lymphopoiesis can be rescued by transplantation into a WT microenvironment. These findings confirm that osteoblasts are an important component of the B lymphocyte niche and demonstrate in vivo that G(s)alpha-dependent signaling pathways in cells of the osteoblast lineage extrinsically regulate bone marrow B lymphopoiesis, at least partially in an IL-7-dependent manner. -
Raaijmakers MH, Scadden DT. 2008. Divided within: heterogeneity within adult stem cell pools. Cell. 135(6):1006-8. Pubmed: 19070570 DOI:10.1016/j.cell.2008.11.034 Raaijmakers MH, Scadden DT. 2008. Divided within: heterogeneity within adult stem cell pools. Cell. 135(6):1006-8. Pubmed: 19070570 DOI:10.1016/j.cell.2008.11.034 In this issue, Wilson et al. (2008) demonstrate that there are two functional subsets of hematopoietic stem cells that have distinctive kinetics of cell cycling. They present evidence that cells may transition between the two kinetic states, establishing one subpopulation that is ready to proliferate and another that is a deeply quiescent reserve. -
Scadden DT, Komaroff AL. 2008. Will stem cells finally deliver?. Newsweek. 152(24):57-8. Pubmed: 19105310 Scadden DT, Komaroff AL. 2008. Will stem cells finally deliver?. Newsweek. 152(24):57-8. Pubmed: 19105310 -
Fleming HE, Janzen V, Lo Celso C, Guo J, Leahy KM, Kronenberg HM, Scadden DT. 2008. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell stem cell. 2(3):274-83. Pubmed: 18371452 DOI:10.1016/j.stem.2008.01.003 Fleming HE, Janzen V, Lo Celso C, Guo J, Leahy KM, Kronenberg HM, Scadden DT. 2008. Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo. Cell stem cell. 2(3):274-83. Pubmed: 18371452 DOI:10.1016/j.stem.2008.01.003 Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Wnt effects are highly context dependent, and varying effects of Wnt signaling on hematopoietic stem cells (HSCs) have been reported. We explored the impact of Wnt signaling in vivo, specifically in the context of the HSC niche by using an osteoblast-specific promoter driving expression of the paninhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1). Here we report that Wnt signaling was markedly inhibited in HSCs and, unexpectedly given prior reports, reduction in HSC Wnt signaling resulted in reduced p21Cip1 expression, increased cell cycling, and a progressive decline in regenerative function after transplantation. This effect was microenvironment determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to limit HSC proliferation and preserve the reconstituting function of endogenous hematopoietic stem cells. -
Friel AM, Sergent PA, Patnaude C, Szotek PP, Oliva E, Scadden DT, Seiden MV, Foster R, Rueda BR. 2008. Functional analyses of the cancer stem cell-like properties of human endometrial tumor initiating cells. Cell cycle (Georgetown, Tex.). 7(2):242-9. Pubmed: 18256549 Friel AM, Sergent PA, Patnaude C, Szotek PP, Oliva E, Scadden DT, Seiden MV, Foster R, Rueda BR. 2008. Functional analyses of the cancer stem cell-like properties of human endometrial tumor initiating cells. Cell cycle (Georgetown, Tex.). 7(2):242-9. Pubmed: 18256549 Recent data suggest that rare stem cell populations with the capacity to self renew and drive tumor formation are a feature of solid tumors. Several investigators have identified putative stem cells from solid tumors and cancer cell lines following isolation of a side population (SP) defined by dye exclusion. We investigated this parameter in our efforts to identify an endometrial cancer (EnCa) stem cell population. Multiple EnCa cell lines were assessed and verapamil sensitive SP and non-SP cells were isolated from two human EnCa cell lines. The functional significance of the SP and non-SP derived from AN3CA was evaluated in vitro and in vivo. SP cells proliferated at a significantly slower rate than the non-SP fraction, and a larger proportion of the SP cells were in the G(1) phase of the cell cycle as compared to the non-SP fraction. The SP fraction was more resistant to the chemotherapeutic agent paclitaxel. The SP comprised -0.02% of the initial AN3CA cell population and this proportion of SP cells was maintained within the larger heterogeneous population following repeated passages of purified SP cells. These findings suggest that SP cells derived from the An3CA cell line have the stem cell properties of low proliferative activity, chemoresistance and self-renewal. We also tested relative tumor formation activity of the SP and non-SP fractions. Only the SP fraction was tumorigenic. Additionally, we identified SP fractions in primary EnCa. Together these results are consistent with the hypothesis that EnCa contain a subpopulation of tumor initiating cells with stem like properties. -
Daley GQ, Scadden DT. 2008. Prospects for stem cell-based therapy. Cell. 132(4):544-8. Pubmed: 18295571 DOI:10.1016/j.cell.2008.02.009 Daley GQ, Scadden DT. 2008. Prospects for stem cell-based therapy. Cell. 132(4):544-8. Pubmed: 18295571 DOI:10.1016/j.cell.2008.02.009 Resident pools of somatic stem cells in many organs are responsible for tissue maintenance and repair. The goal of regenerative medicine is to exploit these cells either by transplanting them from an exogenous source or by activating endogenous stem cells pharmacologically. For diseases caused by mutations in a single gene, the therapeutic goal is tissue replacement using stem cells engineered to correct the genetic defect. However, a number of technical hurdles must be overcome before therapies based on pluripotent human stem cells can enter the clinic. -
Mukherjee S, Raje N, Schoonmaker JA, Liu JC, Hideshima T, Wein MN, Jones DC, Vallet S, Bouxsein ML, Pozzi S, Chhetri S, Seo YD, Aronson JP, Patel C, Fulciniti M, Purton LE, Glimcher LH, Lian JB, Stein G, Anderson KC, Scadden DT. 2008. Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice. The Journal of clinical investigation. 118(2):491-504. Pubmed: 18219387 DOI:10.1172/JCI33102 Mukherjee S, Raje N, Schoonmaker JA, Liu JC, Hideshima T, Wein MN, Jones DC, Vallet S, Bouxsein ML, Pozzi S, Chhetri S, Seo YD, Aronson JP, Patel C, Fulciniti M, Purton LE, Glimcher LH, Lian JB, Stein G, Anderson KC, Scadden DT. 2008. Pharmacologic targeting of a stem/progenitor population in vivo is associated with enhanced bone regeneration in mice. The Journal of clinical investigation. 118(2):491-504. Pubmed: 18219387 DOI:10.1172/JCI33102 Drug targeting of adult stem cells has been proposed as a strategy for regenerative medicine, but very few drugs are known to target stem cell populations in vivo. Mesenchymal stem/progenitor cells (MSCs) are a multipotent population of cells that can differentiate into muscle, bone, fat, and other cell types in context-specific manners. Bortezomib (Bzb) is a clinically available proteasome inhibitor used in the treatment of multiple myeloma. Here, we show that Bzb induces MSCs to preferentially undergo osteoblastic differentiation, in part by modulation of the bone-specifying transcription factor runt-related transcription factor 2 (Runx-2) in mice. Mice implanted with MSCs showed increased ectopic ossicle and bone formation when recipients received low doses of Bzb. Furthermore, this treatment increased bone formation and rescued bone loss in a mouse model of osteoporosis. Thus, we show that a tissue-resident adult stem cell population in vivo can be pharmacologically modified to promote a regenerative function in adult animals. -
Orford KW, Scadden DT. 2008. Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nature reviews. Genetics. 9(2):115-28. Pubmed: 18202695 DOI:10.1038/nrg2269 Orford KW, Scadden DT. 2008. Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nature reviews. Genetics. 9(2):115-28. Pubmed: 18202695 DOI:10.1038/nrg2269 The regulation of stem cell self-renewal must balance the regenerative needs of tissues that persist throughout life with the potential for cell overgrowth, transformation and cancer. Here, we attempt to deconstruct the relationship that exists between cell-cycle progression and the self-renewal versus commitment cell-fate decision in embryonic and adult stem cells. Recent genetic studies in mice have provided insights into the regulation of the cell cycle in stem cells, including its potential modulation by the stem cell niche. Although the dynamics of the embryonic and adult stem cell cycles are profoundly dissimilar, we suggest that shared principles underlie the governance of this important decision point in diverse stem cell types. -
Spitzer TR, Ambinder RF, Lee JY, Kaplan LD, Wachsman W, Straus DJ, Aboulafia DM, Scadden DT. 2008. Dose-reduced busulfan, cyclophosphamide, and autologous stem cell transplantation for human immunodeficiency virus-associated lymphoma: AIDS Malignancy Consortium study 020. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 14(1):59-66. Pubmed: 18158962 Spitzer TR, Ambinder RF, Lee JY, Kaplan LD, Wachsman W, Straus DJ, Aboulafia DM, Scadden DT. 2008. Dose-reduced busulfan, cyclophosphamide, and autologous stem cell transplantation for human immunodeficiency virus-associated lymphoma: AIDS Malignancy Consortium study 020. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 14(1):59-66. Pubmed: 18158962 Intensive chemotherapy for human immunodeficiency virus (HIV)-associated non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL) has resulted in durable remissions in a substantial proportion of patients. High-dose chemotherapy and autologous stem cell transplantation (AuSCT), moreover, has resulted in sustained complete remissions in selected patients with recurrent chemosensitive disease. Based on a favorable experience with dose-reduced high-dose busulfan, cyclophosphamide, and AuSCT for older patients with non-HIV-associated aggressive lymphomas, an AIDS Malignancy Consortium multicenter trial was undertaken using the same dose-reduced busulfan and cyclophosphamide preparative regimen with AuSCT for recurrent HIV-associated NHL and HL. Of the 27 patients in the study, 20 received an AuSCT. The median time to achievement of an absolute neutrophil count (ANC) of >or= 0.5 x 10(9)/L was 11 days (range, 9-16 days). The median time to achievement of an unsupported platelet count of >or= 20 x 10(9)/L was 13 days (range, 6-57 days). One patient died on day +33 posttransplantation from hepatic veno-occlusive disease (VOD) and multiorgan failure. No other fatal regimen-related toxicity occurred. Ten of 19 patients (53%) were in complete remission at the time of their day +100 post-AuSCT evaluation. Of the 20 patients, 10 were alive and event-free at a median of 23 weeks post-AuSCT. Median overall survival (OS) was not reached by 13 of the 20 patients alive at the time of last follow-up. This multi-institutional trial demonstrates that a regimen of dose-reduced high-dose busulfan, cyclophosphamide, and AuSCT is well tolerated and is associated with favorable disease-free survival (DFS) and OS probabilities for selected patients with HIV-associated NHL and HL. -
Craiu A, Scadden D. 2008. Flow electroporation with pulsed electric fields for purging tumor cells. Methods in molecular biology (Clifton, N.J.). 423:301-10. Pubmed: 18370208 DOI:10.1007/978-1-59745-194-9_22 Craiu A, Scadden D. 2008. Flow electroporation with pulsed electric fields for purging tumor cells. Methods in molecular biology (Clifton, N.J.). 423:301-10. Pubmed: 18370208 DOI:10.1007/978-1-59745-194-9_22 Electroporation has been used in biological laboratories for many years to transiently porate cell membranes and permit plasmid or protein transfection. It has been shown that the application of pulsed electric fields (PEFs) of defined strength will kill off larger cells and select for viable small cells, in samples containing heterogeneous cells. This permits the selective killing of several blood and bone marrow-resident tumor cells. PEF technology is being applied to tumor purging of progenitor-cell transfusions, in support of high-dose chemotherapy, for the treatment of cancers such as lymphoma and multiple myeloma. Autologous stem-cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Progenitor cells are preserved or enriched. To overcome issues of electrical resistance, purging fidelity, and large sample volume, a flowing chamber PEF apparatus was designed and constructed for large-scale purging of clinical quantities of progenitor-cell transfusions. The specifics of this technique are described here. Treatment of greater than 10(9) cells is achieved in 30 min, under optimized flow conditions designed to overcome surface area or resistance issues and to optimize exposure of cells to electric fields. Efficient, large volume tumor purging of greater than 3 logs, for mixtures of tumor cells and mononuclear cells, is routinely achieved under defined conditions. -
Adams GB, Scadden DT. 2008. A niche opportunity for stem cell therapeutics. Gene therapy. 15(2):96-9. Pubmed: 18004404 Adams GB, Scadden DT. 2008. A niche opportunity for stem cell therapeutics. Gene therapy. 15(2):96-9. Pubmed: 18004404 The success of hematopoietic stem cell (HSC)-based therapies relies on the ability of the stem cells to both engraft and self-renew sufficiently in the bone marrow microenvironment. Previous studies identified that a number of components of bone contribute to the regulation of HSCs indicating that they participate in a stem cell 'niche'. This niche is a dynamic microenvironment that changes during development and with varying physiologic states. Components of it, such as the osteoblast, can be modulated through pharmacological treatment. Reasoning that the stem cell niche may be manipulated to augment the effectiveness of stem cell therapies, we demonstrated that daily treatment with parathyroid hormone (a clinically approved method for increasing osteoblast function) resulted in therapeutic benefit in three clinically relevant models of stem cell therapy. These results suggest that the niche may be a pharmacological target for altering stem cell function in settings of regenerative medicine. -
Kim WJ, Okimoto RA, Purton LE, Goodwin M, Haserlat SM, Dayyani F, Sweetser DA, McClatchey AI, Bernard OA, Look AT, Bell DW, Scadden DT, Haber DA. 2008. Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias. Blood. 111(9):4716-22. Pubmed: 18299447 DOI:10.1182/blood-2007-10-113068 Kim WJ, Okimoto RA, Purton LE, Goodwin M, Haserlat SM, Dayyani F, Sweetser DA, McClatchey AI, Bernard OA, Look AT, Bell DW, Scadden DT, Haber DA. 2008. Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias. Blood. 111(9):4716-22. Pubmed: 18299447 DOI:10.1182/blood-2007-10-113068 Ceramide is a lipid second messenger derived from the hydrolysis of sphingomyelin by sphingomyelinases (SMases) and implicated in diverse cellular responses, including growth arrest, differentiation, and apoptosis. Defects in the neutral SMase (nSMase) gene Smpd3, the primary regulator of ceramide biosynthesis, are responsible for developmental defects of bone; regulation of ceramide levels have been implicated in macrophage differentiation, but this pathway has not been directly implicated in human cancer. In a genomic screen for gene copy losses contributing to tumorigenesis in a mouse osteosarcoma model, we identified a somatic homozygous deletion specifically targeting Smpd3. Reconstitution of SMPD3 expression in mouse tumor cells lacking the endogenous gene enhanced tumor necrosis factor (TNF)-induced reduction of cell viability. Nucleotide sequencing of the highly conserved SMPD3 gene in a large panel of human cancers revealed mutations in 5 (5%) of 92 acute myeloid leukemias (AMLs) and 8 (6%) of 131 acute lymphoid leukemias (ALLs), but not in other tumor types. In a subset of these mutations, functional analysis indicated defects in protein stability and localization. Taken together, these observations suggest that disruption of the ceramide pathway may contribute to a subset of human leukemias. -
Scadden DT. 2008. Circadian rhythms: stem cells traffic in time. Nature. 452(7186):416-7. Pubmed: 18368105 DOI:10.1038/452416a Scadden DT. 2008. Circadian rhythms: stem cells traffic in time. Nature. 452(7186):416-7. Pubmed: 18368105 DOI:10.1038/452416a -
Janzen V, Fleming HE, Riedt T, Karlsson G, Riese MJ, Lo Celso C, Reynolds G, Milne CD, Paige CJ, Karlsson S, Woo M, Scadden DT. 2008. Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3. Cell stem cell. 2(6):584-94. Pubmed: 18522851 DOI:10.1016/j.stem.2008.03.012 Janzen V, Fleming HE, Riedt T, Karlsson G, Riese MJ, Lo Celso C, Reynolds G, Milne CD, Paige CJ, Karlsson S, Woo M, Scadden DT. 2008. Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3. Cell stem cell. 2(6):584-94. Pubmed: 18522851 DOI:10.1016/j.stem.2008.03.012 Limited responsiveness to inflammatory cytokines is a feature of adult hematopoietic stem cells and contributes to the relative quiescence and durability of the stem cell population in vivo. Here we report that the executioner Caspase, Caspase-3, unexpectedly participates in that process. Mice deficient in Caspase-3 had increased numbers of immunophenotypic long-term repopulating stem cells in association with multiple functional changes, most prominently cell cycling. Though these changes were cell autonomous, they reflected altered activation by exogenous signals. Caspase-3(-/-) cells exhibited cell type-specific changes in phosphorylated members of the Ras-Raf-MEK-ERK pathway in response to specific cytokines, while notably, members of other pathways, such as pSTAT3, pSTAT5, pAKT, pp38 MAPK, pSmad2, and pSmad3, were unaffected. Caspase-3 contributes to stem cell quiescence, dampening specific signaling events and thereby cell responsiveness to microenvironmental stimuli. -
Lu J, Guo S, Ebert BL, Zhang H, Peng X, Bosco J, Pretz J, Schlanger R, Wang JY, Mak RH, Dombkowski DM, Preffer FI, Scadden DT, Golub TR. 2008. MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors. Developmental cell. 14(6):843-53. Pubmed: 18539114 DOI:10.1016/j.devcel.2008.03.012 Lu J, Guo S, Ebert BL, Zhang H, Peng X, Bosco J, Pretz J, Schlanger R, Wang JY, Mak RH, Dombkowski DM, Preffer FI, Scadden DT, Golub TR. 2008. MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors. Developmental cell. 14(6):843-53. Pubmed: 18539114 DOI:10.1016/j.devcel.2008.03.012 Lineage specification is a critical issue in developmental and regenerative biology. We hypothesized that microRNAs (miRNAs) are important participants in those processes and used the poorly understood regulation of megakaryocyte-erythrocyte progenitors (MEPs) in hematopoiesis as a model system. We report here that miR-150 modulates lineage fate in MEPs. Using a novel methodology capable of profiling miRNA expression in small numbers of primary cells, we identify miR-150 as preferentially expressed in the megakaryocytic lineage. Through gain- and loss-of-function experiments, we demonstrate that miR-150 drives MEP differentiation toward megakaryocytes at the expense of erythroid cells in vitro and in vivo. Moreover, we identify the transcription factor MYB as a critical target of miR-150 in this regulation. These experiments show that miR-150 regulates MEP fate, and thus establish a role for miRNAs in lineage specification of mammalian multipotent cells. -
Orford K, Kharchenko P, Lai W, Dao MC, Worhunsky DJ, Ferro A, Janzen V, Park PJ, Scadden DT. 2008. Differential H3K4 methylation identifies developmentally poised hematopoietic genes. Developmental cell. 14(5):798-809. Pubmed: 18477461 DOI:10.1016/j.devcel.2008.04.002 Orford K, Kharchenko P, Lai W, Dao MC, Worhunsky DJ, Ferro A, Janzen V, Park PJ, Scadden DT. 2008. Differential H3K4 methylation identifies developmentally poised hematopoietic genes. Developmental cell. 14(5):798-809. Pubmed: 18477461 DOI:10.1016/j.devcel.2008.04.002 Throughout development, cell fate decisions are converted into epigenetic information that determines cellular identity. Covalent histone modifications are heritable epigenetic marks and are hypothesized to play a central role in this process. In this report, we assess the concordance of histone H3 lysine 4 dimethylation (H3K4me2) and trimethylation (H3K4me3) on a genome-wide scale in erythroid development by analyzing pluripotent, multipotent, and unipotent cell types. Although H3K4me2 and H3K4me3 are concordant at most genes, multipotential hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3-). These genes are transcriptionally silent, highly enriched in lineage-specific hematopoietic genes, and uniquely susceptible to differentiation-induced H3K4 demethylation. Self-renewing embryonic stem cells, which restrict H3K4 methylation to genes that contain CpG islands (CGIs), lack H3K4me2+/me3- genes. These data reveal distinct epigenetic regulation of CGI and non-CGI genes during development and indicate an interactive relationship between DNA sequence and differential H3K4 methylation in lineage-specific differentiation. -
Fernandez L, Rodriguez S, Huang H, Chora A, Fernandes J, Mumaw C, Cruz E, Pollok K, Cristina F, Price JE, Ferkowicz MJ, Scadden DT, Clauss M, Cardoso AA, Carlesso N. 2008. Tumor necrosis factor-alpha and endothelial cells modulate Notch signaling in the bone marrow microenvironment during inflammation. Experimental hematology. 36(5):545-558. Pubmed: 18439488 DOI:10.1016/j.exphem.2007.12.012 Fernandez L, Rodriguez S, Huang H, Chora A, Fernandes J, Mumaw C, Cruz E, Pollok K, Cristina F, Price JE, Ferkowicz MJ, Scadden DT, Clauss M, Cardoso AA, Carlesso N. 2008. Tumor necrosis factor-alpha and endothelial cells modulate Notch signaling in the bone marrow microenvironment during inflammation. Experimental hematology. 36(5):545-558. Pubmed: 18439488 DOI:10.1016/j.exphem.2007.12.012 Array -
Papayannopoulou T, Scadden DT. 2008. Stem-cell ecology and stem cells in motion. Blood. 111(8):3923-30. Pubmed: 18398055 DOI:10.1182/blood-2007-08-078147 Papayannopoulou T, Scadden DT. 2008. Stem-cell ecology and stem cells in motion. Blood. 111(8):3923-30. Pubmed: 18398055 DOI:10.1182/blood-2007-08-078147 This review highlights major scientific developments over the past 50 years or so in concepts related to stem-cell ecology and to stem cells in motion. Many thorough and eloquent reviews have been presented in the last 5 years updating progress in these issues. Some paradigms have been challenged, others validated, or new ones brought to light. In the present review, we will confine our remarks to the historical development of progress. In doing so, we will refrain from a detailed analysis of controversial data, emphasizing instead widely accepted views and some challenging novel ones. -
Au P, Daheron LM, Duda DG, Cohen KS, Tyrrell JA, Lanning RM, Fukumura D, Scadden DT, Jain RK. 2008. Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels. Blood. 111(3):1302-5. Pubmed: 17993613 Au P, Daheron LM, Duda DG, Cohen KS, Tyrrell JA, Lanning RM, Fukumura D, Scadden DT, Jain RK. 2008. Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels. Blood. 111(3):1302-5. Pubmed: 17993613 Tissue engineering requires formation of a de novo stable vascular network. Because of their ability to proliferate, differentiate into endothelial cells, and form new vessels, blood-derived endothelial progenitor cells (EPCs) are attractive source of cells for use in engineering blood vessels. However, the durability and function of EPC-derived vessels implanted in vivo are unclear. To this end, we directly compared formation and functions of tissue-engineered blood vessels generated by peripheral blood- and umbilical cord blood-derived EPCs in a model of in vivo vasculogenesis. We found that adult peripheral blood EPCs form blood vessels that are unstable and regress within 3 weeks. In contrast, umbilical cord blood EPCs form normal-functioning blood vessels that last for more than 4 months. These vessels exhibit normal blood flow, perm-selectivity to macromolecules, and induction of leukocyte-endothelial interactions in response to cytokine activation similar to normal vessels. Thus, umbilical cord blood EPCs hold great therapeutic potential, and their use should be pursued for vascular engineering. 2007
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Adams GB, Martin RP, Alley IR, Chabner KT, Cohen KS, Calvi LM, Kronenberg HM, Scadden DT. 2007. Therapeutic targeting of a stem cell niche. Nature biotechnology. 25(2):238-43. Pubmed: 17237769 Adams GB, Martin RP, Alley IR, Chabner KT, Cohen KS, Calvi LM, Kronenberg HM, Scadden DT. 2007. Therapeutic targeting of a stem cell niche. Nature biotechnology. 25(2):238-43. Pubmed: 17237769 The specialized microenvironment or niche where stem cells reside provides regulatory input governing stem cell function. We tested the hypothesis that targeting the niche might improve stem cell-based therapies using three mouse models that are relevant to clinical uses of hematopoietic stem (HS) cells. We and others previously identified the osteoblast as a component of the adult HS cell niche and established that activation of the parathyroid hormone (PTH) receptor on osteoblasts increases stem cell number. Here we show that pharmacologic use of PTH increases the number of HS cells mobilized into the peripheral blood for stem cell harvests, protects stem cells from repeated exposure to cytotoxic chemotherapy and expands stem cells in transplant recipients. These data provide evidence that the niche may be an attractive target for drug-based stem cell therapeutics. -
Chen T, Bai H, Shao Y, Arzigian M, Janzen V, Attar E, Xie Y, Scadden DT, Wang ZZ. 2007. Stromal cell-derived factor-1/CXCR4 signaling modifies the capillary-like organization of human embryonic stem cell-derived endothelium in vitro. Stem cells (Dayton, Ohio). 25(2):392-401. Pubmed: 17038674 Chen T, Bai H, Shao Y, Arzigian M, Janzen V, Attar E, Xie Y, Scadden DT, Wang ZZ. 2007. Stromal cell-derived factor-1/CXCR4 signaling modifies the capillary-like organization of human embryonic stem cell-derived endothelium in vitro. Stem cells (Dayton, Ohio). 25(2):392-401. Pubmed: 17038674 The molecular mechanisms that regulate human blood vessel formation during early development are largely unknown. Here we used human ESCs (hESCs) as an in vitro model to explore early human vasculogenesis. We demonstrated that stromal cell-derived factor-1 (SDF-1) and CXCR4 were expressed concurrently with hESC-derived embryonic endothelial differentiation. Human ESC-derived embryonic endothelial cells underwent dose-dependent chemotaxis to SDF-1, which enhanced vascular network formation in Matrigel. Blocking of CXCR4 signaling abolished capillary-like structures induced by SDF-1. Inhibition of the SDF-1/CXCR4 signaling pathway by AMD3100, a CXCR4 antagonist, disrupted the endothelial sprouting outgrowth from human embryoid bodies, suggesting that the SDF-1/CXCR4 axis plays a critical role in regulating initial vessel formation, and may function as a morphogen during human embryonic vascular development. -
Duda DG, Cohen KS, Scadden DT, Jain RK. 2007. A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood. Nature protocols. 2(4):805-10. Pubmed: 17446880 Duda DG, Cohen KS, Scadden DT, Jain RK. 2007. A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood. Nature protocols. 2(4):805-10. Pubmed: 17446880 Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2-2.5 h, and is expected to detect 0.1-6.0% viable CECs and 0.01-0.20% CPCs within blood mononuclear cell population. -
Batchelor TT, Sorensen AG, di Tomaso E, Zhang WT, Duda DG, Cohen KS, Kozak KR, Cahill DP, Chen PJ, Zhu M, Ancukiewicz M, Mrugala MM, Plotkin S, Drappatz J, Louis DN, Ivy P, Scadden DT, Benner T, Loeffler JS, Wen PY, Jain RK. 2007. AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer cell. 11(1):83-95. Pubmed: 17222792 Batchelor TT, Sorensen AG, di Tomaso E, Zhang WT, Duda DG, Cohen KS, Kozak KR, Cahill DP, Chen PJ, Zhu M, Ancukiewicz M, Mrugala MM, Plotkin S, Drappatz J, Louis DN, Ivy P, Scadden DT, Benner T, Loeffler JS, Wen PY, Jain RK. 2007. AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer cell. 11(1):83-95. Pubmed: 17222792 Using MRI techniques, we show here that normalization of tumor vessels in recurrent glioblastoma patients by daily administration of AZD2171-an oral tyrosine kinase inhibitor of VEGF receptors-has rapid onset, is prolonged but reversible, and has the significant clinical benefit of alleviating edema. Reversal of normalization began by 28 days, though some features persisted for as long as four months. Basic FGF, SDF1alpha, and viable circulating endothelial cells (CECs) increased when tumors escaped treatment, and circulating progenitor cells (CPCs) increased when tumors progressed after drug interruption. Our study provides insight into different mechanisms of action of this class of drugs in recurrent glioblastoma patients and suggests that the timing of combination therapy may be critical for optimizing activity against this tumor. -
Walkley CR, Olsen GH, Dworkin S, Fabb SA, Swann J, McArthur GA, Westmoreland SV, Chambon P, Scadden DT, Purton LE. 2007. A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency. Cell. 129(6):1097-110. Pubmed: 17574023 Walkley CR, Olsen GH, Dworkin S, Fabb SA, Swann J, McArthur GA, Westmoreland SV, Chambon P, Scadden DT, Purton LE. 2007. A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency. Cell. 129(6):1097-110. Pubmed: 17574023 Myeloproliferative syndromes (MPS) are a heterogeneous subclass of nonlymphoid hematopoietic neoplasms which are considered to be intrinsic to hematopoietic cells. The causes of MPS are largely unknown. Here, we demonstrate that mice deficient for retinoic acid receptor gamma (RARgamma), develop MPS induced solely by the RARgamma-deficient microenvironment. RARgamma(-/-) mice had significantly increased granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, and spleen. The MPS phenotype continued for the lifespan of the mice and was more pronounced in older mice. Unexpectedly, transplant studies revealed this disease was not intrinsic to the hematopoietic cells. BM from wild-type mice transplanted into mice with an RARgamma(-/-) microenvironment rapidly developed the MPS, which was partially caused by significantly elevated TNFalpha in RARgamma(-/-) mice. These data show that loss of RARgamma results in a nonhematopoietic cell-intrinsic MPS, revealing the capability of the microenvironment to be the sole cause of hematopoietic disorders. -
