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Wagers Lab
Our publications
Featured Publications
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2019. In Situ Modification of Tissue Stem and Progenitor Cell Genomes. Cell reports. 27(4):1254-1264.e7. Pubmed: 31018138 DOI:S2211-1247(19)30452-8 Goldstein JM, Tabebordbar M, Zhu K, Wang LD, Messemer KA, Peacker B, Ashrafi Kakhki S, Gonzalez-Celeiro M, Shwartz Y, Cheng JKW, Xiao R, Barungi T, Albright C, Hsu YC, Vandenberghe LH, Wagers AJ. 2019. In Situ Modification of Tissue Stem and Progenitor Cell Genomes. Cell reports. 27(4):1254-1264.e7. Pubmed: 31018138 DOI:S2211-1247(19)30452-8 In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. -
Oh J, Sinha I, Tan KY, Rosner B, Dreyfuss JM, Gjata O, Tran P, Shoelson SE, Wagers AJ. 2016. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging. 8(11):2871-2896. Pubmed: 27852976 DOI:10.18632/aging.101098 Oh J, Sinha I, Tan KY, Rosner B, Dreyfuss JM, Gjata O, Tran P, Shoelson SE, Wagers AJ. 2016. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging. 8(11):2871-2896. Pubmed: 27852976 DOI:10.18632/aging.101098 Skeletal muscle is a highly regenerative tissue, but muscle repair potential is increasingly compromised with advancing age. In this study, we demonstrate that increased NF-κB activity in aged muscle fibers contributes to diminished myogenic potential of their associated satellite cells. We further examine the impact of genetic modulation of NF-κB signaling in muscle satellite cells or myofibers on recovery after damage. These studies reveal that NF-κB activity in differentiated myofibers is sufficient to drive dysfunction of muscle regenerative cells via cell-non-autonomous mechanisms. Inhibition of NF-κB, or its downstream target Phospholipase A2, in myofibers rescued muscle regenerative potential in aged muscle. Moreover, systemic administration of sodium salicylate, an FDA-approved NF-κB inhibitor, decreased inflammatory gene expression and improved repair in aged muscle. Together, these studies identify a unique NF-κB regulated, non-cell autonomous mechanism by which stem cell function is linked to lipid signaling and homeostasis, and provide important new targets to stimulate muscle repair in aged individuals. -
Poggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, Gonzalez E, Walker RG, Thompson TB, Wagers AJ, Fong YW, Lee RT. 2016. Circulating Growth Differentiation Factor 11/8 Levels Decline With Age. Circulation research. 118(1):29-37. Pubmed: 26489925 DOI:10.1161/CIRCRESAHA.115.307521 Poggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, Gonzalez E, Walker RG, Thompson TB, Wagers AJ, Fong YW, Lee RT. 2016. Circulating Growth Differentiation Factor 11/8 Levels Decline With Age. Circulation research. 118(1):29-37. Pubmed: 26489925 DOI:10.1161/CIRCRESAHA.115.307521 Array© 2015 American Heart Association, Inc. -
Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. 2016. A multifunctional AAV-CRISPR-Cas9 and its host response. Nature methods. 13(10):868-74. Pubmed: 27595405 DOI:10.1038/nmeth.3993 Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. 2016. A multifunctional AAV-CRISPR-Cas9 and its host response. Nature methods. 13(10):868-74. Pubmed: 27595405 DOI:10.1038/nmeth.3993 CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics. -
Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. 2016. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science (New York, N.Y.). 351(6271):407-411. Pubmed: 26721686 DOI:10.1126/science.aad5177 Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. 2016. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science (New York, N.Y.). 351(6271):407-411. Pubmed: 26721686 DOI:10.1126/science.aad5177 Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.Copyright © 2016, American Association for the Advancement of Science.
All Publications
2025
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Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL, Cyster JG, Engleman EG. 2025. Addendum: Langerhans cells renew in the skin throughout life under steady-state conditions. Nature immunology. 26(5):805. Pubmed: 40186071 DOI:10.1038/s41590-025-02131-9 Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL, Cyster JG, Engleman EG. 2025. Addendum: Langerhans cells renew in the skin throughout life under steady-state conditions. Nature immunology. 26(5):805. Pubmed: 40186071 DOI:10.1038/s41590-025-02131-9 -
Hu Y, Horlbeck MA, Zhang R, Ma S, Shrestha R, Kartha VK, Duarte FM, Hock C, Savage RE, Labade A, Kletzien H, Meliki A, Castillo A, Durand NC, Mattei E, Anderson LJ, Tay T, Earl AS, Shoresh N, Epstein CB, Wagers AJ, Buenrostro JD. 2025. Multiscale footprints reveal the organization of cis-regulatory elements. Nature. 638(8051):779-786. Pubmed: 39843737 DOI:10.1038/s41586-024-08443-4 Hu Y, Horlbeck MA, Zhang R, Ma S, Shrestha R, Kartha VK, Duarte FM, Hock C, Savage RE, Labade A, Kletzien H, Meliki A, Castillo A, Durand NC, Mattei E, Anderson LJ, Tay T, Earl AS, Shoresh N, Epstein CB, Wagers AJ, Buenrostro JD. 2025. Multiscale footprints reveal the organization of cis-regulatory elements. Nature. 638(8051):779-786. Pubmed: 39843737 DOI:10.1038/s41586-024-08443-4 Cis-regulatory elements (CREs) control gene expression and are dynamic in their structure and function, reflecting changes in the composition of diverse effector proteins over time. However, methods for measuring the organization of effector proteins at CREs across the genome are limited, hampering efforts to connect CRE structure to their function in cell fate and disease. Here we developed PRINT, a computational method that identifies footprints of DNA-protein interactions from bulk and single-cell chromatin accessibility data across multiple scales of protein size. Using these multiscale footprints, we created the seq2PRINT framework, which uses deep learning to allow precise inference of transcription factor and nucleosome binding and interprets regulatory logic at CREs. Applying seq2PRINT to single-cell chromatin accessibility data from human bone marrow, we observe sequential establishment and widening of CREs centred on pioneer factors across haematopoiesis. We further discover age-associated alterations in the structure of CREs in murine haematopoietic stem cells, including widespread reduction of nucleosome footprints and gain of de novo identified Ets composite motifs. Collectively, we establish a method for obtaining rich insights into DNA-binding protein dynamics from chromatin accessibility data, and reveal the architecture of regulatory elements across differentiation and ageing.© 2025. The Author(s). -
Cohen OS, Sinha M, Wang Y, Daman T, Li PC, Deatherage C, Charrez B, Deshpande A, Jordan S, Makoni NJ, LeDonne K, Dale CJ, Ben Driss L, Pan C, Gasperini C, Wagers AJ, Rubin LL, Finklestein SP, Allen M, Lee RT, Sandrasagra A. 2025. Recombinant GDF11 Promotes Recovery in a Rat Permanent Ischemia Model of Subacute Stroke. Stroke. 56(4):996-1009. Pubmed: 39909827 DOI:10.1161/STROKEAHA.124.049908 Cohen OS, Sinha M, Wang Y, Daman T, Li PC, Deatherage C, Charrez B, Deshpande A, Jordan S, Makoni NJ, LeDonne K, Dale CJ, Ben Driss L, Pan C, Gasperini C, Wagers AJ, Rubin LL, Finklestein SP, Allen M, Lee RT, Sandrasagra A. 2025. Recombinant GDF11 Promotes Recovery in a Rat Permanent Ischemia Model of Subacute Stroke. Stroke. 56(4):996-1009. Pubmed: 39909827 DOI:10.1161/STROKEAHA.124.049908 Array -
Horwitz N, Florea M, Medha KC, Liu T, Garcia V, Kim R, Lam A, Messemer K, Rios C, Almada AE, Wagers AJ. 2025. Soluble RAGE enhances muscle regeneration after cryoinjury in aged and diseased mice. PloS one. 20(2):e0318754. Pubmed: 39999114 DOI:10.1371/journal.pone.0318754 Horwitz N, Florea M, Medha KC, Liu T, Garcia V, Kim R, Lam A, Messemer K, Rios C, Almada AE, Wagers AJ. 2025. Soluble RAGE enhances muscle regeneration after cryoinjury in aged and diseased mice. PloS one. 20(2):e0318754. Pubmed: 39999114 DOI:10.1371/journal.pone.0318754 The Receptor for Advanced Glycation End Products (RAGE), classically considered a mediator of acute and chronic inflammatory responses, has recently been implicated by genetic knockout studies as a regulator of skeletal muscle physiology during development and following acute injury. Yet, the role of its soluble isoform, soluble RAGE (sRAGE), in