Scadden DT. 2007. The stem cell niche in health and leukemic disease. Best practice & research. Clinical haematology. 20(1):19-27. Pubmed: 17336251 Scadden DT. 2007. The stem cell niche in health and leukemic disease. Best practice & research. Clinical haematology. 20(1):19-27. Pubmed: 17336251 That adult stem cells live in a highly specialized complex microenvironment, also known as a niche, is a pedestrian concept about 30 years old. It may, however, represent a relatively novel approach to being able to modify either normal or abnormal stem cells. Our emphasis in the past has been focused on identifying autonomous regulators of the stem cells and in attempting to modify them through the use of exogenous agents like cytokines. The body modulates these cells largely through the complex system that is embodied in the niche. This report discusses studies in which the niche components are modified to observe their effect on stem cells. The niches being investigated lie in the gut, skin, brain and bone. Other sites for hematopoiesis exist in the body, but these specific microenvironments can be localized and each component can be carefully evaluated using mouse models. Studies are ongoing as to how the stem cell microenvironment can support or propagate malignancies. By understanding the signals of this particular microenvironment, we may be able to adapt them to achieve a therapeutic benefit. -
Bihl F, Narayan M, Chisholm JV, Henry LM, Suscovich TJ, Brown EE, Welzel TM, Kaufmann DE, Zaman TM, Dollard S, Martin JN, Wang F, Scadden DT, Kaye KM, Brander C. 2007. Lytic and latent antigens of the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus induce T-cell responses with similar functional properties and memory phenotypes. Journal of virology. 81(9):4904-8. Pubmed: 17329344 Bihl F, Narayan M, Chisholm JV, Henry LM, Suscovich TJ, Brown EE, Welzel TM, Kaufmann DE, Zaman TM, Dollard S, Martin JN, Wang F, Scadden DT, Kaye KM, Brander C. 2007. Lytic and latent antigens of the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus induce T-cell responses with similar functional properties and memory phenotypes. Journal of virology. 81(9):4904-8. Pubmed: 17329344 The cellular immunity against Kaposi's sarcoma-associated herpesvirus (KSHV) is poorly characterized and has not been compared to T-cell responses against other human herpesviruses. Here, novel and dominant targets of KSHV-specific cellular immunity are identified and compared to T cells specific for lytic and latent antigens in a second human gammaherpesvirus, Epstein-Barr virus. The data identify a novel HLA-B57- and HLA-B58-restricted epitope in the Orf57 protein and show consistently close parallels in immune phenotypes and functional response patterns between cells targeting lytic or latent KSHV- and EBV-encoded antigens, suggesting common mechanisms in the induction of these responses. -
Scadden DT. 2007. The weight of cell identity. The Journal of clinical investigation. 117(12):3653-5. Pubmed: 18060026 Scadden DT. 2007. The weight of cell identity. The Journal of clinical investigation. 117(12):3653-5. Pubmed: 18060026 Recent studies involving molecular modification of adult somatic cells have pointed to a remarkable plasticity in cell identity. In this issue of the JCI, Koh and colleagues assessed whether bone marrow-derived cells could alter their fate under circumstances conducive to adipocyte generation in vivo (see the related article beginning on page 3684). These cells remained true to their roots, indicating how difficult it will be to exploit cell plasticity for therapeutic purposes. -
Lo Celso C, Klein RJ, Scadden DT. 2007. Analysis of the hematopoietic stem cell niche. Current protocols in stem cell biology. Chapter 2:Unit 2A.5. Pubmed: 18785177 DOI:10.1002/9780470151808.sc02a05s3 Lo Celso C, Klein RJ, Scadden DT. 2007. Analysis of the hematopoietic stem cell niche. Current protocols in stem cell biology. Chapter 2:Unit 2A.5. Pubmed: 18785177 DOI:10.1002/9780470151808.sc02a05s3 Hematopoietic stem cells (HSCs) continuously replenish all blood cell lineages not only to maintain the normal rapid turnover of differentiated cells but also to respond to injury and stress. Cell-extrinsic mechanisms are critical determinants of the fine balance between HSC self-renewal and differentiation. The bone marrow microenvironment has emerged as a new area of intense study to identify which of its many components constitute the HSC niche and regulate HSC fate. While HSCs have been isolated, characterized and used in clinical practice for many years thanks to the development of very specific assays and technology (i.e., bone marrow transplants and fluorescence activated cell sorting), study of the HSC niche has evolved by combining experimental designs developed in different fields. In this unit we describe a collection of protocols spanning a wide range of techniques that can help every researcher tackling questions regarding the nature of the HSC niche.Copyright 2007 by John Wiley & Sons, Inc. -
Purton LE, Scadden DT. 2007. Limiting factors in murine hematopoietic stem cell assays. Cell stem cell. 1(3):263-70. Pubmed: 18371361 DOI:10.1016/j.stem.2007.08.016 Purton LE, Scadden DT. 2007. Limiting factors in murine hematopoietic stem cell assays. Cell stem cell. 1(3):263-70. Pubmed: 18371361 DOI:10.1016/j.stem.2007.08.016 Hematopoiesis arguably provides the most well-defined role of stem cells in tissue development, maintenance, and repair, largely because of the experimental methods developed over decades of investigation. Assays of hematopoietic stem and progenitor cell potential were developed in the late 1950s-1960s with the first reports of in vivo transplantation into lethally irradiated recipients (Ford et al., 1956; McCulloch and Till, 1960) and clonal growth of hematopoietic bone marrow cells in vitro (Bradley and Metcalf, 1966). These two major assays have undergone substantial refinement but remain the foundation for defining hematopoietic stem cell biology. Here, we provide a brief overview of methods commonly used to analyze hematopoietic stem and progenitor cell content in mice, discuss the limitations of these assays, and provide an in-depth review of the limiting dilution assay (Szilvassy et al., 1990), the best single assay for quantitating HSC content. -
Lo Celso C, Scadden DT. 2007. Stem cells remember their grade. Cell stem cell. 1(2):132-4. Pubmed: 18371344 DOI:10.1016/j.stem.2007.07.011 Lo Celso C, Scadden DT. 2007. Stem cells remember their grade. Cell stem cell. 1(2):132-4. Pubmed: 18371344 DOI:10.1016/j.stem.2007.07.011 The stem cell state is understood based on what cells do in performance assays, crude measures of a highly refined state. In this issue of Cell Stem Cell, Dykstra et al. (2007) reveal stem cell gradation and the extent to which that gradation is retained in stem cell daughters of hematopoietic stem cells. -
Bihl F, Mosam A, Henry LN, Chisholm JV, Dollard S, Gumbi P, Cassol E, Page T, Mueller N, Kiepiela P, Martin JN, Coovadia HM, Scadden DT, Brander C. 2007. Kaposi's sarcoma-associated herpesvirus-specific immune reconstitution and antiviral effect of combined HAART/chemotherapy in HIV clade C-infected individuals with Kaposi's sarcoma. AIDS (London, England). 21(10):1245-52. Pubmed: 17545700 Bihl F, Mosam A, Henry LN, Chisholm JV, Dollard S, Gumbi P, Cassol E, Page T, Mueller N, Kiepiela P, Martin JN, Coovadia HM, Scadden DT, Brander C. 2007. Kaposi's sarcoma-associated herpesvirus-specific immune reconstitution and antiviral effect of combined HAART/chemotherapy in HIV clade C-infected individuals with Kaposi's sarcoma. AIDS (London, England). 21(10):1245-52. Pubmed: 17545700 Array -
Lo Celso C, Scadden D. 2007. Isolation and transplantation of hematopoietic stem cells (HSCs). Journal of visualized experiments : JoVE. Pubmed: 18830434 DOI:10.3791/157 Lo Celso C, Scadden D. 2007. Isolation and transplantation of hematopoietic stem cells (HSCs). Journal of visualized experiments : JoVE. Pubmed: 18830434 DOI:10.3791/157 -
Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H, Arzigian M, Fukumura D, Jain RK, Scadden DT. 2007. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nature biotechnology. 25(3):317-8. Pubmed: 17322871 Wang ZZ, Au P, Chen T, Shao Y, Daheron LM, Bai H, Arzigian M, Fukumura D, Jain RK, Scadden DT. 2007. Endothelial cells derived from human embryonic stem cells form durable blood vessels in vivo. Nature biotechnology. 25(3):317-8. Pubmed: 17322871 We describe the differentiation of human embryonic stem (hES) cells into endothelial cells using a scalable two-dimensional method that avoids an embryoid-body intermediate. After transplantation into severe combined immunodeficient (SCID) mice, the differentiated cells contributed to arborized blood vessels that integrated into the host circulatory system and served as blood conduits for 150 d. -
Zhang J, Scadden DT, Crumpacker CS. 2007. Primitive hematopoietic cells resist HIV-1 infection via p21. The Journal of clinical investigation. 117(2):473-81. Pubmed: 17273559 Zhang J, Scadden DT, Crumpacker CS. 2007. Primitive hematopoietic cells resist HIV-1 infection via p21. The Journal of clinical investigation. 117(2):473-81. Pubmed: 17273559 Hematopoietic stem cells are resistant to HIV-1 infection. Here, we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21(Waf1/Cip1/Sdi1) (p21), a known regulator of stem cell pool size, restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs, p27(Kip1) and p18(INK4C), had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase, SIVmac-251, and the other cell-intrinsic inhibitors of HIV-1, Trim5alpha, PML, Murr1, and IFN-alpha, were unaffected by p21. Therefore, p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected "sanctuary" in HIV disease. 2006
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Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, Kos CH, Pollak MR, Brown EM, Scadden DT. 2006. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature. 439(7076):599-603. Pubmed: 16382241 Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, Kos CH, Pollak MR, Brown EM, Scadden DT. 2006. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature. 439(7076):599-603. Pubmed: 16382241 During mammalian ontogeny, haematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, where haematopoiesis occurs throughout adulthood. Unique features of bone that contribute to a microenvironmental niche for stem cells might include the known high concentration of calcium ions at the HSC-enriched endosteal surface. Cells respond to extracellular ionic calcium concentrations through the seven-transmembrane-spanning calcium-sensing receptor (CaR), which we identified as being expressed on HSCs. Here we show that, through the CaR, the simple ionic mineral content of the niche may dictate the preferential localization of adult mammalian haematopoiesis in bone. Antenatal mice deficient in CaR had primitive haematopoietic cells in the circulation and spleen, whereas few were found in bone marrow. CaR-/- HSCs from fetal liver were normal in number, in proliferative and differentiative function, and in migration and homing to the bone marrow. Yet they were highly defective in localizing anatomically to the endosteal niche, behaviour that correlated with defective adhesion to the extracellular matrix protein, collagen I. CaR has a function in retaining HSCs in close physical proximity to the endosteal surface and the regulatory niche components associated with it. -
Scadden DT, Muse VV, Hasserjian RP. 2006. Case records of the Massachusetts General Hospital. Case 30-2006. A 41-year-old man with dyspnea, fever, and lymphadenopathy. The New England journal of medicine. 355(13):1358-68. Pubmed: 17005954 Scadden DT, Muse VV, Hasserjian RP. 2006. Case records of the Massachusetts General Hospital. Case 30-2006. A 41-year-old man with dyspnea, fever, and lymphadenopathy. The New England journal of medicine. 355(13):1358-68. Pubmed: 17005954 -
Duda DG, Cohen KS, Kozin SV, Perentes JY, Fukumura D, Scadden DT, Jain RK. 2006. Evidence for incorporation of bone marrow-derived endothelial cells into perfused blood vessels in tumors. Blood. 107(7):2774-6. Pubmed: 16339405 Duda DG, Cohen KS, Kozin SV, Perentes JY, Fukumura D, Scadden DT, Jain RK. 2006. Evidence for incorporation of bone marrow-derived endothelial cells into perfused blood vessels in tumors. Blood. 107(7):2774-6. Pubmed: 16339405 Recent studies have demonstrated that the cellular contribution of the bone marrow to tumor neovascularization is highly complex. In this context, the extent to which bone marrow-derived cells incorporate as bona fide endothelial (nonhematopoietic) cells into perfused tumor vessels, or any new vessels formed postnatally (vasculogenesis), is unclear. To this end, we developed models to characterize local vessel-derived and bone marrow-derived endothelial cells (BMD-ECs). Then, we characterized the BMD-ECs based on a set of endothelial markers and morphology. Finally, we quantified their contribution to perfused blood vessels in tumors using transplanted as well as spontaneous primary and metastatic tumor models. We demonstrate that BMD-ECs incorporate in perfused tumor vessels, and that this contribution varies with organ site and mouse strain. -
Robertson P, Means TK, Luster AD, Scadden DT. 2006. CXCR4 and CCR5 mediate homing of primitive bone marrow-derived hematopoietic cells to the postnatal thymus. Experimental hematology. 34(3):308-19. Pubmed: 16543065 Robertson P, Means TK, Luster AD, Scadden DT. 2006. CXCR4 and CCR5 mediate homing of primitive bone marrow-derived hematopoietic cells to the postnatal thymus. Experimental hematology. 34(3):308-19. Pubmed: 16543065 Factors governing the entry of cells into the postnatal thymus are poorly understood. We aimed to define molecular mechanisms mediating the homing of bone marrow cells to the thymus using a sublethally irradiated in vivo murine model. Entry of unfractionated and lineage-depleted bone marrow cells to the thymus, but not bone marrow, was a Galphai-mediated phenomenon. Lineage-depleted cells that had homed to the thymus expressed abundant CXCR4 and CCR5 mRNA, alone of 17 chemokine receptors evaluated by QPCR. Thymic-homed cells were distinct from cells that had homed to bone marrow in expression of CXCR4 and CCR5 by mRNA quantification and cell-surface expression of protein. Abrogation of CXCR4 and CCR5 function by genetic, antibody, or pharmacologic means impaired homing of lineage-depleted cells to the thymus, although not in a synergistic manner, implying interdependency of these receptors in the homing process. Competitive repopulation experiments demonstrated that inhibiting CXCR4-mediated homing adversely affected the double-negative cell pool at 2 weeks, suggesting that cells with prothymocytic activity may in part home via CXCR4. Overall, our data demonstrate differential homing mechanisms governing entry of unfractionated and lineage-depleted cells to irradiated bone marrow or thymus, with thymic homing of immature cells being pertussis-sensitive and mediated by the chemokine receptors CXCR4 and CCR5. -
Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT. 2006. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature. 443(7110):421-6. Pubmed: 16957735 Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT. 2006. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature. 443(7110):421-6. Pubmed: 16957735 Stem-cell ageing is thought to contribute to altered tissue maintenance and repair. Older humans experience increased bone marrow failure and poorer haematologic tolerance of cytotoxic injury. Haematopoietic stem cells (HSCs) in older mice have decreased per-cell repopulating activity, self-renewal and homing abilities, myeloid skewing of differentiation, and increased apoptosis with stress. Here we report that the cyclin-dependent kinase inhibitor p16INK4a, the level of which was previously noted to increase in other cell types with age, accumulates and modulates specific age-associated HSC functions. Notably, in the absence of p16INK4a, HSC repopulating defects and apoptosis were mitigated, improving the stress tolerance of cells and the survival of animals in successive transplants, a stem-cell-autonomous tissue regeneration model. Inhibition of p16INK4a may ameliorate the physiological impact of ageing on stem cells and thereby improve injury repair in aged tissue. -
Larsson J, Scadden D. 2006. Nervous activity in a stem cell niche. Cell. 124(2):253-5. Pubmed: 16439198 Larsson J, Scadden D. 2006. Nervous activity in a stem cell niche. Cell. 124(2):253-5. Pubmed: 16439198 In this issue of Cell, report a new regulatory axis for the mobilization of hematopoietic stem cells that links these cells to the nervous system and bone in an unanticipated way. The new findings suggest that the nervous system, which has the inherent ability to integrate information from throughout the organism, may govern the local relationship between stem cells and their niches. -
Adams GB, Scadden DT. 2006. The hematopoietic stem cell in its place. Nature immunology. 7(4):333-7. Pubmed: 16550195 Adams GB, Scadden DT. 2006. The hematopoietic stem cell in its place. Nature immunology. 7(4):333-7. Pubmed: 16550195 A signature characteristic of stem cells is their ability to self-renew, affording a theoretically limitless ability to produce daughter cells and their descendents. This near-timeless dimension of stem cell function is not free of the constraints of place. The idea that highly specialized 'microenvironmental' cues participate in the regulation of stem cells has evidence in classic embryology and more recently in adult stem cells through the use of model organisms. There is now ample evidence that an anatomically defined, specifically constituted place represents the niche for hematopoietic and other tissue-specific stem cells. This review provides a conceptual framework and detailed account of the hematopoietic stem cell niche as defined at present. The components are assembling into a more complex view of the niche and may now be amenable to examination as a system and possibly to alteration to affect outcomes in immune regeneration. -
Miyake N, Brun AC, Magnusson M, Miyake K, Scadden DT, Karlsson S. 2006. HOXB4-induced self-renewal of hematopoietic stem cells is significantly enhanced by p21 deficiency. Stem cells (Dayton, Ohio). 24(3):653-61. Pubmed: 16210402 Miyake N, Brun AC, Magnusson M, Miyake K, Scadden DT, Karlsson S. 2006. HOXB4-induced self-renewal of hematopoietic stem cells is significantly enhanced by p21 deficiency. Stem cells (Dayton, Ohio). 24(3):653-61. Pubmed: 16210402 Enforced expression of the HOXB4 transcription factor and downregulation of p21(Cip1/Waf) (p21) can each independently increase proliferation of murine hematopoietic stem cells (HSCs). We asked whether the increase in HSC self-renewal generated by overexpression of HOXB4 is enhanced in p21-deficient HSCs. HOXB4 was overexpressed in hematopoietic cells from wild-type (wt) and p21-/- mice. Bone marrow (BM) cells were transduced with a retroviral vector expressing HOXB4 together with GFP (MIGB4), or a control vector containing GFP alone (MIG) and maintained in liquid culture for up to 11 days. At day 11 of the expansion culture, the number of primary CFU-GM (colony-forming unit granulocyte-macrophage) colonies and the repopulating ability were significantly increased in MIGB4 p21-/- BM (p21B4) cells compared with MIGB4-transduced wt BM (wtB4) cells. To test proliferation of HSCs in vivo, we performed competitive repopulation experiments and obtained significantly higher long-term engraftment of expanded p21B4 cells compared with wtB4 cells. The 5-day expansion of p21B4 HSCs generated 100-fold higher numbers of competitive repopulating units compared with wtMIG and threefold higher numbers compared with wtB4. The findings demonstrate that increased expression of HOXB4, in combination with suppression of p21 expression, could be a useful strategy for effective and robust expansion of HSCs. -
Purton LE, Scadden DT. 2006. Osteoclasts eat stem cells out of house and home. Nature medicine. 12(6):610-1. Pubmed: 16761001 Purton LE, Scadden DT. 2006. Osteoclasts eat stem cells out of house and home. Nature medicine. 12(6):610-1. Pubmed: 16761001 -
Aghi M, Cohen KS, Klein RJ, Scadden DT, Chiocca EA. 2006. Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer research. 66(18):9054-64. Pubmed: 16982747 Aghi M, Cohen KS, Klein RJ, Scadden DT, Chiocca EA. 2006. Tumor stromal-derived factor-1 recruits vascular progenitors to mitotic neovasculature, where microenvironment influences their differentiated phenotypes. Cancer research. 66(18):9054-64. Pubmed: 16982747 Mechanisms underlying tumor vasculogenesis, the homing and engraftment of bone marrow-derived vascular progenitors, remain undefined. We hypothesized that tumor cell-secreted factors regulate vasculogenesis. We studied vasculogenic and nonvasculogenic intracranial murine gliomas. A PCR screen identified stromal-derived factor-1 (SDF-1/CXCL12) and vascular endothelial growth factor (VEGF) expression by vasculogenic glioma cells and spontaneously arising vasculogenic tumors in NF1+/-:Trp53+/- mice, but not by nonvasculogenic glioma cells. Enforced SDF-1, not VEGF, expression in nonvasculogenic cells caused vasculogenesis. Combined SDF-1 and VEGF expression augmented vasculogenesis over SDF-1 expression alone. Blocking SDF-1 receptor CXCR4 reduced short-term homing and long-term engraftment of vascular progenitors. Implanting tumor cells secreting SDF-1 was therefore necessary and sufficient to incorporate marrow-derived precursors into tumor endothelium. SDF-1 seemed to exert these effects by acting locally intratumorally and did not cause an efflux of marrow-derived progenitors into circulation. Tumor microenvironment determined additional fates of marrow-derived cells. Hypoxia, observed with ectopic s.c. murine tumors at levels approximating that of intracranial human glioblastoma, interacted with tumor-secreted SDF-1 to expand engrafted vascular progenitor differentiated phenotypes to include pericytes as well as endothelium. In contrast, less hypoxic orthotopic intracranial murine gliomas contained only marrow-derived endothelium without marrow-derived pericytes. Furthermore, we found that vasculogenesis is significant for tumors because it generates endothelium with a higher mitotic index than endothelium derived from local sources. Although CXCR4 blockade selectively targeted endothelium generated by vasculogenesis, completely inhibiting vessel formation may require combination therapy targeting locally derived and marrow-derived endothelium. -
Duda DG, Cohen KS, di Tomaso E, Au P, Klein RJ, Scadden DT, Willett CG, Jain RK. 2006. Differential CD146 expression on circulating versus tissue endothelial cells in rectal cancer patients: implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 24(9):1449-53. Pubmed: 16549839 Duda DG, Cohen KS, di Tomaso E, Au P, Klein RJ, Scadden DT, Willett CG, Jain RK. 2006. Differential CD146 expression on circulating versus tissue endothelial cells in rectal cancer patients: implications for circulating endothelial and progenitor cells as biomarkers for antiangiogenic therapy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 24(9):1449-53. Pubmed: 16549839 Array -
Scadden DT. 2006. The stem-cell niche as an entity of action. Nature. 441(7097):1075-9. Pubmed: 16810242 Scadden DT. 2006. The stem-cell niche as an entity of action. Nature. 441(7097):1075-9. Pubmed: 16810242 Stem-cell populations are established in 'niches'--specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics. -
Janzen V, Scadden DT. 2006. Stem cells: good, bad and reformable. Nature. 441(7092):418-9. Pubmed: 16724049 Janzen V, Scadden DT. 2006. Stem cells: good, bad and reformable. Nature. 441(7092):418-9. Pubmed: 16724049 -
Fleming HE, Scadden DT. 2006. Embryonic stem cells make human T cells. Proceedings of the National Academy of Sciences of the United States of America. 103(33):12213-4. Pubmed: 16894150 Fleming HE, Scadden DT. 2006. Embryonic stem cells make human T cells. Proceedings of the National Academy of Sciences of the United States of America. 103(33):12213-4. Pubmed: 16894150 2005
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Zhang J, Attar E, Cohen K, Crumpacker C, Scadden D. 2005. Silencing p21(Waf1/Cip1/Sdi1) expression increases gene transduction efficiency in primitive human hematopoietic cells. Gene therapy. 12(19):1444-52. Pubmed: 15877047 Zhang J, Attar E, Cohen K, Crumpacker C, Scadden D. 2005. Silencing p21(Waf1/Cip1/Sdi1) expression increases gene transduction efficiency in primitive human hematopoietic cells. Gene therapy. 12(19):1444-52. Pubmed: 15877047 Adult hematopoietic and other tissue stem cells have highly constrained cell cycling that limits their susceptibility to standard gene therapy vectors, which depend upon chromosomal integration. Using cytokine cocktails to increase transduction efficiency often compromises subsequent stem cell function in vivo. We previously showed that p21(Waf1/Cip1/Sdi1) (p21) mediates stem cell quiescence in vivo and decreasing its expression ex vivo leads to an expansion of stem cell pool in vivo. Here, we report that application of p21 specific siRNA increased the gene transduction efficiency in hematopoietic stem cells while preserving cell multipotentiality. Both types of siRNA, synthesized siRNA and transcribed shRNA, reduced p21 expression in target cells by 85-98%. The effect of RNAi in these cells was transient and the level of p21 mRNA returned to base line 14-28 days after siRNA treatment. This brief interval of reduction, however, was sufficient to increase transduction efficiency to two- to four-fold in cell cultures, and followed by a seven- to eight-fold increase in mice. The RNAi treated, lentivector-transduced CD34+ cells retained multipotentiality as assessed in vitro by colony formation assay and in vivo by NOD/SCID mouse transplantation assay. Reduction of p21 resulted in an increased chromosomal integration of lentivector into target cellular DNA. Taken together, both synthesized and transcribed siRNA knocked down p21 expression in human CD34+ hematopoietic stem/progenitor cells. Silencing p21 expression increased gene transduction efficiency and vector integration while retaining stem cell multipotentiality. Thus, RNAi targeting of p21 is a useful strategy to increase stem cell gene transfer efficiency. Decreasing p21 expression transiently while increasing gene-transfer vector integration may ultimately facilitate clinical applications of gene therapy. -
Woodberry T, Suscovich TJ, Henry LM, Davis JK, Frahm N, Walker BD, Scadden DT, Wang F, Brander C. 2005. Differential targeting and shifts in the immunodominance of Epstein-Barr virus--specific CD8 and CD4 T cell responses during acute and persistent infection. The Journal of infectious diseases. 192(9):1513-24. Pubmed: 16206065 Woodberry T, Suscovich TJ, Henry LM, Davis JK, Frahm N, Walker BD, Scadden DT, Wang F, Brander C. 2005. Differential targeting and shifts in the immunodominance of Epstein-Barr virus--specific CD8 and CD4 T cell responses during acute and persistent infection. The Journal of infectious diseases. 192(9):1513-24. Pubmed: 16206065 The evolution of Epstein-Barr virus (EBV)-specific T cell responses that occurs during the acute and persistent stages of infection remains poorly characterized despite its importance for developing immune interventions for EBV-associated disorders. This study assessed T cell responses to 113 EBV-derived epitopes in 40 subjects with acute or persistent EBV infection. Although no significant differences were seen in the breadth of CD8 and CD4 T cell responses, their magnitude differed significantly over time; acutely infected subjects generated especially strong responses to lytic viral antigens. The cross-sectional shift in immunodominance was also confirmed in subjects followed longitudinally from acute to persistent infection. In addition, human leukocyte antigen-matched siblings with discordant histories of symptomatic EBV infection showed no significant differences in their response patterns, suggesting that symptomatic EBV infection does not lead to unique persistent-stage responses. These data provide an assessment of immunodominance patterns and guidance for developing immunotherapeutic interventions for EBV-associated disorders. -
Johnson J, Bagley J, Skaznik-Wikiel M, Lee HJ, Adams GB, Niikura Y, Tschudy KS, Tilly JC, Cortes ML, Forkert R, Spitzer T, Iacomini J, Scadden DT, Tilly JL. 2005. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell. 122(2):303-15. Pubmed: 16051153 Johnson J, Bagley J, Skaznik-Wikiel M, Lee HJ, Adams GB, Niikura Y, Tschudy KS, Tilly JC, Cortes ML, Forkert R, Spitzer T, Iacomini J, Scadden DT, Tilly JL. 2005. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell. 122(2):303-15. Pubmed: 16051153 It has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood. -