muscle regeneration remains relatively unexplored. To address this knowledge gap, Adeno-Associated Virus (AAV) mediated and genetic knockin supplementation strategies were developed to specifically assess the effects of changing levels of sRAGE on muscle regeneration. We evaluated general muscle physiology and histology, including central nucleation, and myofiber size. We found that acute induction of sRAGE in aged and atherosclerotic animals accelerates muscle repair after cryoinjury. Similarly, genetic modification of the endogenous Ager gene locus to favor production of sRAGE over transmembrane RAGE accelerates repair of cryo-damaged skeletal muscle. However, increasing sRAGE via AAV delivery or using our transgenic mouse lines had no impact on muscle repair in aged or diseased mice after barium chloride (BaCl2) injury. Together, these studies identify a unique muscle regulatory activity of sRAGE that is variable across injury models and may be targeted in a context-specific manner to alter the skeletal muscle microenvironment and boost muscle regenerative output.Copyright: © 2025 Horwitz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. -
Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2025. Author Correction: Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 5(2):333. Pubmed: 39881191 DOI:10.1038/s43587-025-00804-6 Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2025. Author Correction: Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 5(2):333. Pubmed: 39881191 DOI:10.1038/s43587-025-00804-6 2024
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Lin KH, Hibbert JE, Flynn CG, Lemens JL, Torbey MM, Steinert ND, Flejsierowicz PM, Melka KM, Lindley GT, Lares M, Setaluri V, Wagers AJ, Hornberger TA. 2024. Satellite cell-derived TRIM28 is pivotal for mechanical load- and injury-induced myogenesis. EMBO reports. 25(9):3812-3841. Pubmed: 39143258 DOI:10.1038/s44319-024-00227-1 Lin KH, Hibbert JE, Flynn CG, Lemens JL, Torbey MM, Steinert ND, Flejsierowicz PM, Melka KM, Lindley GT, Lares M, Setaluri V, Wagers AJ, Hornberger TA. 2024. Satellite cell-derived TRIM28 is pivotal for mechanical load- and injury-induced myogenesis. EMBO reports. 25(9):3812-3841. Pubmed: 39143258 DOI:10.1038/s44319-024-00227-1 Satellite cells are skeletal muscle stem cells that contribute to postnatal muscle growth, and they endow skeletal muscle with the ability to regenerate after a severe injury. Here we discover that this myogenic potential of satellite cells requires a protein called tripartite motif-containing 28 (TRIM28). Interestingly, different from the role reported in a previous study based on C2C12 myoblasts, multiple lines of both in vitro and in vivo evidence reveal that the myogenic function of TRIM28 is not dependent on changes in the phosphorylation of its serine 473 residue. Moreover, the functions of TRIM28 are not mediated through the regulation of satellite cell proliferation or differentiation. Instead, our findings indicate that TRIM28 regulates the ability of satellite cells to progress through the process of fusion. Specifically, we discover that TRIM28 controls the expression of a fusogenic protein called myomixer and concomitant fusion pore formation. Collectively, the outcomes of this study expose the framework of a novel regulatory pathway that is essential for myogenesis.© 2024. The Author(s). -
Braun P, Alawi M, Saygi C, Pantel K, Wagers AJ. 2024. Expression profiling by high-throughput sequencing reveals GADD45, SMAD7, EGR-1 and HOXA3 activation in Myostatin (MSTN) and GDF11 treated myoblasts. Genetics and molecular biology. 47(2):e20230304. Pubmed: 39012095 DOI:10.1590/1678-4685-GMB-2023-0304 Braun P, Alawi M, Saygi C, Pantel K, Wagers AJ. 2024. Expression profiling by high-throughput sequencing reveals GADD45, SMAD7, EGR-1 and HOXA3 activation in Myostatin (MSTN) and GDF11 treated myoblasts. Genetics and molecular biology. 47(2):e20230304. Pubmed: 39012095 DOI:10.1590/1678-4685-GMB-2023-0304 Growth differentiation factor 11 (GDF11) and myostatin (MSTN/GDF8) are closely related members of the transforming growth factor β (TGFβ) superfamily, sharing structural homology. Despite these structural similarities, recent research has shed light on the distinct roles these ligands play within muscle tissue. This study aims to uncover both the differences and similarities in gene expression at the transcriptome level by utilizing RNA sequencing. We conducted experiments involving five distinct groups, each with three biological replicates, using C2C12 cell cultures. The cells were subjected to high-throughput profiling to investigate disparities in gene expression patterns following preconditioning with either GDF11 or MSTN at concentrations of 1 nM and 10 nM, respectively. In addition, control groups were established. Our research revealed concentration-dependent gene expression patterns, with 38 genes showing significant differences when compared to the control groups. Notably, GADD45, SMAD7, EGR-1, and HOXA3 exhibited significant differential expression. We also conducted an over-representation analysis, highlighting the activation of MAPK and JNK signaling pathways, along with GO-terms related to genes that negatively regulate metabolic processes, biosynthesis, and protein phosphorylation. This study unveiled the activation of several genes not previously discussed in existing literature whose full biological implications are yet to be determined in future research. -
Yang VK, Rush JE, Bhasin S, Wagers AJ, Lee RT. 2024. Corrigendum to "Plasma growth differentiation factors 8 and 11 levels in cats with congestive heart failure secondary to hypertrophic cardiomyopathy" [J Vet Cardiol 25 (2019 Oct) 41-51]. Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology. 54:86. Pubmed: 38582690 DOI:S1760-2734(24)00024-9 Yang VK, Rush JE, Bhasin S, Wagers AJ, Lee RT. 2024. Corrigendum to "Plasma growth differentiation factors 8 and 11 levels in cats with congestive heart failure secondary to hypertrophic cardiomyopathy" [J Vet Cardiol 25 (2019 Oct) 41-51]. Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology. 54:86. Pubmed: 38582690 DOI:S1760-2734(24)00024-9 -
Yang JH, Hayano M, Griffin PT, Amorim JA, Bonkowski MS, Apostolides JK, Salfati EL, Blanchette M, Munding EM, Bhakta M, Chew YC, Guo W, Yang X, Maybury-Lewis S, Tian X, Ross JM, Coppotelli G, Meer MV, Rogers-Hammond R, Vera DL, Lu YR, Pippin JW, Creswell ML, Dou Z, Xu C, Mitchell SJ, Das A, O'Connell BL, Thakur S, Kane AE, Su Q, Mohri Y, Nishimura EK, Schaevitz L, Garg N, Balta AM, Rego MA, Gregory-Ksander M, Jakobs TC, Zhong L, Wakimoto H, El Andari J, Grimm D, Mostoslavsky R, Wagers AJ, Tsubota K, Bonasera SJ, Palmeira CM, Seidman JG, Seidman CE, Wolf NS, Kreiling JA, Sedivy JM, Murphy GF, Green RE, Garcia BA, Berger SL, Oberdoerffer P, Shankland SJ, Gladyshev VN, Ksander BR, Pfenning AR, Rajman LA, Sinclair DA. 2024. Loss of epigenetic information as a cause of mammalian aging. Cell. 187(5):1312-1313. Pubmed: 38428398 DOI:S0092-8674(24)00118-1 Yang JH, Hayano M, Griffin PT, Amorim JA, Bonkowski MS, Apostolides JK, Salfati EL, Blanchette M, Munding EM, Bhakta M, Chew YC, Guo W, Yang X, Maybury-Lewis S, Tian X, Ross JM, Coppotelli G, Meer MV, Rogers-Hammond R, Vera DL, Lu YR, Pippin JW, Creswell ML, Dou Z, Xu C, Mitchell SJ, Das A, O'Connell BL, Thakur S, Kane AE, Su Q, Mohri Y, Nishimura EK, Schaevitz L, Garg N, Balta AM, Rego MA, Gregory-Ksander M, Jakobs TC, Zhong L, Wakimoto H, El Andari J, Grimm D, Mostoslavsky R, Wagers AJ, Tsubota K, Bonasera SJ, Palmeira CM, Seidman JG, Seidman CE, Wolf NS, Kreiling JA, Sedivy JM, Murphy GF, Green RE, Garcia BA, Berger SL, Oberdoerffer P, Shankland SJ, Gladyshev VN, Ksander BR, Pfenning AR, Rajman LA, Sinclair DA. 2024. Loss of epigenetic information as a cause of mammalian aging. Cell. 187(5):1312-1313. Pubmed: 38428398 DOI:S0092-8674(24)00118-1 2023
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Driss LB, Lian J, Walker RG, Howard JA, Thompson TB, Rubin LL, Wagers AJ, Lee RT. 2023. GDF11 and aging biology - controversies resolved and pending. The journal of cardiovascular aging. 3(4). Pubmed: 38235060 DOI:10.20517/jca.2023.23 Driss LB, Lian J, Walker RG, Howard JA, Thompson TB, Rubin LL, Wagers AJ, Lee RT. 2023. GDF11 and aging biology - controversies resolved and pending. The journal of cardiovascular aging. 3(4). Pubmed: 38235060 DOI:10.20517/jca.2023.23 Since the exogenous administration of GDF11, a TGF-ß superfamily member, was reported to have beneficial effects in some models of human disease, there have been many research studies in GDF11 biology. However, many studies have now confirmed that exogenous administration of GDF11 can improve physiology in disease models, including cardiac fibrosis, experimental stroke, and disordered metabolism. GDF11 is similar to GDF8 (also called Myostatin), differing only by 11 amino acids in their mature signaling domains. These two proteins are now known to be biochemically different both and . GDF11 is much more potent than GDF8 and induces more strongly SMAD2 phosphorylation in the myocardium compared to GDF8. GDF8 and GDF11 prodomain are only 52% identical and are cleaved by different Tolloid proteases to liberate the mature signaling domain from inhibition of the prodomain. Here, we review the state of GDF11 biology, highlighting both resolved and remaining controversies. -