Woodberry T, Suscovich TJ, Henry LM, Martin JN, Dollard S, O'Connor PG, Davis JK, Osmond D, Lee TH, Kedes DH, Khatri A, Lee J, Walker BD, Scadden DT, Brander C. 2005. Impact of Kaposi sarcoma-associated herpesvirus (KSHV) burden and HIV coinfection on the detection of T cell responses to KSHV ORF73 and ORF65 proteins. The Journal of infectious diseases. 192(4):622-9. Pubmed: 16028131 Woodberry T, Suscovich TJ, Henry LM, Martin JN, Dollard S, O'Connor PG, Davis JK, Osmond D, Lee TH, Kedes DH, Khatri A, Lee J, Walker BD, Scadden DT, Brander C. 2005. Impact of Kaposi sarcoma-associated herpesvirus (KSHV) burden and HIV coinfection on the detection of T cell responses to KSHV ORF73 and ORF65 proteins. The Journal of infectious diseases. 192(4):622-9. Pubmed: 16028131 Cellular immune responses to Kaposi sarcoma-associated herpesvirus (KSHV), the etiological agent of KS and several other malignancies, are incompletely characterized. We assessed KSHV-specific interferon- gamma enzyme-linked immunospot responses in a cohort of 154 individuals, using overlapping peptide sets spanning the KSHV-encoded latency-associated nuclear antigen (ORF73) and the minor capsid glycoprotein (ORF65). Among KSHV-seropositive subjects, ORF73-specific responses dominated over responses to ORF65 and were preferentially detected in human immunodeficiency virus-coinfected individuals who had elevated levels of cell-associated KSHV DNA, indicating that the viral antigen burden may have been driving these responses. Responses to both ORF73 and ORF65 were also detected in several KSHV-seronegative subjects who were at increased risk for KSHV infection, which demonstrates that cellular immunity can be found in the absence of detectable humoral responses. These data have implications for the reliable identification of KSHV infection and may help guide the design of immune-based therapeutic and prophylactic interventions. -
Woodberry T, Suscovich TJ, Henry LM, August M, Waring MT, Kaur A, Hess C, Kutok JL, Aster JC, Wang F, Scadden DT, Brander C. 2005. Alpha E beta 7 (CD103) expression identifies a highly active, tonsil-resident effector-memory CTL population. Journal of immunology (Baltimore, Md. : 1950). 175(7):4355-62. Pubmed: 16177076 Woodberry T, Suscovich TJ, Henry LM, August M, Waring MT, Kaur A, Hess C, Kutok JL, Aster JC, Wang F, Scadden DT, Brander C. 2005. Alpha E beta 7 (CD103) expression identifies a highly active, tonsil-resident effector-memory CTL population. Journal of immunology (Baltimore, Md. : 1950). 175(7):4355-62. Pubmed: 16177076 The characterization of antiviral CTL responses has largely been limited to assessing Ag-specific immune responses in the peripheral blood. Consequently, there is an incomplete understanding of the cellular immune responses at mucosal sites where many viruses enter and initially replicate and how the Ag specificity and activation status of CTL derived from these mucosal sites may differ from that of blood-derived CTL. In this study, we show that EBV-specific CTL responses in the tonsils are of comparable specificity and breadth but of a significantly higher magnitude compared with responses in the peripheral blood. EBV-specific, tonsil-resident, but not PBMC-derived, T cells expressed the integrin/activation marker CD103 (alphaEbeta7), consistent with the detection of its ligand, E-cadherin, on tonsillar squamous cells. These CD8-positive, CD103-positive, tonsil-derived CTL were largely CCR7- and CD45RA- negative effector-memory cells and responded to lower Ag concentrations in in vitro assays than their CD103-negative PBMC-derived counterparts. Thus, EBV-specific CTL in the tonsil, a crucial site for EBV entry and replication, are of greater magnitude and phenotypically distinct from CTL in the peripheral blood and may be important for effective control of this orally transmitted virus. -
Stroh M, Zimmer JP, Duda DG, Levchenko TS, Cohen KS, Brown EB, Scadden DT, Torchilin VP, Bawendi MG, Fukumura D, Jain RK. 2005. Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo. Nature medicine. 11(6):678-82. Pubmed: 15880117 Stroh M, Zimmer JP, Duda DG, Levchenko TS, Cohen KS, Brown EB, Scadden DT, Torchilin VP, Bawendi MG, Fukumura D, Jain RK. 2005. Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo. Nature medicine. 11(6):678-82. Pubmed: 15880117 A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrow-derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment. -
Kaplan LD, Lee JY, Ambinder RF, Sparano JA, Cesarman E, Chadburn A, Levine AM, Scadden DT. 2005. Rituximab does not improve clinical outcome in a randomized phase 3 trial of CHOP with or without rituximab in patients with HIV-associated non-Hodgkin lymphoma: AIDS-Malignancies Consortium Trial 010. Blood. 106(5):1538-43. Pubmed: 15914552 Kaplan LD, Lee JY, Ambinder RF, Sparano JA, Cesarman E, Chadburn A, Levine AM, Scadden DT. 2005. Rituximab does not improve clinical outcome in a randomized phase 3 trial of CHOP with or without rituximab in patients with HIV-associated non-Hodgkin lymphoma: AIDS-Malignancies Consortium Trial 010. Blood. 106(5):1538-43. Pubmed: 15914552 The addition of rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy results in significant improvement in clinical outcome for individuals with non-HIV-associated aggressive B-cell lymphoma. To assess the potential risks and benefits of the addition of rituximab to CHOP for HIV-associated non-Hodgkin lymphoma (HIV-NHL) 150 patients receiving CHOP for HIV-NHL were randomized (2:1) to receive 375 mg/m(2) rituximab with each chemotherapy cycle (n = 99) or no immunotherapy (n = 50) in a multicenter phase 3 trial. The complete response rate (CR + CRu) was 57.6% for R-CHOP and 47% for CHOP (P = .147). With a median follow-up of 137 weeks, time to progression, progression-free survival, and overall survival times were 125, 45, and 139 weeks, respectively, for R-CHOP and 85, 38, and 110 weeks, respectively, for CHOP (P = not significant, all comparisons). Treatment-related infectious deaths occurred in 14% of patients receiving R-CHOP compared with 2% in the chemotherapy-alone group (P = .035). Of these deaths, 60% occurred in patients with CD4 counts less than 50/mm(3). Progression-free survival was significantly influenced by CD4(+) count (P < .001) and International Prognostic Index score (P = .022), but not bcl-2 status. The addition of rituximab to CHOP in patients with HIV-NHL may be associated with improved tumor responses. However, these benefits may be offset by an increase in infectious deaths, particularly in those individuals with CD4(+) lymphocyte counts less than 50/mm(3). -
Li ZJ, Wang ZZ, Zheng YZ, Xu B, Yang RC, Scadden DT, Han ZC. 2005. Kinetic expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) during embryonic stem cell differentiation. Journal of cellular biochemistry. 95(3):559-70. Pubmed: 15786495 Li ZJ, Wang ZZ, Zheng YZ, Xu B, Yang RC, Scadden DT, Han ZC. 2005. Kinetic expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) during embryonic stem cell differentiation. Journal of cellular biochemistry. 95(3):559-70. Pubmed: 15786495 Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as a marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. However, the expression of PECAM-1 isoforms in ES cells has not been determined. The present study was designed to determine the role of PECAM-1 isoforms during in vitro endothelial differentiation of ES cells. It was found that undifferentiated ES cells expressed high level of PECAM-1, which primarily located at cell-cell junction, but the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. In addition, undifferentiated ES cells were found the expressed all eight known alternatively spliced PECAM-1 isoforms, among them the expression of PECAM-1 isoforms lacking exon 15 or 14&15 was predominant. Quantitative analysis revealed a significant increase in the expression of PECAM-1 isoform lacking exon 12&14&15 as vascular development of ES cells. These results indicate a constitutive expression of PECAM-1 in undifferentiated murine ES cells and suggest a developmental role of PECAM-1 isoform changes during vasculogenesis and angiogenesis.(c) 2005 Wiley-Liss, Inc. -
Duffield JS, Park KM, Hsiao LL, Kelley VR, Scadden DT, Ichimura T, Bonventre JV. 2005. Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. The Journal of clinical investigation. 115(7):1743-55. Pubmed: 16007251 Duffield JS, Park KM, Hsiao LL, Kelley VR, Scadden DT, Ichimura T, Bonventre JV. 2005. Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. The Journal of clinical investigation. 115(7):1743-55. Pubmed: 16007251 Ischemia causes kidney tubular cell damage and abnormal renal function. The kidney is capable of morphological restoration of tubules and recovery of function. Recently, it has been suggested that cells repopulating the ischemically injured tubule derive from bone marrow stem cells. We studied kidney repair in chimeric mice expressing GFP or bacterial beta-gal or harboring the male Y chromosome exclusively in bone marrow-derived cells. In GFP chimeras, some interstitial cells but not tubular cells expressed GFP after ischemic injury. More than 99% of those GFP interstitial cells were leukocytes. In female mice with male bone marrow, occasional tubular cells (0.06%) appeared to be positive for the Y chromosome, but deconvolution microscopy revealed these to be artifactual. In beta-gal chimeras, some tubular cells also appeared to express beta-gal as assessed by X-gal staining, but following suppression of endogenous (mammalian) beta-gal, no tubular cells could be found that stained with X-gal after ischemic injury. Whereas there was an absence of bone marrow-derived tubular cells, many tubular cells expressed proliferating cell nuclear antigen, which is reflective of a high proliferative rate of endogenous surviving tubular cells. Upon i.v. injection of bone marrow mesenchymal stromal cells, postischemic functional renal impairment was reduced, but there was no evidence of differentiation of these cells into tubular cells of the kidney. Thus, our data indicate that bone marrow-derived cells do not make a significant contribution to the restoration of epithelial integrity after an ischemic insult. It is likely that intrinsic tubular cell proliferation accounts for functionally significant replenishment of the tubular epithelium after ischemia. -
Craiu A, Saito Y, Limon A, Eppich HM, Olson DP, Rodrigues N, Adams GB, Dombkowski D, Richardson P, Schlossman R, Choi PS, Grogins J, O'Connor PG, Cohen K, Attar EC, Freshman J, Rich R, Mangano JA, Gribben JG, Anderson KC, Scadden DT. 2005. Flowing cells through pulsed electric fields efficiently purges stem cell preparations of contaminating myeloma cells while preserving stem cell function. Blood. 105(5):2235-8. Pubmed: 15292069 Craiu A, Saito Y, Limon A, Eppich HM, Olson DP, Rodrigues N, Adams GB, Dombkowski D, Richardson P, Schlossman R, Choi PS, Grogins J, O'Connor PG, Cohen K, Attar EC, Freshman J, Rich R, Mangano JA, Gribben JG, Anderson KC, Scadden DT. 2005. Flowing cells through pulsed electric fields efficiently purges stem cell preparations of contaminating myeloma cells while preserving stem cell function. Blood. 105(5):2235-8. Pubmed: 15292069 Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations. -
Noy A, Scadden DT, Lee J, Dezube BJ, Aboulafia D, Tulpule A, Walmsley S, Gill P. 2005. Angiogenesis inhibitor IM862 is ineffective against AIDS-Kaposi's sarcoma in a phase III trial, but demonstrates sustained, potent effect of highly active antiretroviral therapy: from the AIDS Malignancy Consortium and IM862 Study Team. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 23(5):990-8. Pubmed: 15598977 Noy A, Scadden DT, Lee J, Dezube BJ, Aboulafia D, Tulpule A, Walmsley S, Gill P. 2005. Angiogenesis inhibitor IM862 is ineffective against AIDS-Kaposi's sarcoma in a phase III trial, but demonstrates sustained, potent effect of highly active antiretroviral therapy: from the AIDS Malignancy Consortium and IM862 Study Team. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 23(5):990-8. Pubmed: 15598977 Array -
Kutok JL, Yang X, Folkerth RD, Imitola J, Raddassi K, Yano Y, Salahuddin S, Lawitts J, Imboden H, Chinami M, Shirakawa T, Turner H, Khoury S, Sayegh MH, Scadden D, Adra C. 2005. The cell cycle associated protein, HTm4, is expressed in differentiating cells of the hematopoietic and central nervous system in mice. Journal of molecular histology. 36(1-2):77-87. Pubmed: 15704002 Kutok JL, Yang X, Folkerth RD, Imitola J, Raddassi K, Yano Y, Salahuddin S, Lawitts J, Imboden H, Chinami M, Shirakawa T, Turner H, Khoury S, Sayegh MH, Scadden D, Adra C. 2005. The cell cycle associated protein, HTm4, is expressed in differentiating cells of the hematopoietic and central nervous system in mice. Journal of molecular histology. 36(1-2):77-87. Pubmed: 15704002 HTm4 is a member of a newly defined family of human and murine proteins, the MS4 (membrane-spanning four) protein group, which has a distinctive four-transmembrane structure. MS4 protein functions include roles as cell surface signaling receptors and intracellular adapter proteins. We have previously demonstrated that HTm4 regulates the function of the KAP phosphatase, a key regulator of cell cycle progression. In humans, the expression of HTm4 is largely restricted to cells of the hematopoietic lineage, possibly reflecting a causal role for this molecule in differentiation/proliferation of hematopoietic lineage cells. In this study, we show that, like the human homologue, murine HTm4 is also predominantly a hematopoietic protein with distinctive expression patterns in developing murine embryos and in adult animals. In addition, we observed that murine HTm4 is highly expressed in the developing and adult murine nervous system, suggesting a previously unrecognized role in central and peripheral nervous system development. -
Fangman JJ, Scadden DT. 2005. Anemia in HIV-infected adults: epidemiology, pathogenesis, and clinical management. Current hematology reports. 4(2):95-102. Pubmed: 15720957 Fangman JJ, Scadden DT. 2005. Anemia in HIV-infected adults: epidemiology, pathogenesis, and clinical management. Current hematology reports. 4(2):95-102. Pubmed: 15720957 Anemia is the most common cytopenia seen in people with HIV. Independent of CD4 count and HIV-viral load, anemia has been shown to correlate with increased mortality. Furthermore, successful treatment of anemia has been shown to reduce this risk of death in a comparison with patients with similar immunologic and virologic parameters who are not treated. Women, blacks, injection drug users, and people with advanced disease suffer disproportionally from anemia and should be screened. The pathogenesis of anemia in HIV is complex and may result from opportunistic infections, nutritional deficiencies, AIDS-associated malignancies, medications, or alteration in hematopoeisis induced by HIV itself. A careful review of the patient's past medical history, medications, symptoms, and basic laboratory studies often leads to a treatable cause(s). For patients without secondary causes of anemia, a combination of highly active antiretroviral therapy (HAART) and supplemental erythropoietin leads to improved outcomes. Given the importance of completing therapy on adequate doses of both interferon and ribavirin, effective management of anemia in HIV/Hepatitis C (HCV)-coinfected patients is particularly important. -
Mosam A, Cassol E, Page T, Bodasing U, Cassol S, Dawood H, Friedland GH, Scadden DT, Aboobaker J, Jordaan JP, Lalloo UG, Esterhuizen TM, Coovadia HM. 2005. Generic antiretroviral efficacy in AIDS-associated Kaposi's sarcoma in sub-Saharan Africa. AIDS (London, England). 19(4):441-3. Pubmed: 15750399 Mosam A, Cassol E, Page T, Bodasing U, Cassol S, Dawood H, Friedland GH, Scadden DT, Aboobaker J, Jordaan JP, Lalloo UG, Esterhuizen TM, Coovadia HM. 2005. Generic antiretroviral efficacy in AIDS-associated Kaposi's sarcoma in sub-Saharan Africa. AIDS (London, England). 19(4):441-3. Pubmed: 15750399 Generic antiretroviral drugs are pivotal in the implementation of WHO's '3 by 5' programme. However, clinical experience with generics in sub-Saharan Africa is insufficiently documented. We report on 50 patients with HIV-associated Kaposi's sarcoma treated with generic fixed-dose highly active antiretroviral therapy. At 52 weeks, 74% achieved an undetectable viral load of < 50 copies/ml, 86% achieved < 400 copies/ml, and a 3.1 log10 decline from baseline. Side-effects were minimal. The outcomes support the use of generic antiretroviral therapy. -
Rodrigues NP, Janzen V, Forkert R, Dombkowski DM, Boyd AS, Orkin SH, Enver T, Vyas P, Scadden DT. 2005. Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis. Blood. 106(2):477-84. Pubmed: 15811962 Rodrigues NP, Janzen V, Forkert R, Dombkowski DM, Boyd AS, Orkin SH, Enver T, Vyas P, Scadden DT. 2005. Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis. Blood. 106(2):477-84. Pubmed: 15811962 The zinc finger transcription factor GATA-2 plays a fundamental role in generating hematopoietic stem-cells in mammalian development. Less well defined is whether GATA-2 participates in adult stem-cell regulation, an issue we addressed using GATA-2 heterozygote mice that express reduced levels of GATA-2 in hematopoietic cells. While GATA-2+/- mice demonstrated decreases in some colony-forming progenitors, the most prominent changes were observed within the stem-cell compartment. Heterozygote bone marrow had a lower abundance of Lin(-)c-kit(+)Sca-1(+)CD34- cells and performed poorly in competitive transplantation and quantitative week-5 cobblestone area-forming cell (CAFC) assays. Furthermore, a stem-cell-enriched population from GATA1+/- marrow was more quiescent and exhibited a greater frequency of apoptotic cells associated with decreased expression of the anti-apoptotic gene Bcl-xL. Yet the self-renewal potential of the +/- stem-cell compartment, as judged by serial transplantations, was unchanged. These data indicate compromised primitive cell proliferation and survival in the setting of a lower GATA-2 gene dose without a change in the differentiation or self-renewal capacity of the stem-cells that remain. Thus, GATA-2 dose regulates adult stem-cell homeostasis by affecting select aspects of stem cell function. -
Stier S, Ko Y, Forkert R, Lutz C, Neuhaus T, Grünewald E, Cheng T, Dombkowski D, Calvi LM, Rittling SR, Scadden DT. 2005. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. The Journal of experimental medicine. 201(11):1781-91. Pubmed: 15928197 Stier S, Ko Y, Forkert R, Lutz C, Neuhaus T, Grünewald E, Cheng T, Dombkowski D, Calvi LM, Rittling SR, Scadden DT. 2005. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. The Journal of experimental medicine. 201(11):1781-91. Pubmed: 15928197 Stem cells reside in a specialized niche that regulates their abundance and fate. Components of the niche have generally been defined in terms of cells and signaling pathways. We define a role for a matrix glycoprotein, osteopontin (OPN), as a constraining factor on hematopoietic stem cells within the bone marrow microenvironment. Osteoblasts that participate in the niche produce varying amounts of OPN in response to stimulation. Using studies that combine OPN-deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell number and function in a stem cell-nonautonomous manner. The OPN-null microenvironment was sufficient to increase the number of stem cells associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive hematopoietic cell apoptosis. The activation of the stem cell microenvironment with parathyroid hormone induced a superphysiologic increase in stem cells in the absence of OPN. Therefore, OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation. -
Sipkins DA, Wei X, Wu JW, Runnels JM, Côté D, Means TK, Luster AD, Scadden DT, Lin CP. 2005. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature. 435(7044):969-73. Pubmed: 15959517 Sipkins DA, Wei X, Wu JW, Runnels JM, Côté D, Means TK, Luster AD, Scadden DT, Lin CP. 2005. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature. 435(7044):969-73. Pubmed: 15959517 The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells. -
Dong HY, Scadden DT, de Leval L, Tang Z, Isaacson PG, Harris NL. 2005. Plasmablastic lymphoma in HIV-positive patients: an aggressive Epstein-Barr virus-associated extramedullary plasmacytic neoplasm. The American journal of surgical pathology. 29(12):1633-41. Pubmed: 16327436 Dong HY, Scadden DT, de Leval L, Tang Z, Isaacson PG, Harris NL. 2005. Plasmablastic lymphoma in HIV-positive patients: an aggressive Epstein-Barr virus-associated extramedullary plasmacytic neoplasm. The American journal of surgical pathology. 29(12):1633-41. Pubmed: 16327436 AIDS-associated aggressive B-cell lymphomas often have plasmacytoid features. Plasma cell neoplasms in HIV patients were commonly described to have atypical morphology and an aggressive clinical course in the literature. We reviewed 14 cases of neoplasms with marked plasmacytic differentiation in HIV-positive patients to determine their clinicopathologic features. Of these, 13 of 14 had homogeneous morphology and were generally CD45(+), CD20-, PAX-5-, and CD138(+). All were positive for Epstein-Barr virus-encoded RNA (EBER) but lacked EBV late membrane proteins (LMP). Human herpes virus 8 (HHV8) DNA was detected in 6 of 10 cases by nested PCR, but HHV8 latent nuclear antigen (LNA) was absent. The 13 patients ranged in age from 28 to 44 years (median, 41 years) (11 male patients; 2 female patients). All patients had extramedullary and 11 of 13 had extranodal tumor at the initial presentation; 2 had distant marrow involvement. The most commonly involved location was the oral cavity (6 of 13 cases), followed by bone and soft tissue (4 of 13), and the gastrointestinal tract (3 of 13). All 11 patients with follow-up died within 34 months (median, 7 months). The 14th patient who had a nodal disease with more undifferentiated morphology and expression of the HHV8 LNA protein was alive without disease at last follow-up (>72 months), probably representing a novel HHV8(+) lymphoma. We conclude that most plasmacytic tumors in HIV-positive individuals are extramedullary, clinically aggressive EBV(+) tumors identical to plasmablastic lymphoma that does not have the clinical features of plasma cell myeloma. -
Willett CG, Boucher Y, Duda DG, di Tomaso E, Munn LL, Tong RT, Kozin SV, Petit L, Jain RK, Chung DC, Sahani DV, Kalva SP, Cohen KS, Scadden DT, Fischman AJ, Clark JW, Ryan DP, Zhu AX, Blaszkowsky LS, Shellito PC, Mino-Kenudson M, Lauwers GY. 2005. Surrogate markers for antiangiogenic therapy and dose-limiting toxicities for bevacizumab with radiation and chemotherapy: continued experience of a phase I trial in rectal cancer patients. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 23(31):8136-9. Pubmed: 16258121 Willett CG, Boucher Y, Duda DG, di Tomaso E, Munn LL, Tong RT, Kozin SV, Petit L, Jain RK, Chung DC, Sahani DV, Kalva SP, Cohen KS, Scadden DT, Fischman AJ, Clark JW, Ryan DP, Zhu AX, Blaszkowsky LS, Shellito PC, Mino-Kenudson M, Lauwers GY. 2005. Surrogate markers for antiangiogenic therapy and dose-limiting toxicities for bevacizumab with radiation and chemotherapy: continued experience of a phase I trial in rectal cancer patients. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 23(31):8136-9. Pubmed: 16258121 -
Robertson P, Scadden DT. 2005. Differentiation and characterization of T cells. Current protocols in immunology. Chapter 22:22F.8.1-22F.8.8. Pubmed: 18432955 DOI:10.1002/0471142735.im22f08s69 Robertson P, Scadden DT. 2005. Differentiation and characterization of T cells. Current protocols in immunology. Chapter 22:22F.8.1-22F.8.8. Pubmed: 18432955 DOI:10.1002/0471142735.im22f08s69 This paper outlines current standard methods of inducing T lymphopoiesis in vitro and in vivo. Reference is made to both murine and human systems. In addition to differentiation assays, methods to optimally characterize output cells are discussed. In bringing together a number of existing protocols, many techniques important in investigating T cell development can be reviewed in one place. -
Scadden D. 2005. Interview with David Scadden. Interview by Joseph Glorioso and Jenny Jacoby. Gene therapy. 12(23):1663-6. Pubmed: 16121202 Scadden D. 2005. Interview with David Scadden. Interview by Joseph Glorioso and Jenny Jacoby. Gene therapy. 12(23):1663-6. Pubmed: 16121202 2004
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Scadden DT. 2004. The malignant side of successful transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 4(2):153-4. Pubmed: 14974933 Scadden DT. 2004. The malignant side of successful transplantation. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 4(2):153-4. Pubmed: 14974933 -
Willett CG, Boucher Y, di Tomaso E, Duda DG, Munn LL, Tong RT, Chung DC, Sahani DV, Kalva SP, Kozin SV, Mino M, Cohen KS, Scadden DT, Hartford AC, Fischman AJ, Clark JW, Ryan DP, Zhu AX, Blaszkowsky LS, Chen HX, Shellito PC, Lauwers GY, Jain RK. 2004. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nature medicine. 10(2):145-7. Pubmed: 14745444 Willett CG, Boucher Y, di Tomaso E, Duda DG, Munn LL, Tong RT, Chung DC, Sahani DV, Kalva SP, Kozin SV, Mino M, Cohen KS, Scadden DT, Hartford AC, Fischman AJ, Clark JW, Ryan DP, Zhu AX, Blaszkowsky LS, Chen HX, Shellito PC, Lauwers GY, Jain RK. 2004. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nature medicine. 10(2):145-7. Pubmed: 14745444 The effects of vascular endothelial growth factor (VEGF) blockade on the vascular biology of human tumors are not known. Here we show here that a single infusion of the VEGF-specific antibody bevacizumab decreases tumor perfusion, vascular volume, microvascular density, interstitial fluid pressure and the number of viable, circulating endothelial and progenitor cells, and increases the fraction of vessels with pericyte coverage in rectal carcinoma patients. These data indicate that VEGF blockade has a direct and rapid antivascular effect in human tumors. -
Wang Z, Cohen K, Shao Y, Mole P, Dombkowski D, Scadden DT. 2004. Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vessels. Blood. 103(1):100-9. Pubmed: 12958066 Wang Z, Cohen K, Shao Y, Mole P, Dombkowski D, Scadden DT. 2004. Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vessels. Blood. 103(1):100-9. Pubmed: 12958066 Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development, however, must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor, EphB4, known to be spatially restricted in expression and critical for organized vessel formation, modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast, blood cell, cardiomyocyte, and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated, but not neuroectoderm-associated, genes. Therefore, EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization. -
Attar EC, Scadden DT. 2004. Regulation of hematopoietic stem cell growth. Leukemia. 18(11):1760-8. Pubmed: 15457182 Attar EC, Scadden DT. 2004. Regulation of hematopoietic stem cell growth. Leukemia. 18(11):1760-8. Pubmed: 15457182 Hematopoietic stem cells (HSC) must balance self-renewal and differentiation to provide sufficient primitive cells to sustain hematopoiesis, while generating more mature cells with specialized capabilities. The enhanced self-renewal capacity of primitive HSCs enables their ability to sustain hematopoiesis throughout decades of life and their ability to repopulate a host when used therapeutically in bone marrow transplantation. However, hematopoietic cell perturbations resulting in unchecked self-renewal participate in leukemogenesis. While mechanisms governing self-renewal are still being uncovered, they are thought to bear relationship to the malignant process in a variety of tumor types and may therefore provide useful therapeutic targets in putative cancer stem cells. This review discusses molecular mechanisms recently defined to participate in HSC governance and highlights features of stem cell interactions with the microenvironment that may help guide therapies directed at HSCs. -
Scadden DT. 2004. Cancer stem cells refined. Nature immunology. 5(7):701-3. Pubmed: 15224098 Scadden DT. 2004. Cancer stem cells refined. Nature immunology. 5(7):701-3. Pubmed: 15224098 -
Chabner KT, Adams GB, Qiu J, Moskowitz M, Marsters ES, Topulos GP, Scadden DT. 2004. Direct vascular delivery of primitive hematopoietic cells to bone marrow improves localization but not engraftment. Blood. 103(12):4685-6. Pubmed: 15178586 Chabner KT, Adams GB, Qiu J, Moskowitz M, Marsters ES, Topulos GP, Scadden DT. 2004. Direct vascular delivery of primitive hematopoietic cells to bone marrow improves localization but not engraftment. Blood. 103(12):4685-6. Pubmed: 15178586 -
Suscovich TJ, Paulose-Murphy M, Harlow JD, Chen Y, Thomas SY, Mellott TJ, Walker BD, Scadden DT, Zeichner S, Brander C. 2004. Defective immune function of primary effusion lymphoma cells is associated with distinct KSHV gene expression profiles. Leukemia & lymphoma. 45(6):1223-38. Pubmed: 15360006 Suscovich TJ, Paulose-Murphy M, Harlow JD, Chen Y, Thomas SY, Mellott TJ, Walker BD, Scadden DT, Zeichner S, Brander C. 2004. Defective immune function of primary effusion lymphoma cells is associated with distinct KSHV gene expression profiles. Leukemia & lymphoma. 45(6):1223-38. Pubmed: 15360006 Primary effusion lymphomas (PEL) are uniformly infected with Kaposi's sarcoma-associated herpesvirus (KSHV), and thus likely present both tumor and viral antigens to the immune system. In order to grow unrestricted and cause disease, multiple immune evasion strategies may be utilized by PEL to evade immune surveillance. Using six well-established PEL cell lines and comparing these to Epstein-Barr virus-transformed B cell lines and peripheral blood B cells, significant differences were found in the surface expression of molecules involved in antigen presentation, T cell activation and cell-cell adhesion. Significantly reduced stimulation of cytotoxic T lymphocytes, lowered sensitivity to natural killer cell-mediated lysis and impaired function as antigen presenting cells in mixed leukocyte reactions were found for three PEL cell lines with particularly low CD54, CD58 and CD81 expression. Comparative microarray analysis demonstrated specific patterns of KSHV-encoded gene expression that were associated with the different immune functions of these cell lines. Thus, the present data suggest that distinct patterns of KSHV gene expression may be associated with particular phenotypic and functional characteristics of PEL cells, which may influence PEL pathogenesis. -
Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. 2004. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nature cell biology. 6(5):436-42. Pubmed: 15122268 Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. 2004. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nature cell biology. 6(5):436-42. Pubmed: 15122268 Self-renewal of stem cells is critical for tissue repair and maintenance of organ integrity in most mammalian systems. The relative asymmetry between self-renewal and differentiation in balance with apoptosis determines the size and durability of a stem-cell pool. Regulation of the cell cycle is one of the fundamental mechanisms underlying determination of cell fate. Absence of p21(Cip1/Waf1), a late G1-phase cyclin-dependent kinase inhibitor (CKI), has previously been shown to enable cell-cycle entry of haematopoietic stem cells, but leads to premature exhaustion of the stem cells under conditions of stress. We show here that deletion of an early G1-phase CKI, p18(INK4C), results in strikingly improved long-term engraftment, largely by increasing self-renewing divisions of the primitive cells in murine transplant models. Therefore, different CKIs have highly distinct effects on the kinetics of stem cells, possibly because of their active position in the cell cycle, and p18(INK4C) appears to be a strong inhibitor limiting the potential of stem-cell self-renewal in vivo. -
Qiu J, Takagi Y, Harada J, Rodrigues N, Moskowitz MA, Scadden DT, Cheng T. 2004. Regenerative response in ischemic brain restricted by p21cip1/waf1. The Journal of experimental medicine. 199(7):937-45. Pubmed: 15067031 Qiu J, Takagi Y, Harada J, Rodrigues N, Moskowitz MA, Scadden DT, Cheng T. 2004. Regenerative response in ischemic brain restricted by p21cip1/waf1. The Journal of experimental medicine. 199(7):937-45. Pubmed: 15067031 Neural precursor cells from adults have exceptional proliferative and differentiative capability in vitro yet respond minimally to in vivo brain injury due to constraining mechanisms that are poorly defined. We assessed whether cell cycle inhibitors that restrict stem cell populations in other tissues may participate in limiting neural stem cell reactivity in vivo. The cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21), maintains hematopoietic stem cell quiescence, and we evaluated its role in the regenerative response of neural tissue after ischemic injury using the mice deficient in p21. Although steady-state conditions revealed no increase in primitive cell proliferation in p21-null mice, a significantly larger fraction of quiescent neural precursors was activated in the hippocampus and subventricular zone after brain ischemia. The hippocampal precursors migrated and differentiated into a higher number of neurons after injury. Therefore, p21 is an intrinsic suppressor to neural regeneration after brain injury and may serve as a common molecular regulator restricting proliferation among stem cell pools from distinct tissue types. -
Adams G, Scadden D. 2004. Defining the hematopoietic stem cell niche. Discovery medicine. 4(21):118-9. Pubmed: 20705006 Adams G, Scadden D. 2004. Defining the hematopoietic stem cell niche. Discovery medicine. 4(21):118-9. Pubmed: 20705006 Extract: Stem cells modulate tissue formation and repair based on a complex interaction of cell autonomous and non-autonomous regulatory mechanisms. While reductionist approaches to understanding stem cell control continue to be extremely productive, understanding the physiological contexts in which stem cells function, will ultimately require definition of the microenvironments in which they live. The location of stem or precursor populations within numerous solid tissues has been described, but delineating specific associated cells and how they participate in regulating stem cell function has generally been lacking for mammalian tissues. However, the use of invertebrate-based models has created particularly productive systems in which to examine the niche context of stem cells. Gonadal tissue from C. elegans and D. melanogaster has permitted the definition and identification of ancillary niche cells, physical interactions and the molecular pathways such as Notch paralogues that govern the interplay between the stem cell and its local environment. We sought to determine a niche component for a mammalian tissue and focused on the hematopoietic system. We focused on hematopoiesis for multiple reasons, but in particular because of the potential for applying the information gained to a medical context. 2003
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Fukushima T, Miyazaki Y, Tsushima H, Tsutsumi C, Taguchi J, Yoshida S, Kuriyama K, Scadden D, Nimer S, Tomonaga M. 2003. The level of MEF but not ELF-1 correlates with FAB subtype of acute myeloid leukemia and is low in good prognosis cases. Leukemia research. 27(5):387-92. Pubmed: 12620289 Fukushima T, Miyazaki Y, Tsushima H, Tsutsumi C, Taguchi J, Yoshida S, Kuriyama K, Scadden D, Nimer S, Tomonaga M. 2003. The level of MEF but not ELF-1 correlates with FAB subtype of acute myeloid leukemia and is low in good prognosis cases. Leukemia research. 27(5):387-92. Pubmed: 12620289 ETS proteins (such as PU.1, Fli-1 and ETS-1) have been shown to play important roles in normal and abnormal hematopoiesis. We examined the expression of the ELF subfamily of ETS genes (ELF-1, MEF and NERF) in acute myeloid leukemia (AML) cells using Northern blot analysis. ELF-1 and MEF were expressed in all samples, whereas NERF was not. The relative expression (RE) of MEF, but not ELF-1, was significantly lower (P<0.0001) in AML with t(8;21) and t(15;17) compared with AML with normal karyotype. The pattern of MEF expression was not uniform among cells with CD34(+)/CD33(+). It is suggested that the low RE of MEF might be part of a gene expression profile characterizing AML with a good prognosis. -
Robertson P, Scadden DT. 2003. Immune reconstitution in HIV infection and its relationship to cancer. Hematology/oncology clinics of North America. 17(3):703-16, vi. Pubmed: 12852652 Robertson P, Scadden DT. 2003. Immune reconstitution in HIV infection and its relationship to cancer. Hematology/oncology clinics of North America. 17(3):703-16, vi. Pubmed: 12852652 HIV infection results in formidable immune dysfunction, widely affecting the immune system, but typified by T lymphopenia. This dysfunction includes a perturbed immune response to several persistent viruses that have a propensity to cause tumors. Effective control of HIV replication by highly active antiretroviral therapy (HAART) results in regeneration of the damaged immune system, and recent advances have allowed this immune reconstitution to be better defined. This article describes the immunodeficiency caused by HIV and the response of the immune system to HAART, with specific reference to the immune response to cancers associated with HIV infection. -
Lee BC, Cheng T, Adams GB, Attar EC, Miura N, Lee SB, Saito Y, Olszak I, Dombkowski D, Olson DP, Hancock J, Choi PS, Haber DA, Luster AD, Scadden DT. 2003. P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells. Genes & development. 17(13):1592-604. Pubmed: 12842911 Lee BC, Cheng T, Adams GB, Attar EC, Miura N, Lee SB, Saito Y, Olszak I, Dombkowski D, Olson DP, Hancock J, Choi PS, Haber DA, Luster AD, Scadden DT. 2003. P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells. Genes & development. 17(13):1592-604. Pubmed: 12842911 Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y14), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G alpha i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment. -
Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT. 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 425(6960):841-6. Pubmed: 14574413 Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT. 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. 425(6960):841-6. Pubmed: 14574413 Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies. -
Scadden DT. 2003. Stem cells and immune reconstitution in AIDS. Blood reviews. 17(4):227-31. Pubmed: 14556777 Scadden DT. 2003. Stem cells and immune reconstitution in AIDS. Blood reviews. 17(4):227-31. Pubmed: 14556777 The hematopoietic system generally has reserve sufficient to tolerate significant insult and regenerative capacity to overcome most damage due to infectious agents. However, HIV infection results in a progressive decline in hematopoietic function and even in the context of potent, anti-retroviral therapy is able to only incompletely reconstitute immune function. The ability of the immune system to respond to HIV itself remains compromised, a defect that leaves infected individuals with a lifelong dependence on medications. The capability of stem cells and the thymus to restore function and their limitations in the context of HIV infection are discussed in this review. -
Adams GB, Chabner KT, Foxall RB, Weibrecht KW, Rodrigues NP, Dombkowski D, Fallon R, Poznansky MC, Scadden DT. 2003. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment. Blood. 101(1):45-51. Pubmed: 12393569 Adams GB, Chabner KT, Foxall RB, Weibrecht KW, Rodrigues NP, Dombkowski D, Fallon R, Poznansky MC, Scadden DT. 2003. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment. Blood. 101(1):45-51. Pubmed: 12393569 T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8(+) but not CD4(+) T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34(+) cells in a beta(2)-microglobulin-deficient nonobese diabetic/severe combined immunodeficiency (beta(2)m(-/-) NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8(+) cells but not CD4(+) cells. This correlated with CD8(+) augmentation of CD34(+) cell homing to the bone marrow in vivo and CD8(+) cell-associated increases of CD34(+) cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D-induced inhibition of CD8(+) cytoskeletal rearrangements abrogated CD34(+) transendothelial migration and impaired CD34(+) cell homing in vivo. CD8(+) cells did not migrate in tandem with CD34(+) cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34(+) cells in response to the chemokine stromal derived factor-1alpha (SDF-1alpha). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8(+) cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization. -
Christmas P, Carlesso N, Shang H, Cheng SM, Weber BM, Preffer FI, Scadden DT, Soberman RJ. 2003. Myeloid expression of cytochrome P450 4F3 is determined by a lineage-specific alternative promoter. The Journal of biological chemistry. 278(27):25133-42. Pubmed: 12709424 Christmas P, Carlesso N, Shang H, Cheng SM, Weber BM, Preffer FI, Scadden DT, Soberman RJ. 2003. Myeloid expression of cytochrome P450 4F3 is determined by a lineage-specific alternative promoter. The Journal of biological chemistry. 278(27):25133-42. Pubmed: 12709424 The cytochrome P450 4F3 (CYP4F3) gene encodes two functionally distinct enzymes that differ only by the selection of exon 4 (CYP4F3A) or exon 3 (CYP4F3B). CYP4F3A inactivates leukotriene B4, a reaction that has significance for controlling inflammation. CYP4F3B converts arachidonic acid to 20-hydroxyeicosatetraenoic acid, a potent activator of protein kinase C. We have previously shown that mRNAs coding for CYP4F3A and CYP4F3B are generated from distinct transcription start sites in neutrophils and liver. We therefore investigated mechanisms that regulate the cell-specific expression of these two isoforms. Initially, we analyzed the distribution of CYP4F3 in human leukocytes and determined a lineage-specific pattern of isoform expression. CYP4F3A is expressed in myeloid cells and is coordinate with myeloid differentiation markers such as CD11b and myeloperoxidase during development in the bone marrow. In contrast, CYP4F3B expression is restricted to a small population of CD3+ T lymphocytes. We identified distinct transcriptional features in myeloid, lymphoid, and hepatic cells that indicate the presence of multiple promoters in the CYP4F3 gene. The hepatic promoter depends on a cluster of hepatocyte nuclear factor sites 123-155 bp upstream of the initiator ATG codon. The myeloid promoter spans 400 bp in a region 468-872 bp upstream of the ATG codon; it is associated with clusters of CACCT sites and can be activated by ZEB-2, a factor primarily characterized as a transcriptional repressor in cells that include lymphocytes. ZEB-2 interacts with C-terminal binding protein and Smads, and this would provide opportunities for integrating environmental signals in myelopoiesis and inflammation. -
Stier S, Cheng T, Forkert R, Lutz C, Dombkowski DM, Zhang JL, Scadden DT. 2003. Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells. Blood. 102(4):1260-6. Pubmed: 12702511 Stier S, Cheng T, Forkert R, Lutz C, Dombkowski DM, Zhang JL, Scadden DT. 2003. Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells. Blood. 102(4):1260-6. Pubmed: 12702511 Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. -
Anton PA, Mitsuyasu RT, Deeks SG, Scadden DT, Wagner B, Huang C, Macken C, Richman DD, Christopherson C, Borellini F, Lazar R, Hege KM. 2003. Multiple measures of HIV burden in blood and tissue are correlated with each other but not with clinical parameters in aviremic subjects. AIDS (London, England). 17(1):53-63. Pubmed: 12478069 Anton PA, Mitsuyasu RT, Deeks SG, Scadden DT, Wagner B, Huang C, Macken C, Richman DD, Christopherson C, Borellini F, Lazar R, Hege KM. 2003. Multiple measures of HIV burden in blood and tissue are correlated with each other but not with clinical parameters in aviremic subjects. AIDS (London, England). 17(1):53-63. Pubmed: 12478069 Array -
Scadden DT. 2003. AIDS-related malignancies. Annual review of medicine. 54:285-303. Pubmed: 12525676 Scadden DT. 2003. AIDS-related malignancies. Annual review of medicine. 54:285-303. Pubmed: 12525676 Immunodeficiency alters the risk of cancer. Specific types of immune dysfunction are associated with different tumor risks, but most tumors are related to oncogenic viruses. In acquired immunodeficiency due to the human immunodeficiency virus (HIV), HIV itself rarely directly causes cancer; rather, it provides the immunologic background against which other viruses can escape immune control and induce tumors. The most common malignancies are Kaposi's sarcoma and non-Hodgkin's lymphoma. This chapter discusses the pathophysiologic background of these tumors, how they have been affected by the use of anti-HIV medications, and their clinical management. 2002
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Scadden DT. 2002. Toward cellular-based therapies for HIV infection. Journal of hematotherapy & stem cell research. 11(5):759-64. Pubmed: 12427282 Scadden DT. 2002. Toward cellular-based therapies for HIV infection. Journal of hematotherapy & stem cell research. 11(5):759-64. Pubmed: 12427282 Infection with HIV-1 progressively erodes immune function, leading ultimately to multiple hematopoietic cytopenias. In advanced HIV disease, anemia, neutropenia, and thrombocytopenia occur in a large fraction of patients and are reflective of a striking inability of the bone marrow to accomplish a compensatory increase in production. No failure of compensatory production is greater than that of the T lymphoid system, where despite apparently effective control of HIV replication, restoration of anti-HIV immunity does not occur. Recent technical developments have provided considerable insight into hematopoietic dysfunction in HIV disease and, in particular, in vivo T cell generation. This article will review some of the mechanisms participating in immune regeneration, the stakes involved in effectively accomplishing full reconstitution, and potential approaches to enhance the limited endogenous regenerative process. -
Olson DP, Scadden DT, D'Aquila RT, De Pasquale MP. 2002. The protease inhibitor ritonavir inhibits the functional activity of the multidrug resistance related-protein 1 (MRP-1). AIDS (London, England). 16(13):1743-7. Pubmed: 12218384 Olson DP, Scadden DT, D'Aquila RT, De Pasquale MP. 2002. The protease inhibitor ritonavir inhibits the functional activity of the multidrug resistance related-protein 1 (MRP-1). AIDS (London, England). 16(13):1743-7. Pubmed: 12218384 ArrayCopyright 2002 Lippincott Williams & Wilkins -
Preffer FI, Dombkowski D, Sykes M, Scadden D, Yang YG. 2002. Lineage-negative side-population (SP) cells with restricted hematopoietic capacity circulate in normal human adult blood: immunophenotypic and functional characterization. Stem cells (Dayton, Ohio). 20(5):417-27. Pubmed: 12351812 Preffer FI, Dombkowski D, Sykes M, Scadden D, Yang YG. 2002. Lineage-negative side-population (SP) cells with restricted hematopoietic capacity circulate in normal human adult blood: immunophenotypic and functional characterization. Stem cells (Dayton, Ohio). 20(5):417-27. Pubmed: 12351812 Side-population (SP) cells are a recently described rare cell population detected in selected tissues of various mammalian species, but not yet described in the peripheral circulation. In the present study, we have identified for the first time SP cells in lineage-negative adult human blood and have provided an extensive functional and immunophenotypic characterization of these cells. Adult peripheral blood was depleted of mature leukocyte cell types by density gradient and immunomagnetic separation. The SP cell population was identified by its characteristic Hoechst 33342 profile. Immunophenotypic analysis revealed that blood SP cells expressed high levels of CD45, CD59, CD43, CD49d, CD31, and integrin markers and lacked CD34. Highly purified SP cells were put into cobblestone area-forming cell (CAFC), long-term culture-initiating cell (LTC-IC), and liquid cell culture assays; repopulating assays were performed utilizing nonobese diabetic/severe combined immunodeficient mice. Circulating SP cells were shown to exhibit verapamil sensitivity and a low growth rate. LTC-IC, CAFC, and engraftment assays indicated that circulating SP cells had lost the multipotentiality described in murine bone marrow SP cells. However, outgrowth of mature cell types from liquid cell culture suggests the presence of common lymphoid (T and natural killer) and dendritic cell precursor(s) within circulating SP cell populations. The absence of SP cell growth in the LTC-IC, CAFC, and repopulating assays might be intrinsic to the tissue source (marrow versus blood) or species (mouse versus human) tested. Thus, human blood SP cells, although rare, may serve as a source of selected leukocyte progenitor cells. The immunophenotype of circulating SP cells may provide clues to their seeding and homing capacity. -
Brander C, Raje N, O'Connor PG, Davies F, Davis J, Chauhan D, Hideshima T, Martin J, Osmond D, Kedes DH, Walker BD, Scadden DT, Anderson KC. 2002. Absence of biologically important Kaposi sarcoma-associated herpesvirus gene products and virus-specific cellular immune responses in multiple myeloma. Blood. 100(2):698-700. Pubmed: 12091368 Brander C, Raje N, O'Connor PG, Davies F, Davis J, Chauhan D, Hideshima T, Martin J, Osmond D, Kedes DH, Walker BD, Scadden DT, Anderson KC. 2002. Absence of biologically important Kaposi sarcoma-associated herpesvirus gene products and virus-specific cellular immune responses in multiple myeloma. Blood. 100(2):698-700. Pubmed: 12091368 Kaposi sarcoma-associated herpesvirus (KSHV) has been associated with several diseases, but the association between KSHV and multiple myeloma (MM) remains controversial. To address this issue, we studied patients with MM for the presence of viral RNA transcripts as well as KSHV-specific cellular immune responses. Highly sensitive reverse transcription-polymerase chain reaction assays for detection of viral transcripts of KSHV open reading frame (ORF) 26, ORF72, and ORF74 did not detect viral gene transcripts in long-term cultures of bone marrow stromal cells from 23 patients with MM. Moreover, sensitive assays for KSHV ORF65-specific and ORF73-specific cytotoxic T-lymphocyte (CTL) activity that readily and routinely detect CTLs specific for ORF65 and ORF73 in patients positive for human immunodeficiency virus and KSHV did not show any specific responses in 16 patients with MM, despite the presence of positive Epstein-Barr virus-specific CTLs in all cases. These data therefore do not show a biologically important association between ongoing KSHV infection and MM. -
Tulpule A, Groopman J, Saville MW, Harrington W, Friedman-Kien A, Espina BM, Garces C, Mantelle L, Mettinger K, Scadden DT, Gill PS. 2002. Multicenter trial of low-dose paclitaxel in patients with advanced AIDS-related Kaposi sarcoma. Cancer. 95(1):147-54. Pubmed: 12115328 Tulpule A, Groopman J, Saville MW, Harrington W, Friedman-Kien A, Espina BM, Garces C, Mantelle L, Mettinger K, Scadden DT, Gill PS. 2002. Multicenter trial of low-dose paclitaxel in patients with advanced AIDS-related Kaposi sarcoma. Cancer. 95(1):147-54. Pubmed: 12115328 ArrayCopyright 2002 American Cancer Society. -
Miles SA, Dezube BJ, Lee JY, Krown SE, Fletcher MA, Saville MW, Kaplan L, Groopman J, Scadden DT, Cooley T, Von Roenn J, Friedman-Kien A. 2002. Antitumor activity of oral 9-cis-retinoic acid in HIV-associated Kaposi's sarcoma. AIDS (London, England). 16(3):421-9. Pubmed: 11834954 Miles SA, Dezube BJ, Lee JY, Krown SE, Fletcher MA, Saville MW, Kaplan L, Groopman J, Scadden DT, Cooley T, Von Roenn J, Friedman-Kien A. 2002. Antitumor activity of oral 9-cis-retinoic acid in HIV-associated Kaposi's sarcoma. AIDS (London, England). 16(3):421-9. Pubmed: 11834954 Array -
Miles SA, Testa M, Huang J, Wade M, Carden J, Scadden DT. 2002. Lack of antitumor activity and intolerance of interleukin-4 in patients with advanced HIV disease and Kaposi's sarcoma. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 22(11):1143-8. Pubmed: 12513914 Miles SA, Testa M, Huang J, Wade M, Carden J, Scadden DT. 2002. Lack of antitumor activity and intolerance of interleukin-4 in patients with advanced HIV disease and Kaposi's sarcoma. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 22(11):1143-8. Pubmed: 12513914 Kaposi's sarcoma (KS), the most common malignancy associated with HIV infection, is caused by the Kaposi sarcoma herpesvirus (KSHV). Exacerbations of KSHV are associated with increased human interleukin-6 (HuIL-6), and elevated IL-6 could be related to the development of KS. IL-4, a cytokine with pleiotropic effects, suppresses IL-6 in vivo and modestly inhibits AIDS-KS-derived cells in vitro. Suppression of IL-6 by exogenous IL-4 could result in antitumor activity. We report the results of a clinical trial to test this hypothesis. A phase I/II dose escalation safety, tolerance, and efficacy trial was conducted in patients with biopsy-proven AIDS-related KS, at two university medical centers. Patients were scheduled to receive IL-4 (0.5, 1.5, 3.0, or 4.0 microg/kg/day) administered subcutaneously (s.c.) in sequential cohorts. Patients were continued on study as long as the drug was tolerated or the disease progressed. Patients were followed for antitumor activity, effects on viral replication, immune status, and clinical and laboratory toxicity. Seventeen patients were enrolled at two sites over a 21-month period. There were 15 males and 2 females, and 1 patient was Hispanic. All patients had a Karnofsky score >70. Patients enrolled only into the two lower dose cohorts (0.5 and 1.5 microg/kg/day). Both groups had similar baseline characteristics. The median time on treatment was only 7.4 and 8.4 weeks for the 0.5 and 1.5 microg/kg/day dose levels, respectively. There was significant neutropenia, with 6 patients having grade 3 or greater toxicity requiring granulocyte colony-stimulating factor (G-CSF). Three patients on a dose of 1.5 microg/kg/day stopped treatment due to protocol-defined toxicity. There were no appreciable effects on CD4/CD8 counts. HIV viral RNA did not significantly change over time. However, in several people, it appeared to decline with treatment and rebound with discontinuation of treatment. Corresponding changes were noted in the HIV immunocomplex dissociated (ICD) p24 antigen. One patient had a partial response, 11 patients had stable disease, and 5 patients had disease progression during the short period of treatment. The maximum tolerated dose for IL-4 in patients with advanced AIDS-related KS is 1.5 microg/kg/day. At this dose level, IL-4 is poorly tolerated and is not an effective KS treatment. Treatment of the majority of patients is discontinued because of drug-related toxicity or because of disease progression. Future studies of IL-4 should be confined to studies of cytokine manipulation of the underlying HIV infection, as there appears to be little antitumor activity. -
Stier S, Cheng T, Dombkowski D, Carlesso N, Scadden DT. 2002. Notch1 activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome. Blood. 99(7):2369-78. Pubmed: 11895769 Stier S, Cheng T, Dombkowski D, Carlesso N, Scadden DT. 2002. Notch1 activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome. Blood. 99(7):2369-78. Pubmed: 11895769 Hematopoietic stem cells sequentially pass through a series of decision points affecting self-renewal or lineage-specific differentiation. Notch1 receptor is a known modulator of lineage-specific events in hematopoiesis that we assessed in the context of in vivo stem cell kinetics. Using RAG-1(-/-) mouse stems cells, we documented increased stem cell numbers due to decreased differentiation and enhanced stem cell self-renewal induced by Notch1. Unexpectedly, preferential lymphoid over myeloid lineage commitment was noted when differentiation occurred. Therefore, Notch1 affects 2 decision points in stem cell regulation, favoring self-renewal over differentiation and lymphoid over myeloid lineage outcome. Notch1 offers an attractive target for stem cell manipulation strategies, particularly in the context of immunodeficiency and acquired immunodeficiency syndrome. -
Wang Z, Miura N, Bonelli A, Mole P, Carlesso N, Olson DP, Scadden DT. 2002. Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells. Blood. 99(8):2740-7. Pubmed: 11929761 Wang Z, Miura N, Bonelli A, Mole P, Carlesso N, Olson DP, Scadden DT. 2002. Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells. Blood. 99(8):2740-7. Pubmed: 11929761 EphB4 (HTK) and its ligand, ephrinB2, are critical for angiogenesis and result in fatal abnormalities of capillary formation in null mice. EphB4 was originally identified in human bone marrow CD34(+) cells by us and has since been reported to be expressed in erythroid progenitors, whereas the ligand ephrinB2 is expressed in bone marrow stromal cells. Reasoning that the developmental relationship between angiogenesis and hematopoiesis implies common regulatory molecules, we assessed whether EphB4 signaling influences the function and phenotype of primitive human hematopoietic cells. Ectopically expressed EphB4 in cell lines of restricted differentiation potential promoted megakaryocytic differentiation, but not granulocytic or monocytic differentiation. Primary cord blood CD34(+) cells transduced with EphB4 resulted in the elevated expression of megakaryocytic and erythroid specific markers, consistent with EphB4 selectively enhancing some lineage-committed progenitors. In less mature cells, EphB4 depleted primitive cells, as measured by long-term culture-initiating cells or CD34(+)CD38(-) cell numbers, and increased progenitor cells of multiple cell types. Effects of ectopic EphB4 expression could be abrogated by either targeted mutations of select tyrosine residues or by the tyrosine kinase inhibitor, genistein. These data indicate that EphB4 accelerates the differentiation of primitive cells in a nonlineage-restricted manner but alters only select progenitor populations, influencing lineages linked by common ancestry with endothelial cells. EphB4 enforces preferential megakaryocytic and erythroid differentiation and may be a molecular bridge between angiogenesis and hematopoiesis. -
Deeks SG, Wagner B, Anton PA, Mitsuyasu RT, Scadden DT, Huang C, Macken C, Richman DD, Christopherson C, June CH, Lazar R, Broad DF, Jalali S, Hege KM. 2002. A phase II randomized study of HIV-specific T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Molecular therapy : the journal of the American Society of Gene Therapy. 5(6):788-97. Pubmed: 12027564 Deeks SG, Wagner B, Anton PA, Mitsuyasu RT, Scadden DT, Huang C, Macken C, Richman DD, Christopherson C, June CH, Lazar R, Broad DF, Jalali S, Hege KM. 2002. A phase II randomized study of HIV-specific T-cell gene therapy in subjects with undetectable plasma viremia on combination antiretroviral therapy. Molecular therapy : the journal of the American Society of Gene Therapy. 5(6):788-97. Pubmed: 12027564 Highly active antiretroviral therapy (HAART) can suppress HIV replication to undetectable levels in plasma, but it is unlikely to eradicate cellular reservoirs of virus. Immunotherapies that are cytolytic may be useful adjuncts to drug therapies that target HIV replication. We have generated HIV-specific CD4(+) and CD8(+) T cells bearing a chimeric T-cell receptor (CD4zeta) composed of the extracellular and transmembrane domain of human CD4 (which binds HIVgp120) linked to the intracellular-zeta signaling chain of the CD3 T-cell receptor. CD4zeta-modified T cells can inhibit viral replication, kill HIV-infected cells in vitro, and survive for prolonged periods in vivo. We report the results of a phase II randomized trial of CD4zeta gene-modified versus unmodified T cells in 40 HIV-infected subjects on HAART with plasma viral loads <50 copies/ml. Serial analyses of residual blood and tissue HIV reservoirs were done for 6 months postinfusion. No significant between-group differences were noted in viral reservoirs following therapy. However, infusion of gene-modified, but not unmodified, T cells was associated with a decrease from baseline in HIV burden in two of four reservoir assays and a trend toward fewer patients with recurrent viremia. Both groups experienced a treatment-related increase in CD4(+) T-cell counts.(c)2002 Elsevier Science (USA). -
Cheng T, Scadden DT. 2002. Cell cycle entry of hematopoietic stem and progenitor cells controlled by distinct cyclin-dependent kinase inhibitors. International journal of hematology. 75(5):460-5. Pubmed: 12095144 Cheng T, Scadden DT. 2002. Cell cycle entry of hematopoietic stem and progenitor cells controlled by distinct cyclin-dependent kinase inhibitors. International journal of hematology. 75(5):460-5. Pubmed: 12095144 The therapeutic promise of hematopoietic stem cells in medicine has been expanded as broader differentiation potential of the cells has gained experimental support. However, hurdles for stem cell manipulation in vitro and tissue regeneration in vivo remain because of lack of the molecular biology of the stem cells. In particular, elucidating the molecular control of cell cycle entry is necessary for rational stem cell expansion strategies. Understanding how the stem and progenitor cell populations are controlled by negative regulators of cell cycle entry may provide one basis for manipulating these cells. In this mini-review, we focus on the rationale of targeting the cyclin-dependent kinase inhibitors (CKIs) in stem cell biology. Two CKI members, p21(Cip1/Waf1) (p21) and p27kip1 (p27), have been shown to govern the pool sizes of hematopoietic stem and progenitor cells, respectively. Of note, their inhibitory roles in primitive hematopoietic cells are distinct from the action of the inhibitory cytokine, transforming growth factor-beta1 (TGF-beta1). Therefore, the distinct roles of p21, p27, and TGF-beta1 in hematopoietic cells offer attractive targets for specific manipulation of the stem or progenitor cell populations in therapeutic strategies. -
Jiang Z, Adams GB, Hanash AM, Scadden DT, Levy RB. 2002. The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 8(11):588-96. Pubmed: 12463477 Jiang Z, Adams GB, Hanash AM, Scadden DT, Levy RB. 2002. The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 8(11):588-96. Pubmed: 12463477 The present studies were designed for investigation of the requirements for cytotoxic function in donor T-cells transplanted to support engraftment after infusion of allogeneic bone marrow. The experiments examined the capacity of donor CD8 T-cells lacking Fas ligand and/or perforin function to facilitate donor B6 congenic (B6-Ly5.1) BM engraftment across major histocompatibility complex class I/II barriers after transplantation. T-cell-depleted BM cells from B6-Ly5.1 donors were transplanted into sublethally irradiated (5.5 Gy) BALB/c recipients together with different lymphocyte populations from wild-type B6 (B6-wt) donors or donors lacking functional cytotoxic pathways. Early presence of lineage-committed donor progenitor cells was assessed by the presence of day 5 splenic colony-forming units-granulocyte-macrophage (CFU-GM). Recipients of BMT without donor T-cells did not demonstrate significant CFU-GM activity 5 days post-BMT. Lineage-committed progenitor cells in recipient spleens could be supported by addition to the BM of wild-type (B6-wt) and cytotoxically single- (perforin, B6-pko or FasL, B6-gld) or double-deficient (B6-cdd) CD8 T-cells. However, B220+-enriched B-cells could not support the presence of day 5 donor CFU-GM. For further assessment of the capacity of cytotoxically impaired T-cells to participate in the engraftment process, the ability of these and normal CD8 cells to support the homing of donor cells to the BM was examined after infusion of carboxyfluorescein diacetete succinimidyl ester-labeled progenitors. In a syngeneic model lacking resistance, cytotoxically impaired donor T-cells supported increased numbers of progenitor cells in the marrow equivalent to the support provided by wild-type donor T-cells. Examination of peripheral chimerism indicated that during the first month after B6-->BALB/c BMT, donor chimerism was detected in BMT recipients receiving unfractionated T-cells or CD8+ T-cells from B6-wt donors, and chimerism was maintained at least 80 days after BMT. In contrast, B6-cdd unfractionated or CD8+ T-cells failed to maintain long-term B6 donor chimerism in the host. Experiments with highly enriched populations of positively selected CD8+ T-cells from B6-pko, B6-gld, or B6-cdd donors demonstrated that although each of these T-cell populations could promote the initial presence of donor CFU-GM early post-BMT, B6-pko and B6-cdd CD8+ T-cell populations were not able to support long-term peripheral chimerism. These results demonstrate that donor T-cells lacking major cytotoxic effector pathways have functions that support initial donor progenitor cell presence in the host hematopoietic compartment after BMT. They also demonstrate that support of long-term donor BM engraftment requires CD8+ T-cells with intact cytotoxic, that is, perforin, function. Finally, syngeneic B6-->B6 BMT suggests activation of CD8+ T-cells posttransplantation apparently is required to support enhanced progenitor cell activity. This study provides new findings concerning the role of cytotoxic function in the process of facilitating allogeneic donor BM engraftment. -
Cianfrocca M, Cooley TP, Lee JY, Rudek MA, Scadden DT, Ratner L, Pluda JM, Figg WD, Krown SE, Dezube BJ. 2002. Matrix metalloproteinase inhibitor COL-3 in the treatment of AIDS-related Kaposi's sarcoma: a phase I AIDS malignancy consortium study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 20(1):153-9. Pubmed: 11773164 Cianfrocca M, Cooley TP, Lee JY, Rudek MA, Scadden DT, Ratner L, Pluda JM, Figg WD, Krown SE, Dezube BJ. 2002. Matrix metalloproteinase inhibitor COL-3 in the treatment of AIDS-related Kaposi's sarcoma: a phase I AIDS malignancy consortium study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 20(1):153-9. Pubmed: 11773164 Array -
Poznansky MC, Olszak IT, Evans RH, Wang Z, Foxall RB, Olson DP, Weibrecht K, Luster AD, Scadden DT. 2002. Thymocyte emigration is mediated by active movement away from stroma-derived factors. The Journal of clinical investigation. 109(8):1101-10. Pubmed: 11956248 Poznansky MC, Olszak IT, Evans RH, Wang Z, Foxall RB, Olson DP, Weibrecht K, Luster AD, Scadden DT. 2002. Thymocyte emigration is mediated by active movement away from stroma-derived factors. The Journal of clinical investigation. 109(8):1101-10. Pubmed: 11956248 T cells leave the thymus at a specific time during differentiation and do not return despite elaboration of known T cell chemoattractants by thymic stroma. We observed differentiation stage-restricted egress of thymocytes from an artificial thymus in which vascular structures or hemodynamics could not have been playing a role. Hypothesizing that active movement of cells away from a thymic product may be responsible, we demonstrated selective reduction in emigration from primary thymus by inhibitors of active movement down a concentration gradient (chemofugetaxis). Immature intrathymic precursors were insensitive to an emigration signal, whereas mature thymocytes and peripheral blood T cells were sensitive. Thymic stroma was noted to elaborate at least two proteins capable of inducing emigration, one of which was stromal cell-derived factor-1. Thymic emigration is mediated, at least in part, by specific fugetaxis-inducing factors to which only mature cells respond. -
Donato JL, Ko J, Kutok JL, Cheng T, Shirakawa T, Mao XQ, Beach D, Scadden DT, Sayegh MH, Adra CN. 2002. Human HTm4 is a hematopoietic cell cycle regulator. The Journal of clinical investigation. 109(1):51-8. Pubmed: 11781350 Donato JL, Ko J, Kutok JL, Cheng T, Shirakawa T, Mao XQ, Beach D, Scadden DT, Sayegh MH, Adra CN. 2002. Human HTm4 is a hematopoietic cell cycle regulator. The Journal of clinical investigation. 109(1):51-8. Pubmed: 11781350 Proper control of cell cycle progression is critical for the constant self-renewal, differentiation, and homeostasis of the hematopoietic system. Cells of all types share the common cell cycle regulators. The different expression patterns of common regulators, in a broad sense, define cell-type or lineage specificity. However, there remains the possibility of hematopoietic cell cycle regulators tailored to the demands of the hematopoietic system. Here we describe a novel protein, HTm4, which serves as a hematopoietic cell cycle regulator. Our data indicate that HTm4 is expressed in hematopoietic tissues and is tightly regulated during the differentiation of hematopoietic stem cells. It binds to cyclin-dependent kinase-associated (CDK-associated) phosphatase-CDK2 (KAP-CDK2) complexes, and the three proteins demonstrate similar patterns of cellular expression in human lymphoid tissues. HTm4 stimulates the phosphatase activity of KAP, and its C-terminal region is required for binding to KAP-CDK2 complexes and the modulation of KAP activity. Overexpression of HTm4 can cause cell cycle arrest at the G(0)/G(1) phase. Thus, HTm4 is a novel hematopoietic modulator for the G(1)-S cell cycle transition. 2001
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Cohen K, Scadden DT. 2001. Non-Hodgkin's lymphoma: pathogenesis, clinical presentation, and treatment. Cancer treatment and research. 104:201-30. Pubmed: 11191128 Cohen K, Scadden DT. 2001. Non-Hodgkin's lymphoma: pathogenesis, clinical presentation, and treatment. Cancer treatment and research. 104:201-30. Pubmed: 11191128 -
Scadden DT, Shen H, Cheng T. 2001. Hematopoietic stem cells in HIV disease. Journal of the National Cancer Institute. Monographs. Pubmed: 11158203 Scadden DT, Shen H, Cheng T. 2001. Hematopoietic stem cells in HIV disease. Journal of the National Cancer Institute. Monographs. Pubmed: 11158203 The hematopoietic stem cell has long been hypothesized to be a target of human immunodeficiency virus type-1 (HIV) infection that limits the potential for compensatory immune cell production. Data have recently emerged documenting stem cell dysfunction in HIV disease and indicating that immune recovery from potent antiretroviral therapy is partly driven by new T-cell generation. Effects of HIV on stem cell physiology, however, appear to be indirect, as stem cells are highly resistant to HIV infection. Despite the presence of surface receptors for HIV, the hematopoietic stem cell is not infectible with HIV. However, stem transduction can be achieved with HIV constructs in which the envelope glycoproteins have been replaced by vesicular stomatitis virus G protein. Therefore, hematopoietic stem cells are likely participants in HIV-related cytopenias, but they are spared direct infection and can serve as a resource for cellular therapies for AIDS. -
Poznansky MC, Olszak IT, Foxall RB, Piascik A, Adams GB, Evans RH, Cheng T, Scadden DT. 2001. Tissue source dictates lineage outcome of human fetal CD34(+)CD38(-) cells. Experimental hematology. 29(6):766-74. Pubmed: 11378272 Poznansky MC, Olszak IT, Foxall RB, Piascik A, Adams GB, Evans RH, Cheng T, Scadden DT. 2001. Tissue source dictates lineage outcome of human fetal CD34(+)CD38(-) cells. Experimental hematology. 29(6):766-74. Pubmed: 11378272 Array -
Brander C, O'Connor P, Suscovich T, Jones NG, Lee Y, Kedes D, Ganem D, Martin J, Osmond D, Southwood S, Sette A, Walker BD, Scadden DT. 2001. Definition of an optimal cytotoxic T lymphocyte epitope in the latently expressed Kaposi's sarcoma-associated herpesvirus kaposin protein. The Journal of infectious diseases. 184(2):119-26. Pubmed: 11424007 Brander C, O'Connor P, Suscovich T, Jones NG, Lee Y, Kedes D, Ganem D, Martin J, Osmond D, Southwood S, Sette A, Walker BD, Scadden DT. 2001. Definition of an optimal cytotoxic T lymphocyte epitope in the latently expressed Kaposi's sarcoma-associated herpesvirus kaposin protein. The Journal of infectious diseases. 184(2):119-26. Pubmed: 11424007 Cytotoxic T lymphocytes (CTL) recognize and kill virus-infected cells and contribute to immunologic control of viral replication. For many herpesviruses (e.g., Epstein-Barr and cytomegalovirus), virus-specific CTL responses can be readily detected in infected persons, but CTL responses against Kaposi's sarcoma-associated herpesvirus (KSHV) appear to be weak and remain poorly characterized. Using a human leukocyte antigen (HLA) binding motif-based epitope prediction algorithm, we identified 37 HLA-A*0201 binding peptides from 8 KSHV open-reading frames (ORFs). After in vitro stimulation of peripheral blood mononuclear cells from KSHV-infected persons, CTL responses against 1 peptide in the KSHV kaposin protein (ORF K12) were detected in 2 HLA-A*0201-positive subjects. The optimal CTL epitope was identified by HLA restriction analysis and peptide titration assays. These data describe a latent phase viral gene product targeted by CTL that may be relevant for KSHV immunopathogenesis. -
Christmas P, Jones JP, Patten CJ, Rock DA, Zheng Y, Cheng SM, Weber BM, Carlesso N, Scadden DT, Rettie AE, Soberman RJ. 2001. Alternative splicing determines the function of CYP4F3 by switching substrate specificity. The Journal of biological chemistry. 276(41):38166-72. Pubmed: 11461919 Christmas P, Jones JP, Patten CJ, Rock DA, Zheng Y, Cheng SM, Weber BM, Carlesso N, Scadden DT, Rettie AE, Soberman RJ. 2001. Alternative splicing determines the function of CYP4F3 by switching substrate specificity. The Journal of biological chemistry. 276(41):38166-72. Pubmed: 11461919 Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB4 by omega-hydroxylation (Km = 0.68 microm) but has low activity for arachidonic acid (Km = 185 microm); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for omega-hydroxylation (Km = 22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes. -
Cheng T, Shen H, Rodrigues N, Stier S, Scadden DT. 2001. Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21(Cip1/Waf1) or p27(Kip1). Blood. 98(13):3643-9. Pubmed: 11739168 Cheng T, Shen H, Rodrigues N, Stier S, Scadden DT. 2001. Transforming growth factor beta 1 mediates cell-cycle arrest of primitive hematopoietic cells independent of p21(Cip1/Waf1) or p27(Kip1). Blood. 98(13):3643-9. Pubmed: 11739168 The regulation of stem cell proliferation is a poorly understood process balancing rapid, massive blood cell production in times of stress with maintenance of a multipotent stem cell pool over decades of life. Transforming growth factor beta 1 (TGF-beta 1) has pleiotropic effects on hematopoietic cells, including the inhibition of primitive cell proliferation. It was recently demonstrated that the cyclin-dependent kinase inhibitors, p21(Cip1/Waf1) (p21) and p27(Kip1) (p27), can inhibit the proliferation of hematopoietic stem cells and progenitor cells, respectively. The relation of TGF-beta 1 stimulation to p21 and p27 was examined using a fine-mapping approach to gene expression in individual cells. Abundant TGF-beta 1 expression and p21 expression were documented in quiescent, cytokine-resistant hematopoietic stem cells and in terminally differentiated mature blood cells, but not in proliferating progenitor cell populations. TGF-beta 1 receptor (T beta R II) was expressed ubiquitously without apparent modulation. Cell- cycle-synchronized 32D cells exposed to TGF-beta 1 demonstrated a marked antiproliferative effect of TGF-beta 1, yet neither the level of p21 mRNA nor the protein level of either p21 or p27 was altered. To corroborate these observations in primary cells, bone marrow mononuclear cells derived from mice engineered to be deficient in p21 or p27 were assessed. Progenitor and primitive cell function was inhibited by TGF-beta 1 equivalently in -/- and +/+ littermate controls. These data indicate that TGF-beta 1 exerts its inhibition on cell cycling independent of p21 and p27 in hematopoietic cells. TGF-beta 1 and p21 or p27 participate in independent pathways of stem cell regulation, suggesting that targeting each may provide complementary strategies for enhancing stem or progenitor cell expansion and gene transduction. -
Levine AM, Scadden DT, Zaia JA, Krishnan A. 2001. Hematologic Aspects of HIV/AIDS. Hematology. American Society of Hematology. Education Program. Pubmed: 11722999 Levine AM, Scadden DT, Zaia JA, Krishnan A. 2001. Hematologic Aspects of HIV/AIDS. Hematology. American Society of Hematology. Education Program. Pubmed: 11722999 This review addresses various aspects of HIV infection pertinent to hematology, including the consequences of HIV infection on specific aspects of hematopoiesis and an update on the current biologic, epidemiologic and therapeutic aspects of AIDS-related lymphoma and Hodgkin's disease. The results of the expanding use of progenitor cell transplantation in HIV infected patients are also reviewed. In Section I, Dr. Scadden reviews the basis for HIV dysregulation of blood cell production, focusing on the role of the stem cell in HIV disease. T cell production and thymic function are discussed, with emphasis placed upon the mechanisms of immune restoration in HIV infected individuals. Results of clinical and correlative laboratory studies are presented. In Section II, Dr. Levine reviews the recent epidemiologic trends in the incidence of lymphoma, since the widespread availability of highly active anti-retroviral therapy (HAART). The biologic aspects of AIDS-lymphoma and Hodgkin's disease are discussed in terms of pathogenesis of disease. Various treatment options for these disorders and the role of concomitant anti-retroviral and chemotherapeutic intervention are addressed. Drs. Zaia and Krishnan will review the area of stem cell transplantation in patients with AIDS related lymphoma, presenting updated information on clinical results of this procedure. Additionally, they report on the use of gene therapy, with peripheral blood CD34+ cells genetically modified using a murine retrovirus, as a means to treat underlying HIV infection. Results of gene transfer experiments and subsequent gene marking in HIV infected patients are reviewed. -
Ratner L, Lee J, Tang S, Redden D, Hamzeh F, Herndier B, Scadden D, Kaplan L, Ambinder R, Levine A, Harrington W, Grochow L, Flexner C, Tan B, Straus D. 2001. Chemotherapy for human immunodeficiency virus-associated non-Hodgkin's lymphoma in combination with highly active antiretroviral therapy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 19(8):2171-8. Pubmed: 11304769 Ratner L, Lee J, Tang S, Redden D, Hamzeh F, Herndier B, Scadden D, Kaplan L, Ambinder R, Levine A, Harrington W, Grochow L, Flexner C, Tan B, Straus D. 2001. Chemotherapy for human immunodeficiency virus-associated non-Hodgkin's lymphoma in combination with highly active antiretroviral therapy. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 19(8):2171-8. Pubmed: 11304769 Array -
Shen H, Cheng T, Olszak I, Garcia-Zepeda E, Lu Z, Herrmann S, Fallon R, Luster AD, Scadden DT. 2001. CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells. Journal of immunology (Baltimore, Md. : 1950). 166(8):5027-33. Pubmed: 11290783 Shen H, Cheng T, Olszak I, Garcia-Zepeda E, Lu Z, Herrmann S, Fallon R, Luster AD, Scadden DT. 2001. CXCR-4 desensitization is associated with tissue localization of hemopoietic progenitor cells. Journal of immunology (Baltimore, Md. : 1950). 166(8):5027-33. Pubmed: 11290783 The chemokine stroma-derived factor (SDF)-1, and its receptor, CXCR-4, have been shown to be essential for the translocation of hemopoietic stem cells from the fetal liver to the bone marrow (BM). We hypothesized that if CXCR-4 plays a crucial role in the localization of human hemopoiesis, stem cells from distinct tissue sources should demonstrate distinct CXCR-4 expression or signaling profiles. CD34(+) cells from BM were compared with blood: either mobilized peripheral blood or umbilical cord blood. Unexpectedly, significantly higher levels of CXCR-4 surface expression on CD34(+) cells from blood sources, mobilized peripheral blood, or cord blood were observed compared with BM (p = 0.0005 and p = 0.002, respectively). However, despite lower levels of CXCR-4, responsiveness of the cells to SDF-1 as measured by either calcium flux or transmigration was proportionally greatest in cells derived from BM. Further, internalization of CXCR-4 in response to ligand, associated with receptor desensitization, was significantly lower on BM-derived cells. Therefore, preserved chemokine receptor signaling was highly associated with marrow rather than blood localization. To test the functional effects of perturbing CXCR-4 signaling, adult mice were exposed to the methionine-SDF-1beta analog that induces prolonged down-regulation/desensitization of CXCR-4 and observed mobilization of Lin(-), Sca-1(+), Thy-1(low), and c-kit(+) hemopoietic progenitor cells to the peripheral blood with a >30-fold increase compared with PBS control (p = 0.0007 day 1 and p = 0.004 day 2). These data demonstrate that CXCR-4 expression and function can be dissociated in progenitor cells and that desensitization of CXCR-4 induces stem cell entry into the circulation. -
Ellisen LW, Carlesso N, Cheng T, Scadden DT, Haber DA. 2001. The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells. The EMBO journal. 20(8):1897-909. Pubmed: 11296223 Ellisen LW, Carlesso N, Cheng T, Scadden DT, Haber DA. 2001. The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells. The EMBO journal. 20(8):1897-909. Pubmed: 11296223 WT1, a transcription factor implicated in both normal kidney differentiation and tumorigenesis, is also expressed in differentiating hematopoietic progenitors. Most human acute leukemias contain high levels of the wild-type transcript, while a minority have point mutations, raising the possibility that this tumor suppressor might have a paradoxical oncogenic effect in some hematopoietic cells. Using high titer retroviral infection, we demonstrate that WT1 triggers rapid growth arrest and lineage-specific differentiation in primary hematopoietic progenitors and differentiation-competent leukemia cell lines, while it induces cellular quiescence in a primitive subset of primary precursors. Growth arrest by WT1 is associated with induction of p21(CIP1), but expression of this cyclin-dependent kinase inhibitor alone is insufficient for either cellular differentiation or primitive cell preservation. The effects of WT1 are enhanced by co-expression of its naturally occurring isoforms, and are correlated with the physiological expression pattern of WT1 in vivo. Our observations suggest a role for WT1 in the differentiation of human hematopoietic cells, and provide a functional model that supports its capacity as a tumor suppressor in human acute leukemia. 2000
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Cheng T, Rodrigues N, Dombkowski D, Stier S, Scadden DT. 2000. Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nature medicine. 6(11):1235-40. Pubmed: 11062534 Cheng T, Rodrigues N, Dombkowski D, Stier S, Scadden DT. 2000. Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nature medicine. 6(11):1235-40. Pubmed: 11062534 Sustained blood cell production requires preservation of a quiescent, multipotential stem cell pool that intermittently gives rise to progenitors with robust proliferative potential. The ability of cells to shift from a highly constrained to a vigorously active proliferative state is critical for maintaining stem cells while providing the responsiveness necessary for host defense. The cyclin-dependent kinase inhibitor (CDKI), p21(cip1/waf1) (p21) dominates stem cell kinetics. Here we report that another CDKI, p27(kip1) (p27), does not affect stem cell number, cell cycling, or self-renewal, but markedly alters progenitor proliferation and pool size. Therefore, distinct CDKIs govern the highly divergent stem and progenitor cell populations. When competitively transplanted, p27-deficient stem cells generate progenitors that eventually dominate blood cell production. Modulating p27 expression in a small number of stem cells may translate into effects on the majority of mature cells, thereby providing a strategy for potentiating the impact of transduced cells in stem cell gene therapy. -
Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT. 2000. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science (New York, N.Y.). 287(5459):1804-8. Pubmed: 10710306 Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT. 2000. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science (New York, N.Y.). 287(5459):1804-8. Pubmed: 10710306 Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of stem cells has been conjectured to be of critical biologic importance in protecting the stem cell compartment, which we directly assessed using mice engineered to be deficient in the G1 checkpoint regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic stem cell proliferation and absolute number were increased under normal homeostatic conditions. Exposing the animals to cell cycle-specific myelotoxic injury resulted in premature death due to hematopoietic cell depletion. Further, self-renewal of primitive cells was impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death. -
Podestà M, Zocchi E, Pitto A, Usai C, Franco L, Bruzzone S, Guida L, Bacigalupo A, Scadden DT, Walseth TF, De Flora A, Daga A. 2000. Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 14(5):680-90. Pubmed: 10744625 Podestà M, Zocchi E, Pitto A, Usai C, Franco L, Bruzzone S, Guida L, Bacigalupo A, Scadden DT, Walseth TF, De Flora A, Daga A. 2000. Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 14(5):680-90. Pubmed: 10744625 Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells. -
Tulpule A, Scadden DT, Espina BM, Cabriales S, Howard W, Shea K, Gill PS. 2000. Results of a randomized study of IM862 nasal solution in the treatment of AIDS-related Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 18(4):716-23. Pubmed: 10673512 Tulpule A, Scadden DT, Espina BM, Cabriales S, Howard W, Shea K, Gill PS. 2000. Results of a randomized study of IM862 nasal solution in the treatment of AIDS-related Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 18(4):716-23. Pubmed: 10673512 Array -
Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R. 2000. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nature biotechnology. 18(4):410-4. Pubmed: 10748521 Lewin M, Carlesso N, Tung CH, Tang XW, Cory D, Scadden DT, Weissleder R. 2000. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nature biotechnology. 18(4):410-4. Pubmed: 10748521 The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells. -
Poznansky MC, Olszak IT, Foxall R, Evans RH, Luster AD, Scadden DT. 2000. Active movement of T cells away from a chemokine. Nature medicine. 6(5):543-8. Pubmed: 10802710 Poznansky MC, Olszak IT, Foxall R, Evans RH, Luster AD, Scadden DT. 2000. Active movement of T cells away from a chemokine. Nature medicine. 6(5):543-8. Pubmed: 10802710 Movement towards or away from a given stimulus guides the directional migration of prokaryotes, simple eukaryotes and neurons. As bi-directional cues may influence entry and exit of immune effector cells from tissue sites, we evaluated the migratory responses of T-cell subsets to varying concentrations of the chemokine stromal cell derived factor-1 (SDF-1). There was selective repulsion of subpopulations of T cells at high concentrations of recombinant SDF-1 or naturally occurring bone marrow-derived SDF-1, which could be inhibited by pertussis toxin and antibody against the chemokine receptor CXCR4. Distinct sensitivity profiles to genistein, herbimycin and 8-Br-cAMP biochemically distinguished movement of cells towards or away from an SDF-1 gradient. In vivo, antigen-induced T-cell recruitment into the peritoneal cavity was reversed by high but not low concentrations of SDF-1. The phenomenon of movement away from a chemokine represents a previously unknown mechanism regulating the localization of mature T cells. It adds to the functional repertoire of chemokines that may participate in immune physiology and may be applied therapeutically to alter the immune response. -
Brander C, Suscovich T, Lee Y, Nguyen PT, O'Connor P, Seebach J, Jones NG, van Gorder M, Walker BD, Scadden DT. 2000. Impaired CTL recognition of cells latently infected with Kaposi's sarcoma-associated herpes virus. Journal of immunology (Baltimore, Md. : 1950). 165(4):2077-83. Pubmed: 10925292 Brander C, Suscovich T, Lee Y, Nguyen PT, O'Connor P, Seebach J, Jones NG, van Gorder M, Walker BD, Scadden DT. 2000. Impaired CTL recognition of cells latently infected with Kaposi's sarcoma-associated herpes virus. Journal of immunology (Baltimore, Md. : 1950). 165(4):2077-83. Pubmed: 10925292 Kaposi's sarcoma-associated herpes virus (KSHV) is a recently identified human gamma2-herpesvirus associated with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease. We reasoned that CTL responses may provide host defense against this virus, and consequently, KSHV may have evolved strategies to evade the CTL-mediated immune surveillance. In this study six B cell lines latently infected with KSHV were found to express reduced levels of HLA class I surface molecules compared with B cell lines transformed by the related gamma-herpesvirus EBV. KSHV-infected cells also required higher concentrations of soluble peptides to induce efficient CTL-mediated lysis than control cell lines and were unable to process and/or present intracellularly expressed Ag. Incubation of the KSHV-infected cell lines with high concentrations of soluble HLA class I binding peptides did not restore the deficient HLA class I surface expression. To assess the underlying mechanisms of these phenomena, TAP-1 and TAP-2 gene expression was analyzed. While no attenuation in TAP-2 expression was observed, TAP-1 expression was significantly reduced in all KSHV cell lines compared with that in controls. These results indicate that KSHV can modulate HLA class I-restricted Ag presentation to CTL, which may allow latently infected cells to escape CTL recognition and persist in the infected host. -
Eppich HM, Foxall R, Gaynor K, Dombkowski D, Miura N, Cheng T, Silva-Arrieta S, Evans RH, Mangano JA, Preffer FI, Scadden DT. 2000. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants. Nature biotechnology. 18(8):882-7. Pubmed: 10932160 Eppich HM, Foxall R, Gaynor K, Dombkowski D, Miura N, Cheng T, Silva-Arrieta S, Evans RH, Mangano JA, Preffer FI, Scadden DT. 2000. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants. Nature biotechnology. 18(8):882-7. Pubmed: 10932160 Purging of tumor cells and selection of stem cells are key technologies for enabling stem cell transplantation and stem cell gene therapy. Here we report a strategy for cell selection based on physical properties of the cells. Exposing cells to an external pulsed electric field (PEF) increases the natural potential difference across the cell membrane until a critical threshold is reached and pore formation occurs, resulting in fatal perturbation of cell physiology. Attaining this threshold is a function of the applied field intensity and cell size, with larger cells porated at lower field intensities than smaller cells. Since hematopoietic stem cells are smaller than other hematopoietic cells and tumor cells, we found that exposure of peripheral blood mononuclear cells (PBMCs) to PEFs caused stepwise elimination of monocytes without affecting the function of smaller lymphocyte populations. Mobilized peripheral blood exposed to PEFs was enriched for CD34+/CD38- cells and stem cell function was preserved. Furthermore, PEF treatment was able to selectively purge blood preparations of tumor cells and eradicate transplantable tumor. -
Scadden DT. 2000. Epstein-Barr virus, the CNS, and AIDS-related lymphomas: as close as flame to smoke. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 18(19):3323-4. Pubmed: 11013270 Scadden DT. 2000. Epstein-Barr virus, the CNS, and AIDS-related lymphomas: as close as flame to smoke. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 18(19):3323-4. Pubmed: 11013270 -
Poznansky MC, Evans RH, Foxall RB, Olszak IT, Piascik AH, Hartman KE, Brander C, Meyer TH, Pykett MJ, Chabner KT, Kalams SA, Rosenzweig M, Scadden DT. 2000. Efficient generation of human T cells from a tissue-engineered thymic organoid. Nature biotechnology. 18(7):729-34. Pubmed: 10888839 Poznansky MC, Evans RH, Foxall RB, Olszak IT, Piascik AH, Hartman KE, Brander C, Meyer TH, Pykett MJ, Chabner KT, Kalams SA, Rosenzweig M, Scadden DT. 2000. Efficient generation of human T cells from a tissue-engineered thymic organoid. Nature biotechnology. 18(7):729-34. Pubmed: 10888839 Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency. -
Mitsuyasu RT, Anton PA, Deeks SG, Scadden DT, Connick E, Downs MT, Bakker A, Roberts MR, June CH, Jalali S, Lin AA, Pennathur-Das R, Hege KM. 2000. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood. 96(3):785-93. Pubmed: 10910888 Mitsuyasu RT, Anton PA, Deeks SG, Scadden DT, Connick E, Downs MT, Bakker A, Roberts MR, June CH, Jalali S, Lin AA, Pennathur-Das R, Hege KM. 2000. Prolonged survival and tissue trafficking following adoptive transfer of CD4zeta gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects. Blood. 96(3):785-93. Pubmed: 10910888 We have genetically engineered CD4(+) and CD8(+) T cells with human immunodeficiency virus (HIV) specificity by inserting a gene, CD4zeta, containing the extracellular domain of human CD4 (which binds HIV env) linked to the zeta (zeta) chain of the T-cell receptor (which mediates T-cell activation). Twenty-four HIV-positive subjects received a single infusion of 2 to 3 x 10(10) autologous CD4zeta-modified CD4(+) and CD8(+) T cells administered with (n = 11) or without (n = 13) interleukin-2 (IL-2). Subjects had CD4 counts greater than 50/microL and viral loads of at least 1000 copies/mL at entry. T cells were costimulated ex vivo through CD3 and CD28 and expanded for approximately 2 weeks. CD4zeta was detected in 1% to 3% of blood mononuclear cells at 8 weeks and 0.1% at 1 year after infusion, and survival was not enhanced by IL-2. Trafficking of gene-modified T cells to bulk rectal tissue and/or isolated lamina propria lymphocytes was documented in a subset of 5 of 5 patients at 14 days and 2 of 3 at 1 year. A greater than 0.5 log mean decrease in rectal tissue-associated HIV RNA was observed for at least 14 days, suggesting compartmental antiviral activity of CD4zeta T cells. CD4(+) counts increased by 73/microL at 8 weeks in the group receiving IL-2. There was no significant mean change in plasma HIV RNA or blood proviral DNA in either treatment arm. This sustained, high-level persistence of gene-modified T cells demonstrates the feasibility of ex vivo T-cell gene therapy in HIV-infected adults and suggests the importance of providing HIV-specific T-helper function. -
Duvic M, Friedman-Kien AE, Looney DJ, Miles SA, Myskowski PL, Scadden DT, Von Roenn J, Galpin JE, Groopman J, Loewen G, Stevens V, Truglia JA, Yocum RC. 2000. Topical treatment of cutaneous lesions of acquired immunodeficiency syndrome-related Kaposi sarcoma using alitretinoin gel: results of phase 1 and 2 trials. Archives of dermatology. 136(12):1461-9. Pubmed: 11115156 Duvic M, Friedman-Kien AE, Looney DJ, Miles SA, Myskowski PL, Scadden DT, Von Roenn J, Galpin JE, Groopman J, Loewen G, Stevens V, Truglia JA, Yocum RC. 2000. Topical treatment of cutaneous lesions of acquired immunodeficiency syndrome-related Kaposi sarcoma using alitretinoin gel: results of phase 1 and 2 trials. Archives of dermatology. 136(12):1461-9. Pubmed: 11115156 Array -
Olszak IT, Poznansky MC, Evans RH, Olson D, Kos C, Pollak MR, Brown EM, Scadden DT. 2000. Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo. The Journal of clinical investigation. 105(9):1299-305. Pubmed: 10792005 Olszak IT, Poznansky MC, Evans RH, Olson D, Kos C, Pollak MR, Brown EM, Scadden DT. 2000. Extracellular calcium elicits a chemokinetic response from monocytes in vitro and in vivo. The Journal of clinical investigation. 105(9):1299-305. Pubmed: 10792005 Recruitment of macrophages to sites of cell death is critical for induction of an immunologic response. Calcium concentrations in extracellular fluids vary markedly, and are particularly high at sites of injury or infection. We hypothesized that extracellular calcium participates in modulating the immune response, perhaps acting via the seven-transmembrane calcium-sensing receptor (CaR) on mature monocytes/macrophages. We observed a dose-dependent increase in monocyte chemotaxis in response to extracellular calcium or the selective allosteric CaR activator NPS R-467. In contrast, monocytes derived from mice deficient in CaR lacked the normal chemotactic response to a calcium gradient. Notably, CaR activation of monocytes bearing the receptor synergistically augmented the transmigration response of monocytes to the chemokine MCP-1 in association with increased cell-surface expression of its cognate receptor, CCR2. Conversely, stimulation of monocytes with MCP-1 or SDF-1alpha reciprocally increased CaR expression, suggesting a dual-enhancing interaction of Ca(2+) with chemokines in recruiting inflammatory cells. Subcutaneous administration in mice of Ca(2+), MCP-1, or (more potently) the combination of Ca(2+) and MCP-1, elicited an inflammatory infiltrate consisting of monocytes/macrophages. Thus extracellular calcium functions as an ionic chemokinetic agent capable of modulating the innate immune response in vivo and in vitro by direct and indirect actions on monocytic cells. Calcium deposition may be both consequence and cause of chronic inflammatory changes at sites of injury, infection, and atherosclerosis. -
Evans RH, Scadden DT. 2000. Haematological aspects of HIV infection. Bailliere's best practice & research. Clinical haematology. 13(2):215-30. Pubmed: 10942622 Evans RH, Scadden DT. 2000. Haematological aspects of HIV infection. Bailliere's best practice & research. Clinical haematology. 13(2):215-30. Pubmed: 10942622 Multiple interacting factors contribute to the haematological manifestations of HIV disease. The effects of HIV-1 infection influence all haemopoietic cell lineages resulting in a spectrum of haematological abnormalities. Even in the absence of other pathological processes, bone marrow morphology is invariably abnormal, and anaemia, neutropenia and thrombocytopenia are all common during the course of disease. Intercurrent opportunistic infections may cause bone marrow suppression or induce specific cytopenias. Therapies used to treat HIV and its complications are frequently implicated as the cause of haematological dysfunction, and many have significant myelotoxic side-effects. Insights into the molecular basis for many of these abnormalities have permitted a clearer understanding of the pathophysiology of HIV-1 infection. Recombinant human growth factors that may be used to treat isolated cytopenias or to ameliorate the myelotoxic effects of other essential therapies. Lymph opoietic growth factors and the use of gene modified cells provide future therapeutic strategies that may alter the course of HIV disease.Copyright 2000 Harcourt Publishers Ltd. -
O'Connor PG, Scadden DT. 2000. AIDS oncology. Infectious disease clinics of North America. 14(4):945-65, vii. Pubmed: 11144646 O'Connor PG, Scadden DT. 2000. AIDS oncology. Infectious disease clinics of North America. 14(4):945-65, vii. Pubmed: 11144646 Kaposi's sarcoma, non-Hodgkin's lymphoma, Hodgkin's disease, and squamous cell carcinoma are among the malignancies seen with increased frequency in patients infected with HIV. The outlook for patients with these malignancies has improved significantly with the utilization of highly active antiretroviral therapy (HAART) and more aggressive cytotoxic therapies. Novel biologic therapies with lesser side effects are currently being evaluated. This article reviews the current knowledge about HIV malignancies, their epidemiology, pathogenesis, clinical manifestations, and treatment. -
Whelan P, Scadden DT. 2000. New developments in the etiopathogenesis and treatment of HIV-related Kaposi's sarcoma. Clinics in dermatology. 18(4):469-77. Pubmed: 11024314 Whelan P, Scadden DT. 2000. New developments in the etiopathogenesis and treatment of HIV-related Kaposi's sarcoma. Clinics in dermatology. 18(4):469-77. Pubmed: 11024314 1999
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Scadden DT. 1999. Immune reconstitution in AIDS: oncologic implications and hematologic approaches. Current opinion in oncology. 11(6):503-7. Pubmed: 10550015 Scadden DT. 1999. Immune reconstitution in AIDS: oncologic implications and hematologic approaches. Current opinion in oncology. 11(6):503-7. Pubmed: 10550015 Combination anti-retroviral therapy for HIV disease has profoundly altered the nature of the AIDS epidemic. Mitigating the impact of an uncontrollable decline in immune function is no longer the focal point for AIDS therapy, but has evolved to an emphasis on maximizing the potential for immune regeneration. Improved control of HIV replication has diminished, albeit unevenly, the frequency of AIDS-related malignancies and has altered the focus of hematologic and oncologic interventions in HIV disease. Now, with adoptive cellular therapies and the genetic engineering of cells in the clinical arena, the potential for cellular therapeutics in enhancing immune restoration is being tested. These approaches are based on better understanding of the immunobiology of HIV and its impact on hematopoietic tissues. -
Poznansky MC, La Vecchio J, Silva-Arietta S, Porter-Brooks J, Brody K, Olszak IT, Adams GB, Ramstedt U, Marasco WA, Scadden DT. 1999. Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat. Human gene therapy. 10(15):2505-14. Pubmed: 10543615 Poznansky MC, La Vecchio J, Silva-Arietta S, Porter-Brooks J, Brody K, Olszak IT, Adams GB, Ramstedt U, Marasco WA, Scadden DT. 1999. Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells and monocytes derived from CD34+ cells transduced with an intracellular antibody directed against human immunodeficiency virus type 1 Tat. Human gene therapy. 10(15):2505-14. Pubmed: 10543615 Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4+ cell count <400 cells/mm3). We examined the efficiency of transduction and transgene expression in adult bone marrow (BM)- and umbilical cord blood (UCB)-derived CD34+ cells induced to differentiate into T cells and monocytes in vitro with an MuLV-based vector encoding the neomycin resistance gene and an intracellular antibody directed against the Tat protein of HIV-1 (sFvtat1-Ckappa). The expression of the marker gene and the effects of antiviral construct on subsequent challenge with monocytotropic and T cell-tropic HIV-1 isolates were monitored in vitro in purified T cells and monocytes generated in culture from the transduced CD34+ cells. Transduction efficiencies of CD34+ cells ranged between 22 and 27%. Differentiation of CD34+ cells into T cells or monocytes was not significantly altered by the transduction process. HIV-1 replication in monocytes and CD4+ T cells derived from CD34+ cells transduced with the intracellular antibody gene was significantly reduced in comparison with the degree of HIV replication seen in monocytes and CD4+ T cells derived from CD34+ cells transduced with the neomycin resistance gene alone. Further, T cells and monocytes derived from CD34+ cells transduced with the intracellular antibody gene were demonstrated to express the sFvtat1-Ckappa transgene by RT-PCR and had a selective growth advantage in cultures that had been challenged with HIV-1. These data demonstrate that sFvtat1-Ckappa inhibits HIV-1 replication in T cells and monocytes developing from CD34+ cells and supports the continuing development of a stem cell gene therapy for the treatment of HIV-1 infection. -
Gill PS, Tulpule A, Espina BM, Cabriales S, Bresnahan J, Ilaw M, Louie S, Gustafson NF, Brown MA, Orcutt C, Winograd B, Scadden DT. 1999. Paclitaxel is safe and effective in the treatment of advanced AIDS-related Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 17(6):1876-83. Pubmed: 10561228 Gill PS, Tulpule A, Espina BM, Cabriales S, Bresnahan J, Ilaw M, Louie S, Gustafson NF, Brown MA, Orcutt C, Winograd B, Scadden DT. 1999. Paclitaxel is safe and effective in the treatment of advanced AIDS-related Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 17(6):1876-83. Pubmed: 10561228 Array -
Shen H, Cheng T, Preffer FI, Dombkowski D, Tomasson MH, Golan DE, Yang O, Hofmann W, Sodroski JG, Luster AD, Scadden DT. 1999. Intrinsic human immunodeficiency virus type 1 resistance of hematopoietic stem cells despite coreceptor expression. Journal of virology. 73(1):728-37. Pubmed: 9847379 Shen H, Cheng T, Preffer FI, Dombkowski D, Tomasson MH, Golan DE, Yang O, Hofmann W, Sodroski JG, Luster AD, Scadden DT. 1999. Intrinsic human immunodeficiency virus type 1 resistance of hematopoietic stem cells despite coreceptor expression. Journal of virology. 73(1):728-37. Pubmed: 9847379 Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1. -
Turnbull JL, Patchen ML, Scadden DT. 1999. The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines. Acta haematologica. 102(2):66-71. Pubmed: 10529508 Turnbull JL, Patchen ML, Scadden DT. 1999. The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines. Acta haematologica. 102(2):66-71. Pubmed: 10529508 beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.5 ng/ml), PGG-glucan significantly increased BMMC myeloid colony formation comparable to the increase observed with either interleukin-3 (rhIL-3) or stem cell factor (rhSCF). Moreover, the addition of PGG-glucan to cultures containing GM-CSF + IL-3 or GM-CSF + SCF significantly augmented granulocyte-macrophage colony production above baseline, demonstrating that PGG-glucan acts independently of those early-acting cytokines and can enhance their activity in an additive manner. Anti-PGG-glucan monoclonal antibody specifically abrogated the growth-enhancing effect of added PGG-glucan in a saturable manner and other control carbohydrate polymers failed to affect colony formation. Further, PGG-glucan was not associated with induction of IL-6, GM-CSF production and removal of accessory cells by CD34+ cell isolation did not alter the PGG-glucan effect. These data demonstrate that PGG-glucan acts on committed myeloid progenitors to enhance human hematopoietic activity by a mechanism of direct action independent of IL-3 or SCF and independent of secondary cytokine stimulation. -
Carlesso N, Aster JC, Sklar J, Scadden DT. 1999. Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. Blood. 93(3):838-48. Pubmed: 9920832 Carlesso N, Aster JC, Sklar J, Scadden DT. 1999. Notch1-induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. Blood. 93(3):838-48. Pubmed: 9920832 Hematopoiesis is a balance between proliferation and differentiation that may be modulated by environmental signals. Notch receptors and their ligands are highly conserved during evolution and have been shown to regulate cell fate decisions in multiple developmental systems. To assess whether Notch1 signaling may regulate human hematopoiesis to maintain cells in an immature state, we transduced a vesicular stomatitis virus G-protein (VSV-G) pseudo-typed bicistronic murine stem cell virus (MSCV)-based retroviral vector expressing a constitutively active form of Notch1 (ICN) and green fluorescence protein into the differentiation competent HL-60 cell line and primary cord blood-derived CD34(+) cells. In addition, we observed endogenous Notch1 expression on the surface of both HL-60 cells and primary CD34(+) cells, and therefore exposed cells to Notch ligand Jagged2, expressed on NIH3T3 cells. Both ligand-independent and ligand-dependent activation of Notch resulted in delayed acquisition of differentiation markers by HL-60 cells and cord blood CD34(+) cells. In addition, primary CD34(+) cells retained their ability to form immature colonies, colony-forming unit-mix (CFU-mix), whereas control cells lost this capacity. Activation of Notch1 correlated with a decrease in the fraction of HL-60 cells that were in G0/G1 phase before acquisition of a mature cell phenotype. This enhanced progression through G1 was noted despite preservation of the proliferative rate of the cells and the overall length of the cell cycle. These findings show that Notch1 activation delays human hematopoietic differentiation and suggest a link of Notch differentiation effects with altered cell cycle kinetics. -
Schwartz JD, Howard W, Scadden DT. 1999. Potential interaction of antiretroviral therapy with paclitaxel in patients with AIDS-related Kaposi's sarcoma. AIDS (London, England). 13(2):283-4. Pubmed: 10202837 Schwartz JD, Howard W, Scadden DT. 1999. Potential interaction of antiretroviral therapy with paclitaxel in patients with AIDS-related Kaposi's sarcoma. AIDS (London, England). 13(2):283-4. Pubmed: 10202837 -
Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes M. 1999. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. Journal of immunology (Baltimore, Md. : 1950). 162(6):3402-7. Pubmed: 10092795 Nikolic B, Gardner JP, Scadden DT, Arn JS, Sachs DH, Sykes M. 1999. Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC. Journal of immunology (Baltimore, Md. : 1950). 162(6):3402-7. Pubmed: 10092795 The induction of T cell tolerance is likely to play an essential role in successful xenotransplantation in humans. In this study, we show that porcine thymus grafts in immunodeficient mice support normal development of polyclonal, functional human T cells. These T cells were specifically tolerant to MHC Ags of the porcine thymus donor and responded to nondonor porcine xenoantigens and alloantigens. Exogenous IL-2 did not abolish tolerance, suggesting central clonal deletion rather than anergy as the likely tolerance mechanism. Our study suggests that the thymic transplantation approach to achieving tolerance with restoration of immunocompetence may be applicable to xenotransplantation of pig tissues to humans. -
Cavacini LA, Wisnewski A, Peterson JE, Montefiori D, Emes C, Duval M, Kingsbury G, Wang A, Scadden D, Posner MR. 1999. A human anti-HIV autoantibody enhances EBV transformation and HIV infection. Clinical immunology (Orlando, Fla.). 93(3):263-73. Pubmed: 10600338 Cavacini LA, Wisnewski A, Peterson JE, Montefiori D, Emes C, Duval M, Kingsbury G, Wang A, Scadden D, Posner MR. 1999. A human anti-HIV autoantibody enhances EBV transformation and HIV infection. Clinical immunology (Orlando, Fla.). 93(3):263-73. Pubmed: 10600338 A highly specific, human IgG mAb, F223, which reacts with both HIV-1-infected cells and uninfected lymphoid cells, has been derived. F223 reacts with gp120 but fails to neutralize viral infection. The antibody does enhance HIV-1 infection in a complement-dependent manner. The autoantigen recognized by F223 is expressed on a small percentage of T cells and NK cells and the majority of B cells. Immunoprecipitation demonstrates F223 reactivity with an as of yet unidentified 159-kDa protein in uninfected lymphoid cells. This reactivity with uninfected cells is inhibited by free gp120 demonstrating the cross-reactive nature of this antibody. The F223 light chain demonstrates strong homology to VLlambda2 family genes whereas the heavy chain is most homologous (84%) to the germline gene VH3-H.11. In vivo usage of VH3 family genes by F223 and an anti-HIV-1 (gp41) human mAb, 3D6, with related autoreactivity, suggests that VH3 sequences may be important components of potentially pathogenic human anti-HIV-1 envelope autoantibodies. F223 was isolated from an HIV-1 infected individual with lymphoma and in vitro F223 significantly enhances EBV transformation of normal B cells and increases immunoglobulin production without affecting B cell proliferation. Characterization of this antibody response may provide important insights and mechanistic information on HIV pathogenesis.Copyright 1999 Academic Press. -
Adams GB, McMullen M, Turner S, Olszak IT, Scadden DT, McClure MO, Poznansky MC. 1999. Isolation and transduction of CD34+ cells from small quantities of peripheral blood from HIV-1-infected patients not treated with hemopoietic growth factors. Journal of acquired immune deficiency syndromes (1999). 21(1):1-8. Pubmed: 10235508 Adams GB, McMullen M, Turner S, Olszak IT, Scadden DT, McClure MO, Poznansky MC. 1999. Isolation and transduction of CD34+ cells from small quantities of peripheral blood from HIV-1-infected patients not treated with hemopoietic growth factors. Journal of acquired immune deficiency syndromes (1999). 21(1):1-8. Pubmed: 10235508 A proposed hemopoietic stem cell gene therapy for treatment for HIV infection would involve transduction of CD34+ hemopoietic stem cells with vectors encoding anti-HIV constructs. Peripheral blood has proved to be a useful source of these hemopoietic stem cells and this study exploits this finding. Small quantities of peripheral blood were obtained from HIV-negative patients and HIV-positive patients who were and were not receiving hemopoietic growth factors (HGFs). CD34+ cells were obtained from these samples using a simple technique and scored for frequency of colony type. This demonstrated that HIV-negative patients had the highest frequency of colony-forming units (CFUs). HIV-positive patients not treated with HGFs had a lower frequency of CFUs, but the same colony type distribution as HIV-negative patients. HIV-positive patients treated with HGFs had the lowest frequency of CFUs, but their colony type distribution demonstrated that they had responded to treatment. CD34+ cells selected in this way were also transduced with the murine retroviral MFG vector using a technique that demonstrated transduction efficiencies ranging from 2% to 16% (median, 11.5%). This study simplifies the experimental requirements for development of a hemopoietic stem cell gene therapy for HIV infection and offers the possibility that longitudinal studies could be performed on peripheral blood CD34+ cells from HIV-positive or HIV-negative patients without the need for granulocyte colony-stimulating factor mobilization. 1998
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Gardner JP, Rosenzweig M, Marks DF, Harper D, Gaynor K, Fallon RJ, Wall DA, Johnson RP, Scadden DT. 1998. T-lymphopoietic capacity of cord blood-derived CD34+ progenitor cells. Experimental hematology. 26(10):991-9. Pubmed: 9728935 Gardner JP, Rosenzweig M, Marks DF, Harper D, Gaynor K, Fallon RJ, Wall DA, Johnson RP, Scadden DT. 1998. T-lymphopoietic capacity of cord blood-derived CD34+ progenitor cells. Experimental hematology. 26(10):991-9. Pubmed: 9728935 Umbilical cord blood contains an abundance of CD34+ hematopoietic progenitor cells and has been used in transplantation as an alternative to adult bone marrow or mobilized peripheral blood. Although efficient myelomonocytic, erythroid, and B lymphoid differentiation has been shown in highly purified cord blood CD34+ mononuclear cells lacking expression of lineage-specific antigens, generation of functional T cells has not been previously documented. Exploiting two recently developed, complementary thymic stromal monolayer systems, we show here that immature hematopoietic progenitor cells (CD34+lineage-) from human and rhesus monkey cord blood mononuclear cells undergo T lymphopoiesis in a manner that recapitulates T cell ontogeny in vivo. After 2 weeks of proliferation, cultures contained myeloid [corrected] cells and discrete populations of CD4+CD8+ (double-positive) immature T lymphocytes, followed after an additional 2 weeks by the appearance of single-positive CD4+CD8- and CD4-CD8+ T cells that coexpressed CD3. The T lymphoid phenotype was confirmed at the transcriptional level by the presence of the lymphoid-restricted genes RAG-2 and T cell receptor. T cells generated from cord blood progenitors in these systems exhibited immunofunction as assessed by alloreactive responses in mixed lymphocyte reactions. These findings were comparable between human and rhesus progenitor cells and closely resemble previous data using adult bone marrow CD34+ cells in these models. Together, these observations demonstrate that cord blood contains abundant lymphoid progenitors that undergo T lymphopoiesis in vitro, suggesting the full multipotentiality of this stem cell source and its validity in investigating T lymphoid differentiation. -
Tulpule A, Yung RC, Wernz J, Espina BM, Myers A, Scadden DT, Cabriales S, Ilaw M, Boswell W, Gill PS. 1998. Phase II trial of liposomal daunorubicin in the treatment of AIDS-related pulmonary Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 16(10):3369-74. Pubmed: 9779714 Tulpule A, Yung RC, Wernz J, Espina BM, Myers A, Scadden DT, Cabriales S, Ilaw M, Boswell W, Gill PS. 1998. Phase II trial of liposomal daunorubicin in the treatment of AIDS-related pulmonary Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 16(10):3369-74. Pubmed: 9779714 Array -
Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, Tenen DG. 1998. CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Molecular and cellular biology. 18(7):4301-14. Pubmed: 9632814 Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, Tenen DG. 1998. CCAAT/enhancer binding protein alpha is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Molecular and cellular biology. 18(7):4301-14. Pubmed: 9632814 The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) regulates a number of myeloid cell-specific genes. To delineate the role of C/EBPalpha in human granulopoiesis, we studied its expression and function in human primary cells and bipotential (granulocytic/monocytic) myeloid cell lines. We show that the expression of C/EBPalpha initiates with the commitment of multipotential precursors to the myeloid lineage, is specifically upregulated during granulocytic differentiation, and is rapidly downregulated during the alternative monocytic pathway. Conditional expression of C/EBPalpha alone in stably transfected bipotential cells triggers neutrophilic differentiation, concomitant with upregulation of the granulocyte-specific granulocyte colony-stimulating factor receptor and secondary granule protein genes. Moreover, induced expression of C/EBPalpha in bipotential precursors blocks their monocytic differentiation program. These results indicate that C/EBPalpha serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes. -
Scadden DT, Schenkein DP, Bernstein Z, Luskey B, Doweiko J, Tulpule A, Levine AM. 1998. Immunotoxin combined with chemotherapy for patients with AIDS-related non-Hodgkin's lymphoma. Cancer. 83(12):2580-7. Pubmed: 9874466 Scadden DT, Schenkein DP, Bernstein Z, Luskey B, Doweiko J, Tulpule A, Levine AM. 1998. Immunotoxin combined with chemotherapy for patients with AIDS-related non-Hodgkin's lymphoma. Cancer. 83(12):2580-7. Pubmed: 9874466 Array -
Yamaguchi T, Olozak I, Chattopadhyay N, Butters RR, Kifor O, Scadden DT, Brown EM. 1998. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes. Biochemical and biophysical research communications. 246(2):501-6. Pubmed: 9610391 Yamaguchi T, Olozak I, Chattopadhyay N, Butters RR, Kifor O, Scadden DT, Brown EM. 1998. Expression of extracellular calcium (Ca2+o)-sensing receptor in human peripheral blood monocytes. Biochemical and biophysical research communications. 246(2):501-6. Pubmed: 9610391 The calcium-sensing receptor (CaR) is a G protein-coupled receptor playing key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone turnover and may play a role in the "reversal" phase of skeletal remodeling that follows osteoclastic resorption and precedes osteoblastic bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for such mononuclear cells present locally within the bone marrow microenvironment. Indeed, previous studies by other investigators have shown that raising Ca2+o either in vivo or in vitro stimulated the release of interleukin-6 (IL-6) from human peripheral blood monocytes, suggesting that these cells express a Ca2+o-sensing mechanism. In these earlier studies, however, the use of reverse transcription-polymerase chain reaction (RT-PCR) failed to detect transcripts for the CaR previously cloned from parathyroid and kidney in peripheral blood monocytes. Since we recently found that non-specific esterase-positive, putative monocytes isolated from murine bone marrow express the CaR, we reevaluated the expression of this receptor in human peripheral blood monocytes. Immunocytochemistry, flow cytometry, and Western blot analysis, performed using a polyclonal antiserum specific for the CaR, detected CaR protein in human monocytes. In addition, the use of RT-PCR with CaR-specific primers, followed by nucleotide sequencing of the amplified products, identified CaR transcripts in the cells. Therefore, taken together, our data show that human peripheral blood monocytes possess both CaR protein and mRNA very similar if not identical to those expressed in parathyroid and kidney that could mediate the previously described, direct effects of Ca2+o on these cells. Furthermore, since mononuclear cells isolated from bone marrow also express the CaR, the latter might play some role in the "reversal" phase of bone remodeling, sensing local changes in Ca2+o resulting from osteoclastic bone resorption and secreting osteotropic cytokines or performing other Ca2+o-regulated functions that contribute to the control of bone turnover. -
Wu CJ, Scadden DT. 1998. Posttransplant Kaposi's sarcoma treated with paclitaxel. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 16(10):3478-9. Pubmed: 9779728 Wu CJ, Scadden DT. 1998. Posttransplant Kaposi's sarcoma treated with paclitaxel. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 16(10):3478-9. Pubmed: 9779728 -
Cabriales S, Bresnahan J, Testa D, Espina BM, Scadden DT, Ross M, Gill PS. 1998. Extravasation of liposomal daunorubicin in patients with AIDS-associated Kaposi's sarcoma: a report of four cases. Oncology nursing forum. 25(1):67-70. Pubmed: 9460774 Cabriales S, Bresnahan J, Testa D, Espina BM, Scadden DT, Ross M, Gill PS. 1998. Extravasation of liposomal daunorubicin in patients with AIDS-associated Kaposi's sarcoma: a report of four cases. Oncology nursing forum. 25(1):67-70. Pubmed: 9460774 Array -
Scadden DT, Howard WW. 1998. AIDS-Related Malignancies. The oncologist. 3(2):119-123. Pubmed: 10388093 Scadden DT, Howard WW. 1998. AIDS-Related Malignancies. The oncologist. 3(2):119-123. Pubmed: 10388093 The changing face of the AIDS epidemic since the introduction of protease inhibitors has had a significant impact on AIDS-related malignancies. These issues, emerging concepts regarding pathophysiology and current treatment strategies, are the subject of this review. -
Scadden DT. 1998. Mother knows best. Nature medicine. 4(4):390-1. Pubmed: 9546778 Scadden DT. 1998. Mother knows best. Nature medicine. 4(4):390-1. Pubmed: 9546778 -
Scadden DT. 1998. AIDS-related malignancies. Current opinion in oncology. 10(5):403-4. Pubmed: 9800109 Scadden DT. 1998. AIDS-related malignancies. Current opinion in oncology. 10(5):403-4. Pubmed: 9800109 -
Jones D, Ballestas ME, Kaye KM, Gulizia JM, Winters GL, Fletcher J, Scadden DT, Aster JC. 1998. Primary-effusion lymphoma and Kaposi's sarcoma in a cardiac-transplant recipient. The New England journal of medicine. 339(7):444-9. Pubmed: 9700178 Jones D, Ballestas ME, Kaye KM, Gulizia JM, Winters GL, Fletcher J, Scadden DT, Aster JC. 1998. Primary-effusion lymphoma and Kaposi's sarcoma in a cardiac-transplant recipient. The New England journal of medicine. 339(7):444-9. Pubmed: 9700178 1997
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Levine AM, Tulpule A, Tessman D, Kaplan L, Giles F, Luskey BD, Scadden DT, Northfelt DW, Silverberg I, Wernz J, Espina B, Von Hoff D. 1997. Mitoguazone therapy in patients with refractory or relapsed AIDS-related lymphoma: results from a multicenter phase II trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 15(3):1094-103. Pubmed: 9060550 Levine AM, Tulpule A, Tessman D, Kaplan L, Giles F, Luskey BD, Scadden DT, Northfelt DW, Silverberg I, Wernz J, Espina B, Von Hoff D. 1997. Mitoguazone therapy in patients with refractory or relapsed AIDS-related lymphoma: results from a multicenter phase II trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 15(3):1094-103. Pubmed: 9060550 Array -
Scadden DT. 1997. Cytokine use in the management of HIV disease. Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association. 16 Suppl 1:S23-9. Pubmed: 9389312 Scadden DT. 1997. Cytokine use in the management of HIV disease. Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association. 16 Suppl 1:S23-9. Pubmed: 9389312 Multilineage hematopoietic defects occur in patients with human immunodeficiency virus (HIV) infection and affect therapy of the disease and of associated opportunistic infections and neoplasms. Anemia and neutropenia are common in HIV patients, and can occur as a result of HIV-related myelosuppression or complications or may be secondary effects of antiretroviral or other agents used in management of the disease. With the advent of combination drug therapy for the treatment of HIV infection and prophylaxis and treatment of infectious complications, myelosuppression is frequently encountered and may be treated with synthetic hematopoietic growth factors. Erythropoietin has been shown to increase mean hematocrit levels and to reduce transfusion requirements in anemic HIV-infected patients receiving zidovudine. Granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor have been shown to increase neutrophil counts in patients with AIDS-related bone marrow failure and those receiving zidovudine, interferon-alpha, or ganciclovir. Although recent research using interleukin-2 (IL-2) has shown that use of this cytokine in AIDS patients can lead to increases in CD4 cell counts that appear to be functional, further study is needed to determine whether cytokines can play a role other than palliation in HIV-infected patients. -
Gardner JP, Zhu H, Colosi PC, Kurtzman GJ, Scadden DT. 1997. Robust, but transient expression of adeno-associated virus-transduced genes during human T lymphopoiesis. Blood. 90(12):4854-64. Pubmed: 9389702 Gardner JP, Zhu H, Colosi PC, Kurtzman GJ, Scadden DT. 1997. Robust, but transient expression of adeno-associated virus-transduced genes during human T lymphopoiesis. Blood. 90(12):4854-64. Pubmed: 9389702 Recombinant adeno-associated viruses (rAAV) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. We evaluated rAAV vector containing a LacZ reporter gene under the control of a cytomegalovirus (CMV) promoter in the context of primary human CD34+CD2- progenitor cells induced to undergo T-cell differentiation using an in vitro T-lymphopoiesis system. Target cells from either adult bone marrow or umbilical cord blood were efficiently transduced, and 71% to 79% CD2+ cells expressed a LacZ marker gene mRNA and produced LacZ-encoded protein after exposure to rAAV-CMV-LacZ. The impact of transgene expression on the differentiation of T cells was assessed by sequential quantitation of immunophenotypic subsets of virus-exposed cells and no alteration was noted compared with control. The durability of transgene expression was assessed and found to decay by day 35 with kinetics dependent on the multiplicity of infection. In addition, vector DNA was absent from CD4 or CD8 subselected CD3+ cells by DNA-polymerase chain reaction. These data suggest that rAAV vectors may result in robust transgene expression in primitive cells undergoing T-cell lineage commitment without toxicity or alteration in the pattern of T-cell differentiation. However, expression is transient and integration of the transgene unlikely. Recombinant AAV vectors are potentially valuable gene transfer tools for the genetic manipulation of events during T-cell ontogony but their potential in gene therapy strategies for diseases such as acquired immunodeficiency syndrome is limited. 1996
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Scadden DT, Pickus O, Hammer SM, Stretcher B, Bresnahan J, Gere J, McGrath J, Agosti JM. 1996. Lack of in vivo effect of granulocyte-macrophage colony-stimulating factor on human immunodeficiency virus type 1. AIDS research and human retroviruses. 12(12):1151-9. Pubmed: 8844019 Scadden DT, Pickus O, Hammer SM, Stretcher B, Bresnahan J, Gere J, McGrath J, Agosti JM. 1996. Lack of in vivo effect of granulocyte-macrophage colony-stimulating factor on human immunodeficiency virus type 1. AIDS research and human retroviruses. 12(12):1151-9. Pubmed: 8844019 Neutropenia complicates HIV disease or its treatment in a large proportion of patients. Hematopoietic growth factor support has been tested in a number of clinical settings in HIV disease and has been demonstrated to be of benefit for specific parameters. One consideration regarding the use of hematopoietic growth factors in HIV disease is their potential effect on HIV viral burden, since alterations in HIV expression have been documented with certain cytokines in vitro. It has also been reported that some cytokines, notably GM-CSF, potentiate the antiviral properties of thymidine analogs such as zidovudine (AZT) in vitro. We tested these observations in vivo. Twelve HIV-positive patients with a CD4 cell count < or = 200/mm3 or HIV plasma viremia who were receiving a stable dose of zidovudine were enrolled into three dose cohorts of yeast-derived GM-CSF at 50, 125, or 250 micrograms/m2 daily by subcutaneous self-injection for 28 days. Measurements of HIV activity included serum acid-dissociated HIV p24 antigen levels, plasma and peripheral blood mononuclear cell (PBMC) limiting dilution HIV culture, and plasma HIV quantitative competitive polymerase chain reaction (PCR). Serum and intracellular zidovudine levels were measured as well as hematologic, immunologic, and toxicity parameters. Virologic measures showed neither significant upregulation nor downregulation of serum acid-dissociated HIV p24 antigen, plasma and PBMC HIV culture, or PCR in association with GM-CSF administration. A trend toward increased intracellular AZT levels was noted, but this did not achieve statistical significance (p = 0.073). CD4 and CD8 lymphocytes were essentially unaffected while absolute neutrophil counts increased with GM-CSF administration as expected. These data suggest that administration of GM-CSF does not perturb HIV activity or immunologic parameters in patients receiving AZT for advanced HIV disease. No potentiation of AZT antiviral effect was demonstrated. -
Scadden DT. 1996. Hematologic disorders and growth factor support in HIV infection. Hematology/oncology clinics of North America. 10(5):1149-61. Pubmed: 8880202 Scadden DT. 1996. Hematologic disorders and growth factor support in HIV infection. Hematology/oncology clinics of North America. 10(5):1149-61. Pubmed: 8880202 During the course of HIV disease a broad spectrum of hematologic disorders develop including abnormalities in blood cell generation, survival, and function Alterations in coagulation parameters may evolve associated with disruption of immunoglobulin or factor production. This article reviews the manifestations and pathophysiology of these abnormalities and discusses the role for growth factor support. -
Rosenzweig M, Marks DF, Zhu H, Hempel D, Mansfield KG, Sehgal PK, Kalams S, Scadden DT, Johnson RP. 1996. In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells. Blood. 87(10):4040-8. Pubmed: 8639759 Rosenzweig M, Marks DF, Zhu H, Hempel D, Mansfield KG, Sehgal PK, Kalams S, Scadden DT, Johnson RP. 1996. In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells. Blood. 87(10):4040-8. Pubmed: 8639759 Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG-2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome. -
Freedman AR, Zhu H, Levine JD, Kalams S, Scadden DT. 1996. Generation of human T lymphocytes from bone marrow CD34+ cells in vitro. Nature medicine. 2(1):46-51. Pubmed: 8564838 Freedman AR, Zhu H, Levine JD, Kalams S, Scadden DT. 1996. Generation of human T lymphocytes from bone marrow CD34+ cells in vitro. Nature medicine. 2(1):46-51. Pubmed: 8564838 Analysis of the events that regulate development of red blood cells or granulocytes has led to therapies altering clinical conditions associated with anemia or neutropenia. The development of therapeutic approaches to target conditions associated with lymphopenia, such as AIDS, has been thwarted by limited techniques for studying T-lymphocyte development. We describe an in vitro system in which human bone marrow CD34+ cells proliferate, acquire the expression of the lymphoid-specific RAG-2 gene and a broad repertoire of rearranged T-cell receptor genes, develop the ability to produce T cell-specific interleukin-2 and achieve a range of T-cell immunophenotypes. The cells also become susceptible to infection with the T-lymphotropic strain of human immunodeficiency virus-1, HIV-1IIIB. This culture system induces human T lymphopoiesis and may permit further analysis of the events regulating human T-lineage differentiation. It provides a preclinical model for screening stem cell gene therapies directed toward AIDS. -
Gill PS, Wernz J, Scadden DT, Cohen P, Mukwaya GM, von Roenn JH, Jacobs M, Kempin S, Silverberg I, Gonzales G, Rarick MU, Myers AM, Shepherd F, Sawka C, Pike MC, Ross ME. 1996. Randomized phase III trial of liposomal daunorubicin versus doxorubicin, bleomycin, and vincristine in AIDS-related Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 14(8):2353-64. Pubmed: 8708728 Gill PS, Wernz J, Scadden DT, Cohen P, Mukwaya GM, von Roenn JH, Jacobs M, Kempin S, Silverberg I, Gonzales G, Rarick MU, Myers AM, Shepherd F, Sawka C, Pike MC, Ross ME. 1996. Randomized phase III trial of liposomal daunorubicin versus doxorubicin, bleomycin, and vincristine in AIDS-related Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 14(8):2353-64. Pubmed: 8708728 Array -
Franken M, Estabrooks A, Cavacini L, Sherburne B, Wang F, Scadden DT. 1996. Epstein-Barr virus-driven gene therapy for EBV-related lymphomas. Nature medicine. 2(12):1379-82. Pubmed: 8946840 Franken M, Estabrooks A, Cavacini L, Sherburne B, Wang F, Scadden DT. 1996. Epstein-Barr virus-driven gene therapy for EBV-related lymphomas. Nature medicine. 2(12):1379-82. Pubmed: 8946840 Genetic alterations in malignant tissues are potential targets for gene-based cancer therapies. Alternatively, aberrant expression of certain specific genes associated with malignant transformation may be envisioned to enhance the expression of chemosensitizing drugs. Epstein-Barr virus (EBV)-related B-cell lymphomas are fatal complications of immunosuppression due to AIDS, organ transplantation or congenital immune abnormalities. The malignant cells latently infected with EBV typically express the transcription factor EBNA2 as one of nine latent viral genes. We tested whether an EBNA2-responsive EBV promoter may selectively target EBV-related lymphoma cells by virus-regulated expression of a suicide gene. Using the BamC promoter driving a hygromycin-thymidine kinase fusion gene or controls, we demonstrated that sensitivity to ganciclovir was selectively enhanced in cells expressing EBNA2. Further, there was complete macroscopic regression of established B-cell lymphomas in mice with severe combined immunodeficiency disease (SCID mice) treated with a single course of ganciclovir. These data provide in vitro and in vivo support for a model of exploiting the molecular basis of tumor development to enhance the specificity of gene therapy. -
Cheng T, Shen H, Giokas D, Gere J, Tenen DG, Scadden DT. 1996. Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells. Proceedings of the National Academy of Sciences of the United States of America. 93(23):13158-63. Pubmed: 8917561 Cheng T, Shen H, Giokas D, Gere J, Tenen DG, Scadden DT. 1996. Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells. Proceedings of the National Academy of Sciences of the United States of America. 93(23):13158-63. Pubmed: 8917561 A hierarchical order of gene expression has been proposed to control developmental events in hematopoiesis, but direct demonstration of the temporal relationships between regulatory gene expression and differentiation has been difficult to achieve. We modified a single-cell PCR method to detect 2-fold changes in mRNA copies per cell (dynamic range, 250-250,000 copies/cell) and used it to sequentially quantitate gene expression levels as single primitive (CD34+,CD38-) progenitor cells underwent differentiation to become erythrocytes, granulocytes, or monocyte/macrophages. Markers of differentiation such as CD34 or cytokine receptor mRNAs and transcription factors associated with their regulation were assessed. All transcription factors tested were expressed in multipotent progenitors. During lineage-specific differentiation, however, distinct patterns of expression emerged. SCL, GATA-2, and GATA-1 expression sequentially extinguished during erythroid differentiation. PU.1, AML1B, and C/EBP alpha expression profiles and their relationship to cytokine receptor expression in maturing granulocytes could be distinguished from similar profiles in monocytic cells. These data characterize the dynamics of gene expression accompanying blood cell development and define a signature gene expression pattern for specific stages of hematopoietic differentiation. 1995
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Columbyova L, Loda M, Scadden DT. 1995. Thrombopoietin receptor expression in human cancer cell lines and primary tissues. Cancer research. 55(16):3509-12. Pubmed: 7627956 Columbyova L, Loda M, Scadden DT. 1995. Thrombopoietin receptor expression in human cancer cell lines and primary tissues. Cancer research. 55(16):3509-12. Pubmed: 7627956 c-mpl is the receptor for the recently identified megakaryocyte growth and differentiation factor thrombopoietin. Thrombopoietin has been shown to be capable of raising platelet counts in animals and is about to enter clinical trials in humans. In anticipation of its likely use in the care of patients receiving cancer chemotherapy, we evaluated the expression of human c-mpl by reverse transcription PCR on 39 human cell lines and 20 primary human tissue samples derived from both normal and malignant sources. c-mpl transcripts were found in all megakaryocytic cell lines tested (CMK, CMK-2B, CMK-2D, SO, and DAMI), the CD34+ leukemia cell line KMT-2, and a hepatocellular carcinoma cell line (Hep3B). Among primary tissues, fetal liver cells and brain had detectable levels of c-mpl message, and among primary tumors, none were found to express c-mpl. These data support the conclusion that c-mpl has restricted expression that is primarily, but not exclusively, related to megakaryocytopoiesis. These observations suggest that thrombopoietin is unlikely to have direct effects on other malignant or normal tissue should it have a clinical role in the treatment of chemotherapy-induced thrombocytopenia. -
Berardi AC, Wang A, Abraham J, Scadden DT. 1995. Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells. Blood. 86(6):2123-9. Pubmed: 7662960 Berardi AC, Wang A, Abraham J, Scadden DT. 1995. Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells. Blood. 86(6):2123-9. Pubmed: 7662960 Basic fibroblast growth factor or fibroblast growth factor-2 (FGF) has been shown to affect myeloid cell proliferation and hypothesized to stimulate primitive hematopoietic cells. We sought to evaluate the effect of FGF on hematopoietic stem cells and to determine if FGF mediated its effects on progenitor cells directly or through the induction of other cytokines. To address the direct effects of FGF, we investigated whether FGF induced production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, IL-6, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor by two types of accessory cells, bone marrow (BM) fibroblasts and macrophages. We further evaluated whether antibodies to FGF-induced cytokines affected colony formation. To determine if FGF was capable of stimulating multipotent progenitors, we assessed the output of different colony types after stimulation of BM mononuclear cells (BMMC) or CD34+ BMMC and compared the effects of FGF with the stem cell active cytokine, kit ligand (KL). In addition, a subset of CD34+ BMMC with characteristics of hematopoietic stem cells was isolated by functional selection and their response to FGF was evaluated using proliferation, colony-forming, and single-cell polymerase chain reaction (PCR) assays. We determined that FGF had a stimulatory effect on the production of a single cytokine, IL-6, but that the effects of FGF on colony formation were not attributable to that induction. FGF was more restricted in its in vitro effects on BM progenitors than KL was, having no effect on erythroid colony formation. FGF did not stimulate stem cells and FGF receptors were not detected on stem cells as evaluated by single-cell reverse transcription PCR. In contrast, FGF receptor gene expression was detected in myeloid progenitor populations. These data support a directly mediated effect for FGF that appears to be restricted to lineage-committed myeloid progenitor cells. FGF does not appear to modulate the human hematopoietic stem cell. -
Price P, Johnson RP, Scadden DT, Jassoy C, Rosenthal T, Kalams S, Walker BD. 1995. Cytotoxic CD8+ T lymphocytes reactive with human immunodeficiency virus-1 produce granulocyte/macrophage colony-stimulating factor and variable amounts of interleukins 2, 3, and 4 following stimulation with the cognate epitope. Clinical immunology and immunopathology. 74(1):100-6. Pubmed: 7527747 Price P, Johnson RP, Scadden DT, Jassoy C, Rosenthal T, Kalams S, Walker BD. 1995. Cytotoxic CD8+ T lymphocytes reactive with human immunodeficiency virus-1 produce granulocyte/macrophage colony-stimulating factor and variable amounts of interleukins 2, 3, and 4 following stimulation with the cognate epitope. Clinical immunology and immunopathology. 74(1):100-6. Pubmed: 7527747 Infection with human immunodeficiency virus type 1 (HIV-1) induces vigorous and persistent cytotoxic CD8+ T cell responses. CTL clones were derived from peripheral blood or cerebrospinal fluid of three HIV-1 patients, with depressed CD4+ T cell counts. When stimulated with HLA-compatible target cells (B-LCL) presensitized with cognate HIV-1 peptides, all clones produced GM-CSF, TNF-alpha, and IFN-gamma and most produced low amounts of IL2, IL3, and IL4. After nonspecific stimulation with a phorbol ester and calcium ionophore, the clones secreted cytokines at levels similar to those from CD4+ lines from an HIV-1 infected donor. The ability of supernatants from the stimulated CTL clones to support the formation of granulocyte-macrophage colonies in normal bone marrow suggests that the GM-CSF was biologically active. Release of cytokines by activated CTL may influence the immunopathogenesis of HIV disease. -
Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. 1995. Functional isolation and characterization of human hematopoietic stem cells. Science (New York, N.Y.). 267(5194):104-8. Pubmed: 7528940 Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. 1995. Functional isolation and characterization of human hematopoietic stem cells. Science (New York, N.Y.). 267(5194):104-8. Pubmed: 7528940 Hematopoietic cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Human bone marrow cells that were responsive to the early-acting cytokines Kit ligand and interleukin-3 were forced to a metabolic death. The subfraction remaining represented 1 in 10(5) bone marrow mononuclear cells, were determined to be quiescent by cell cycle analysis, and had a stem cell immunophenotype. The cells were highly enriched for long-term culture-initiating cells, were capable of secondary colony formation, and produced both myeloid and lymphoid progeny. Thus, this technically simple strategy led to the efficient purification of cells with characteristics of hematopoietic stem cells. -
Scadden DT, Levine JD, Bresnahan J, Gere J, McGrath J, Wang Z, Resta DJ, Young D, Hammer SM. 1995. In vivo effects of interleukin 3 in HIV type 1-infected patients with cytopenia. AIDS research and human retroviruses. 11(6):731-40. Pubmed: 7576933 Scadden DT, Levine JD, Bresnahan J, Gere J, McGrath J, Wang Z, Resta DJ, Young D, Hammer SM. 1995. In vivo effects of interleukin 3 in HIV type 1-infected patients with cytopenia. AIDS research and human retroviruses. 11(6):731-40. Pubmed: 7576933 Array -
Masood R, Zhang Y, Bond MW, Scadden DT, Moudgil T, Law RE, Kaplan MH, Jung B, Espina BM, Lunardi-Iskandar Y. 1995. Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma. Blood. 85(12):3423-30. Pubmed: 7780129 Masood R, Zhang Y, Bond MW, Scadden DT, Moudgil T, Law RE, Kaplan MH, Jung B, Espina BM, Lunardi-Iskandar Y. 1995. Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma. Blood. 85(12):3423-30. Pubmed: 7780129 Interleukin-10 (IL-10) is an acid-sensitive protein of 35 kD that has pleiotropic effects including inhibition of cytotoxic T-cell response, induction of major histocompatibility complex type II in B lymphocytes, induction of B-cell growth and differentiation, and autocrine growth factor activity in monocytes. We and others have shown that IL-10 is produced spontaneously by blood mononuclear cells from human immunodeficiency virus-seropositive patients. In an attempt to ascertain the potential role of IL-10 in acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma, we evaluated the expression of human IL-10 in both tumor-derived B-cell lines and primary tumor cells. Expression of human IL-10 (hIL-10) mRNA and protein was detected in four of five cell lines examined. An IL-10 antisense oligonucleotide inhibited IL-10 mRNA expression and IL-10 protein production. The proliferation of all B-cell lines was inhibited by an antisense oligonucleotide in a dose-dependent manner that was abrogated by the addition of recombinant hIL-10 protein. No effect of antisense oligonucleotide was observed in the B-cell line not producing hIL-10. Evaluation of primary tumor cells from patients with AIDS-lymphoma cells showed similar production and response to IL-10. These data suggest an autocrine growth mechanism for IL-10 in AIDS-related lymphoma cells and that IL-10 may be important in its pathogenesis. 1994
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Scadden DT, Wang A, Zsebo KM, Groopman JE. 1994. In vitro effects of stem-cell factor or interleukin-3 on myelosuppression associated with AIDS. AIDS (London, England). 8(2):193-6. Pubmed: 7519020 Scadden DT, Wang A, Zsebo KM, Groopman JE. 1994. In vitro effects of stem-cell factor or interleukin-3 on myelosuppression associated with AIDS. AIDS (London, England). 8(2):193-6. Pubmed: 7519020 Array -
Freedman AR, Scadden DT. 1994. Viral activity in early HIV disease. Current opinion in hematology. 1(1):19-23. Pubmed: 9371255 Freedman AR, Scadden DT. 1994. Viral activity in early HIV disease. Current opinion in hematology. 1(1):19-23. Pubmed: 9371255 The commonly accepted paradigm for the natural history of HIV disease has been an acute burst of virus replication followed by years of viral quiescence. A terminal phase of virus activity is accompanied by gradual immunologic failure. Recent studies of the tissue localization of HIV and developments in virus quantitation have prompted a revision of this model. Viral latency has been shown to be only a relative concept by the demonstration of virus accumulation in tissue sites during early disease and quantifiable circulating levels of virus throughout the course of HIV infection. The primacy of HIV infection in AIDS has been further reaffirmed by these studies, but new questions as to its pathogenic effects have been raised. -
Avraham H, Banu N, Scadden DT, Abraham J, Groopman JE. 1994. Modulation of megakaryocytopoiesis by human basic fibroblast growth factor. Blood. 83(8):2126-32. Pubmed: 8161781 Avraham H, Banu N, Scadden DT, Abraham J, Groopman JE. 1994. Modulation of megakaryocytopoiesis by human basic fibroblast growth factor. Blood. 83(8):2126-32. Pubmed: 8161781 Basic fibroblast growth factor (bFGF) may act to modulate hematopoiesis in addition to its effects on mesenchymal cells. We studied the effects of bFGF on human and murine primary marrow megakaryocytes. bFGF modestly enhanced the size of the human megakaryocyte colony-forming unit (CFU-MK) and cell numbers per colony, in combination with interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF). Adhesion of human megakaryocytes to bone marrow (BM) stromal fibroblasts was enhanced when either stromal fibroblasts or megakaryocytes were treated with bFGF. This resulted in significantly increased proliferation of megakaryocytes. In addition, bFGF augmented secretion of the cytokines tumor necrosis factor alpha and IL-6 by human primary BM megakaryocytes. Immature murine megakaryocytes showed a significant growth response to bFGF as measured by the single cell growth assay. This effect was abrogated by specific antibodies for bFGF and combination of anti-IL-6 and anti-IL-1 beta antibodies. bFGF has no effect on murine CFU-MK formation, but significantly potentiated CFU-MK formation in the presence of IL-3 or GM-CSF. These results indicate that the effect of bFGF on various megakaryocyte populations is different and that bFGF may affect megakaryocytopoiesis via modulation of megakaryocyte-stromal interactions and via augmentation of cytokine secretion from megakaryocytes. -
Bennett BD, Wang Z, Kuang WJ, Wang A, Groopman JE, Goeddel DV, Scadden DT. 1994. Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily. The Journal of biological chemistry. 269(19):14211-8. Pubmed: 8188704 Bennett BD, Wang Z, Kuang WJ, Wang A, Groopman JE, Goeddel DV, Scadden DT. 1994. Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily. The Journal of biological chemistry. 269(19):14211-8. Pubmed: 8188704 Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development. -
Mark MR, Scadden DT, Wang Z, Gu Q, Goddard A, Godowski PJ. 1994. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain. The Journal of biological chemistry. 269(14):10720-8. Pubmed: 7511603 Mark MR, Scadden DT, Wang Z, Gu Q, Goddard A, Godowski PJ. 1994. rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain. The Journal of biological chemistry. 269(14):10720-8. Pubmed: 7511603 We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15. -
Gallo KA, Mark MR, Scadden DT, Wang Z, Gu Q, Godowski PJ. 1994. Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity. The Journal of biological chemistry. 269(21):15092-100. Pubmed: 8195146 Gallo KA, Mark MR, Scadden DT, Wang Z, Gu Q, Godowski PJ. 1994. Identification and characterization of SPRK, a novel src-homology 3 domain-containing proline-rich kinase with serine/threonine kinase activity. The Journal of biological chemistry. 269(21):15092-100. Pubmed: 8195146 Protein kinase play important roles in the growth and differentiation of cells. We have isolated cDNA clones from the human megakaryocytic cell line CMK11-5 that encode a novel protein kinase, which we call SPRK (src-homology 3 (SH3) domain-containing proline-rich kinase). The gene sequence predicts an 847-amino acid protein kinase with a unique domain arrangement. An amino-terminal glycine-rich region is followed by an SH3 domain and a kinase domain that is similar to both tyrosine and serine/threonine kinases. Adjacent to the kinase domain are two closely spaced leucine/isoleucine zipper motifs and a stretch of basic amino acids that resembles karyophilic nuclear localization signals. The COOH-terminal half of SPRK is basic, and proline accounts for 24% of the COOH-terminal 216 amino acids. The sprk gene is widely expressed as a 4-kilobase transcript in adult and fetal human tissues. Transfection of 293 cells with a vector encoding an epitope-tagged SPRK results in the expression of a 95-kDa protein. The epitope-tagged SPRK becomes phosphorylated on serine and threonine residues in an in vitro kinase assay, whereas SPRK variants with point mutations in the predicted ATP-binding site fail to become phosphorylated. These data indicate that SPRK has serine/threonine kinase activity. The SH3 domain of SPRK is interrupted by a unique 5-amino acid insert whose location in the SH3 consensus sequence is the same as that of the inserts found in the SH3 domains of neuronal SRC and of the p85 subunit of phosphatidylinositol 3-kinase. -
Lee J, Wang Z, Luoh SM, Wood WI, Scadden DT. 1994. Cloning of FRK, a novel human intracellular SRC-like tyrosine kinase-encoding gene. Gene. 138(1-2):247-51. Pubmed: 7510261 Lee J, Wang Z, Luoh SM, Wood WI, Scadden DT. 1994. Cloning of FRK, a novel human intracellular SRC-like tyrosine kinase-encoding gene. Gene. 138(1-2):247-51. Pubmed: 7510261 We report the cloning of a novel tyrosine kinase (TyK)-encoding gene (TYK) from the human hepatoma cell line Hep3B. Using the polymerase chain reaction (PCR) and oligodeoxyribonucleotide primers based on conserved TYK motifs, a 180-bp fragment was cloned and used to obtain full-length cDNA clones of 2.9 kb, with an open reading frame of 505 amino acids (aa). Restricted expression was detected by Northern blotting or reverse-transcribed PCR in a broad range of cell lines. The predicted aa sequence contains characteristic TyK motifs without a transmembrane region, suggesting an intracellular localization. There was 49% aa sequence identity with human FYN product and 47% with human SRC product; however, several structural differences distinguish this clone from other SRC subfamily members. This clone, FYN-related kinase or FRK, is a novel member of the intracellular TYK gene family. 1993
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Wang Z, Goldberg MA, Scadden DT. 1993. HIV-1 suppresses erythropoietin production in vitro. Experimental hematology. 21(5):683-8. Pubmed: 8390370 Wang Z, Goldberg MA, Scadden DT. 1993. HIV-1 suppresses erythropoietin production in vitro. Experimental hematology. 21(5):683-8. Pubmed: 8390370 Serum erythropoietin (Epo) levels are depressed in anemic AIDS patients relative to controls. The basis for abnormal regulation of Epo has not been defined. The hepatoma cell line Hep3B produces Epo in response to hypoxia and serves as a model for the study of Epo regulation. Hep3B cells are infectible with human immunodeficiency virus-1 (HIV-1) and were used as a model for evaluating potential direct effects of HIV on Epo expression. HIV-1 infected or transfected Hep3B cells produced Epo at significantly lower levels than uninfected Hep3B cells under low O2 conditions or following exposure to cobalt chloride. Epo production induced by hypoxia of HIV-1 infected Hep3B cells was depressed compared with non-HIV containing Hep3B cells when normalized for cell number, total cellular protein or albumin, but not depressed when normalized for alpha-fetoprotein production. The cellular levels of Epo mRNA were not diminished in the HIV-1+ Hep3B cells, indicating a probable posttranscriptional effect of HIV-1 on Epo production. Cellular protein production or secretion rates as measured by precipitable 3H-leucine were not affected by the presence of HIV-1. HIV-1 appeared to depress the production of Epo and some, but not all, other cellular proteins. These results suggest that impaired production of Epo may be a direct effect of HIV-1 infection possibly contributing to anemia in AIDS. -
Scadden DT, Agosti J. 1993. No antibodies to granulocyte macrophage colony-stimulating factor with prolonged use in AIDS. AIDS (London, England). 7(3):438. Pubmed: 8471211 Scadden DT, Agosti J. 1993. No antibodies to granulocyte macrophage colony-stimulating factor with prolonged use in AIDS. AIDS (London, England). 7(3):438. Pubmed: 8471211 1992
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Scadden DT. 1992. The clinical applications of colony-stimulating factors in acquired immunodeficiency syndrome. Seminars in hematology. 29(4 Suppl 3):33-7. Pubmed: 1492232 Scadden DT. 1992. The clinical applications of colony-stimulating factors in acquired immunodeficiency syndrome. Seminars in hematology. 29(4 Suppl 3):33-7. Pubmed: 1492232 -
Avraham H, Scadden DT, Chi S, Broudy VC, Zsebo KM, Groopman JE. 1992. Interaction of human bone marrow fibroblasts with megakaryocytes: role of the c-kit ligand. Blood. 80(7):1679-84. Pubmed: 1382698 Avraham H, Scadden DT, Chi S, Broudy VC, Zsebo KM, Groopman JE. 1992. Interaction of human bone marrow fibroblasts with megakaryocytes: role of the c-kit ligand. Blood. 80(7):1679-84. Pubmed: 1382698 Human kit ligand (KL), also known as stem cell factor (SCF), steel factor, or mast cell growth factor, is a recently identified hematopoietic growth factor whose receptor is the product of the c-kit proto-oncogene. Alternative splicing of the pre-mRNA of KL/SCF results in secreted and membrane-bound forms of the protein. We and others have recently shown that the c-kit gene product is expressed on human megakaryocytes and that soluble KL/SCF in combination with granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), or IL-6 increased megakaryocyte progenitor colony formation (CFU-MEG) and stimulated mature megakaryocytes. Here we show that adhesion of human megakaryocytes to bone marrow stromal fibroblasts, which express the membrane-bound form of KL/SCF (mKL/SCF), is mediated in part by the interaction between mKL/SCF and the c-kit protein. This interaction also results in marrow fibroblast-stimulated proliferation but not an increase in ploidy of megakaryocytes; when the two cell types were separated by a transoluble membrane, proliferation did not occur. Adhesion and proliferation of human megakaryocytes to an immortalized murine stromal cell line SI/SI lacking the KL/SCF gene was impaired, whereas transfection of SI/SI cells with human mKL/SCF significantly increased both adhesion and proliferation. Marrow stromal fibroblast mKL/SCF may serve both as an adhesion structure and as a growth-potentiating factor for megakaryocytes in the bone marrow. -
Scadden DT, Wang Z, Groopman JE. 1992. Quantitation of plasma human immunodeficiency virus type 1 RNA by competitive polymerase chain reaction. The Journal of infectious diseases. 165(6):1119-23. Pubmed: 1583331 Scadden DT, Wang Z, Groopman JE. 1992. Quantitation of plasma human immunodeficiency virus type 1 RNA by competitive polymerase chain reaction. The Journal of infectious diseases. 165(6):1119-23. Pubmed: 1583331 Clinical measures of human immunodeficiency virus (HIV) type 1 activity in vivo are limited and hinder the assessment of antiretroviral therapies. Reported here is a method for quantitating HIV-1 RNA in human plasma using the polymerase chain reaction (PCR). This method uses an internal cRNA standard generated from a cloned 113-bp deletion mutation of a highly conserved HIV-1 gag region sequence. The mutant cRNA (K4) was shown to amplify with efficiency equivalent to that of wild-type HIV-1. Known quantities of K4 cRNA added to wild-type HIV-1 in a competitive PCR strategy using a radiolabeled primer permitted quantitation of wild-type HIV-1 RNA over four orders of magnitude (10(3)-10(6) RNA copies). RNA isolated from plasma from AIDS patients yielded 10(3) to 8 x 10(4) HIV-1 RNA copies/ml of plasma with an average intrasample coefficient of variation of .26. This method offers a sensitive assay with a broad dynamic range for monitoring HIV-1 activity in the plasma of AIDS patients. It may provide a useful tool for assessing the effects of antiretroviral therapy. -
Scadden DT. 1992. The use of GM-CSF in AIDS. Infection. 20 Suppl 2:S103-6. Pubmed: 1283604 Scadden DT. 1992. The use of GM-CSF in AIDS. Infection. 20 Suppl 2:S103-6. Pubmed: 1283604 Hematopoietic growth factors may mitigate the cytopenias that frequently complicate HIV disease or its treatment. Clinical and in vitro studies have indicated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) or erythropoietin (EPO) to overcome the myelosuppression of HIV or many of the drug therapies used in the care of HIV-infected individuals. In addition, neutrophil or monocyte functional abnormalities observed in AIDS patients may be improved by the use of GM-CSF. Issues which may distinguish the use of hematopoietic growth factors in AIDS as compared with in other clinical settings include: 1) interaction of the growth factor with other cytokines which are aberrantly expressed, 2) direct effects of the growth factor on the replicative activity of HIV, and 3) potential interactions of the growth factor with other concurrently administered medications. This review focuses on the potential roles and limitations of growth factor use in AIDS and reviews the clinical studies using GM-CSF in HIV-infected individuals. 1991
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Scadden DT, Feder D, Groopman JE. 1991. Scratching the surface. Current biology : CB. 1(5):301-3. Pubmed: 15336104 Scadden DT, Feder D, Groopman JE. 1991. Scratching the surface. Current biology : CB. 1(5):301-3. Pubmed: 15336104 -
Scadden DT, Bering HA, Levine JD, Bresnahan J, Evans L, Epstein C, Groopman JE. 1991. Granulocyte-macrophage colony-stimulating factor mitigates the neutropenia of combined interferon alfa and zidovudine treatment of acquired immune deficiency syndrome-associated Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 9(5):802-8. Pubmed: 2016623 Scadden DT, Bering HA, Levine JD, Bresnahan J, Evans L, Epstein C, Groopman JE. 1991. Granulocyte-macrophage colony-stimulating factor mitigates the neutropenia of combined interferon alfa and zidovudine treatment of acquired immune deficiency syndrome-associated Kaposi's sarcoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 9(5):802-8. Pubmed: 2016623 The combined use of zidovudine (ZDV) and interferon (IFN) alfa-2a has been shown to have antiretroviral and antitumor potential benefit in the treatment of acquired immune deficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS). However, the clinical use of this combination is frequently complicated by the overlapping myelotoxicity of these agents. We report here the results of a phase I/II study in which granulocyte-macrophage colony-stimulating factor (GM-CSF) was used for those KS patients who became neutropenic while receiving ZDV (1,200 mg/d) and IFN (9 x 10(6) U/d). Nineteen of 29 patients (66%) developed an absolute neutrophil count (ANC) of less than 1,000 cells per cubic millimeter and were begun on GM-CSF. All experienced a prompt increase in the ANC. Those patients receiving GM-CSF/ZDV/IFN alfa-2a had an improved end of study ANC when compared with the ZDV/IFN alfa-2a group, but did not have an increased rate of tumor response, end of study CD4 cell count, or improvement in any other hematologic variable. The use of GM-CSF was not associated with increased toxicity and, in particular, was not associated with a change in serum human immunodeficiency virus (HIV) p24 antigen. Tumor response was noted in 50% of the assessable patients (33% overall) despite "high-risk" characteristics in 80%. Of the responding patients, seven were on GM-CSF and might have otherwise required an alteration in ZDV/IFN alfa-2a dose level. Further study of GM-CSF as an alternate to dose modification of this (ZDV/IFN alfa-2a) and other combination therapies for AIDS patients is warranted. -
Scadden DT, Bering HA, Levine JD, Bresnahan J, Evans L, Epstein C, Groopman JE. 1991. GM-CSF as an alternative to dose modification of the combination zidovudine and interferon-alpha in the treatment of AIDS-associated Kaposi's sarcoma. American journal of clinical oncology. 14 Suppl 1:S40-4. Pubmed: 2048563 Scadden DT, Bering HA, Levine JD, Bresnahan J, Evans L, Epstein C, Groopman JE. 1991. GM-CSF as an alternative to dose modification of the combination zidovudine and interferon-alpha in the treatment of AIDS-associated Kaposi's sarcoma. American journal of clinical oncology. 14 Suppl 1:S40-4. Pubmed: 2048563 Combined zidovudine (ZDV) and interferon-alpha (IFN) is an appealing therapy for AIDS-associated Kaposi's sarcoma because of the antiretroviral as well as antitumor potential of this combination. Overlapping myelotoxicity of these agents, however, frequently complicates their clinical use. This phase I/II study was undertaken to test the safety and efficacy of granulocyte-macrophage colony stimulating factor (GM-CSF) in those patients who became neutropenic while receiving ZDV (1,200 mg/day) and IFN (9 MU/day). Despite a "high-risk" population of patients, the tumor response rate among evaluable patients was 50% (33% overall). Sixty-four percent of patients required GM-CSF and all patients receiving GM-CSF had a prompt improvement in their absolute neutrophil count (ANC). The use of GM-CSF was associated with an improved end of study ANC (p less than 0.05), but was not associated with tumor response, CD4 count improvement, or improved change in hemoglobin concentration. GM-CSF/ZDV/IFN was not associated with increased toxicity over ZDV/IFN; however, two unusual events occurred in the GM-CSF/ZDV/IFN group: erythema multiforme and glucose intolerance. Dose-limiting thrombocytopenia and anemia were seen in two patients and anemia in one patient on GM-CSF/ZDV/IFN. No consistent alterations in serum HIV p24 antigenemia were noted in either group. The use of GM-CSF mitigated the neutropenia of combined ZDV and IFN. Further study evaluating the utility of this hematopoietic growth factor in combination therapies for AIDS patients is warranted. 1990
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Molina JM, Scadden DT, Sakaguchi M, Fuller B, Woon A, Groopman JE. 1990. Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus. Blood. 76(12):2476-82. Pubmed: 2265244 Molina JM, Scadden DT, Sakaguchi M, Fuller B, Woon A, Groopman JE. 1990. Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus. Blood. 76(12):2476-82. Pubmed: 2265244 The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV-1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV-1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells. -
Scadden DT, Zeira M, Woon A, Wang Z, Schieve L, Ikeuchi K, Lim B, Groopman JE. 1990. Human immunodeficiency virus infection of human bone marrow stromal fibroblasts. Blood. 76(2):317-22. Pubmed: 1695109 Scadden DT, Zeira M, Woon A, Wang Z, Schieve L, Ikeuchi K, Lim B, Groopman JE. 1990. Human immunodeficiency virus infection of human bone marrow stromal fibroblasts. Blood. 76(2):317-22. Pubmed: 1695109 The human immunodeficiency virus (HIV) preferentially infects CD4 positive T cells and monocytes. Other human cell types have been reported to be infectable with HIV, including cells of mesenchymal origin. In this report, we show that both primary human bone marrow stromal fibroblasts and an immortalized human stromal fibroblast line are susceptible to HIV infection. These cells are capable of passing HIV to cells of lymphoid or myeloid lineage, and may thereby act as a reservoir of virus. This in vitro system may be a useful model for assessing the pathophysiology of hematopoietic dysfunction in AIDS patients. -
Molina JM, Scadden DT, Amirault C, Woon A, Vannier E, Dinarello CA, Groopman JE. 1990. Human immunodeficiency virus does not induce interleukin-1, interleukin-6, or tumor necrosis factor in mononuclear cells. Journal of virology. 64(6):2901-6. Pubmed: 2335821 Molina JM, Scadden DT, Amirault C, Woon A, Vannier E, Dinarello CA, Groopman JE. 1990. Human immunodeficiency virus does not induce interleukin-1, interleukin-6, or tumor necrosis factor in mononuclear cells. Journal of virology. 64(6):2901-6. Pubmed: 2335821 The production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) by fresh peripheral blood mononuclear cells was evaluated after exposure to human immunodeficiency virus (HIV) or purified recombinant HIV-1 envelope glycoprotein (rgp120). To exclude the role of contaminating endotoxin in this study, all media were subjected to ultrafiltration and reagents contained less than 25 pg of endotoxin per ml by Limulus assay. Under endotoxin-free conditions, no increases in IL-1 beta, IL-6, or TNF-alpha mRNA or protein were detectable in cell cultures exposed to HIV-1, HIV-2, or rgp120 (0.1 to 10 micrograms/ml), as compared with cytokine levels in mock-exposed cultures. However, concentrations of endotoxin (lipopolysaccharide) as low as 0.5 ng/ml induced significant production of mRNA and protein for these three cytokines. Preincubation of mononuclear cells with "shake" HIV-1 preparations and also mock-infected shake preparations prior to lipopolysaccharide stimulation resulted in a two- to threefold increase in IL-1 beta and TNF-alpha production. This priming effect was not observed with rgp120 (0.1 to 10 micrograms/ml) or standard HIV-1 or mock-infected supernatants, suggesting the presence of biologically active material independent of virus in the shake preparations. Our studies indicate that, in the absence of endotoxin, HIV-1, HIV-2, and HIV gp120 do not induce production of IL-1 beta, IL-6, or TNF-alpha by peripheral blood mononuclear cells. -
Scadden DT. 1990. Granulocyte macrophage colony stimulating factor (GM-CSF) in AIDS. Progress in clinical and biological research. 338:163-76. Pubmed: 2189138 Scadden DT. 1990. Granulocyte macrophage colony stimulating factor (GM-CSF) in AIDS. Progress in clinical and biological research. 338:163-76. Pubmed: 2189138 Overall GM-CSF is a well tolerated intervention in patients with HIV associated disease. As in a number of other clinical settings, it is able to improve myelopoiesis and abrogate the myelotoxicity of chemotherapeutic agents. At present, clinical data is insufficient to indicate an ultimate clinical benefit from the use of GM-CSF in terms of opportunistic infection, mortality or quality of life for HIV infected patients. As phase I and phase II trials are completed however comparative clinical trials addressing these issues are anticipated. Hematopoietic growth factors may permit the use of optimal doses of therapeutics and thereby play an adjunctive role in the combination therapies anticipated for the treatment of HIV related disease. -
Scadden DT, Fuller B, Cunningham JM. 1990. Human cells infected with retrovirus vectors acquire an endogenous murine provirus. Journal of virology. 64(1):424-7. Pubmed: 2152828 Scadden DT, Fuller B, Cunningham JM. 1990. Human cells infected with retrovirus vectors acquire an endogenous murine provirus. Journal of virology. 64(1):424-7. Pubmed: 2152828 A 5.2-kilobase mouse RNA is expressed in human cells following infection with recombinant retroviruses propagated in mouse NIH 3T3 cells as psi-2 pseudotypes. This RNA is transcribed from a defective mink cell focus-forming provirus and copackaged into virions and integrated into human target cell DNA at a frequency comparable to that of the recombinant retrovirus genome. 1989
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Groopman JE, Scadden DT. 1989. Interferon therapy for Kaposi sarcoma associated with the acquired immunodeficiency syndrome (AIDS). Annals of internal medicine. 110(5):335-7. Pubmed: 2644884 Groopman JE, Scadden DT. 1989. Interferon therapy for Kaposi sarcoma associated with the acquired immunodeficiency syndrome (AIDS). Annals of internal medicine. 110(5):335-7. Pubmed: 2644884 -
Albritton LM, Tseng L, Scadden D, Cunningham JM. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 57(4):659-66. Pubmed: 2541919 Albritton LM, Tseng L, Scadden D, Cunningham JM. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 57(4):659-66. Pubmed: 2541919 Murine type C ecotropic retrovirus infection is initiated by virus envelope binding to a membrane receptor expressed on mouse cells. We have identified a cDNA clone that may encode for this receptor through a strategy combining gene transfer of mouse NIH 3T3 DNA into nonpermissive human EJ cells, selection of EJ clones that have acquired susceptibility to infection by retrovirus vectors containing drug resistance genes, and identification of the putative receptor cDNA clone through linkage to a mouse repetitive DNA sequence. Human EJ cells that express the cDNA acquire a million-fold increase in MuLV infectivity. The predicted 622 amino acid sequence of the putative receptor protein is extremely hydrophobic; 14 potential membrane-spanning domains have been identified. A computer-based search of sequence data banks did not identify a protein with significant similarity to the putative receptor. We conclude that a novel membrane protein determines susceptibility to ecotropic MuLV infection by binding and/or fusion with the virus envelope. -
Scadden DT, Zon LI, Groopman JE. 1989. Pathophysiology and management of HIV-associated hematologic disorders. Blood. 74(5):1455-63. Pubmed: 2676010 Scadden DT, Zon LI, Groopman JE. 1989. Pathophysiology and management of HIV-associated hematologic disorders. Blood. 74(5):1455-63. Pubmed: 2676010 -
Molina JM, Scadden DT, Byrn R, Dinarello CA, Groopman JE. 1989. Production of tumor necrosis factor alpha and interleukin 1 beta by monocytic cells infected with human immunodeficiency virus. The Journal of clinical investigation. 84(3):733-7. Pubmed: 2474573 Molina JM, Scadden DT, Byrn R, Dinarello CA, Groopman JE. 1989. Production of tumor necrosis factor alpha and interleukin 1 beta by monocytic cells infected with human immunodeficiency virus. The Journal of clinical investigation. 84(3):733-7. Pubmed: 2474573 The production of tumor necrosis factor alpha (TNF alpha) and IL-1 beta by the monocytic cell line THP-1, productively infected with HIV-1, was investigated using specific RIA and Northern blot analysis. HIV-infected cells, like uninfected cells, did not constitutively produce any detectable amounts of protein or mRNA for TNF alpha or IL-1 beta. After stimulation with LPS or a combination of LPS plus IFN-gamma, TNF alpha and IL-1 beta were detected in tissue culture supernatants and cell lysates and transcripts for both cytokines were seen on Northern blots. No significant difference in production of these two cytokines was observed between uninfected and chronically infected cells. Acutely HIV-infected cells, however, showed phenotypic changes compatible with maturation and an increase in TNF alpha and IL-1 beta mRNA production, and released significantly higher levels of TNF alpha and IL-1 beta compared with chronically infected or uninfected cells. Furthermore, LPS stimulation of HIV-infected cells increased virus production. These results suggest that HIV-infected monocytic cells may produce increased amounts of TNF alpha and IL-1 beta in response to stimuli that could be present in vivo. -
Groopman JE, Molina JM, Scadden DT. 1989. Hematopoietic growth factors. Biology and clinical applications. The New England journal of medicine. 321(21):1449-59. Pubmed: 2682244 Groopman JE, Molina JM, Scadden DT. 1989. Hematopoietic growth factors. Biology and clinical applications. The New England journal of medicine. 321(21):1449-59. Pubmed: 2682244 The hematopoietic growth factors are potent regulators of blood-cell proliferation and development. The first phase of clinical trials suggests that they may augment hematopoiesis in a number of different conditions of primary and secondary bone marrow dysfunction. Future clinical use is likely to include combinations of these growth factors, in order to stimulate early marrow progenitors and obtain multilineage effects. An improved understanding of the biologic and clinical effects of hematopoietic growth factors promises future clinical applications for conditions of impaired function and reduced numbers of blood cells. 1984
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Fineberg HV, Scadden D, Goldman L. 1984. Care of patients with a low probability of acute myocardial infarction. Cost effectiveness of alternatives to coronary-care-unit admission. The New England journal of medicine. 310(20):1301-7. Pubmed: 6425687 Fineberg HV, Scadden D, Goldman L. 1984. Care of patients with a low probability of acute myocardial infarction. Cost effectiveness of alternatives to coronary-care-unit admission. The New England journal of medicine. 310(20):1301-7. Pubmed: 6425687 We conducted a cost-effectiveness analysis to examine the clinical and economic consequences of alternatives to admission to a coronary-care unit for patients who have a relatively low probability of acute myocardial infarction. Despite the fact that all our assumptions were slanted to favor the current standard policy of admission to a coronary-care unit, our analysis shows that admission to an intermediate-care unit providing resuscitative facilities and prophylactic lidocaine is highly cost effective. For patients with about a 5 per cent probability of infarction, admission to a coronary-care unit would cost $2.04 million per life saved and $139,000 per year of life saved, as compared with intermediate care. For the expected number of such patients annually in the United States, the cost would be $297 million to save 145 lives. At probabilities of infarction up to about 20 per cent, the incremental cost to save a year of life by choosing a coronary-care unit over an intermediate-care unit would be higher than the estimated cost of saving a year of life by treating a 40-year-old man with mild hypertension. Our results suggest that many patients who have a low risk of acute myocardial infarction would be appropriate candidates for admission to an intermediate-care unit.