Hu Y, Ma S, Kartha VK, Duarte FM, Horlbeck M, Zhang R, Shrestha R, Labade A, Kletzien H, Meliki A, Castillo A, Durand N, Mattei E, Anderson LJ, Tay T, Earl AS, Shoresh N, Epstein CB, Wagers A, Buenrostro JD. 2023. Single-cell multi-scale footprinting reveals the modular organization of DNA regulatory elements. bioRxiv : the preprint server for biology. Pubmed: 37034577 DOI:10.1101/2023.03.28.533945 Hu Y, Ma S, Kartha VK, Duarte FM, Horlbeck M, Zhang R, Shrestha R, Labade A, Kletzien H, Meliki A, Castillo A, Durand N, Mattei E, Anderson LJ, Tay T, Earl AS, Shoresh N, Epstein CB, Wagers A, Buenrostro JD. 2023. Single-cell multi-scale footprinting reveals the modular organization of DNA regulatory elements. bioRxiv : the preprint server for biology. Pubmed: 37034577 DOI:10.1101/2023.03.28.533945 Array -
Rinaldi V, Messemer K, Desevin K, Sun F, Berry BC, Kukreja S, Tapper AR, Wagers AJ, Rando OJ. 2023. Evidence for RNA or protein transport from somatic tissues to the male reproductive tract in mouse. eLife. 12. Pubmed: 36971355 DOI:10.7554/eLife.77733 Rinaldi V, Messemer K, Desevin K, Sun F, Berry BC, Kukreja S, Tapper AR, Wagers AJ, Rando OJ. 2023. Evidence for RNA or protein transport from somatic tissues to the male reproductive tract in mouse. eLife. 12. Pubmed: 36971355 DOI:10.7554/eLife.77733 The development of tools to manipulate the mouse genome, including knockout and transgenic technology, has revolutionized our ability to explore gene function in mammals. Moreover, for genes that are expressed in multiple tissues or at multiple stages of development, the use of tissue-specific expression of the Cre recombinase allows gene function to be perturbed in specific cell types and/or at specific times. However, it is well known that putative tissue-specific promoters often drive unanticipated 'off-target' expression. In our efforts to explore the biology of the male reproductive tract, we unexpectedly found that expression of Cre in the central nervous system resulted in recombination in the epididymis, a tissue where sperm mature for ~1-2 weeks following the completion of testicular development. Remarkably, we not only observed reporter expression in the epididymis when Cre expression was driven from neuron-specific transgenes, but also when Cre expression in the brain was induced from an AAV vector carrying a Cre expression construct. A surprisingly wide range of Cre drivers - including six different neuronal promoters as well as the adipose-specific Cre promoter - exhibited off-target recombination in the epididymis, with a subset of drivers also exhibiting unexpected activity in other tissues such as the reproductive accessory glands. Using a combination of parabiosis and serum transfer experiments, we find evidence supporting the hypothesis that Cre may be trafficked from its cell of origin to the epididymis through the circulatory system. Together, our findings should motivate caution when interpreting conditional alleles, and suggest the exciting possibility of inter-tissue RNA or protein trafficking in modulation of reproductive biology.© 2023, Rinaldi et al. -
Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 3(3):327-345. Pubmed: 37118429 DOI:10.1038/s43587-023-00373-6 Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 3(3):327-345. Pubmed: 37118429 DOI:10.1038/s43587-023-00373-6 Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains after parabiosis. For each cell type, we cataloged alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions and senescence status. Our analyses identified gene signatures, demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest new strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors.© 2023. The Author(s). -
Tavakoli S, Garcia V, Gähwiler E, Adatto I, Rangan A, Messemer KA, Kakhki SA, Yang S, Chan VS, Manning ME, Fotowat H, Zhou Y, Wagers AJ, Zon LI. 2023. Transplantation-based screen identifies inducers of muscle progenitor cell engraftment across vertebrate species. Cell reports. 42(4):112365. Pubmed: 37018075 DOI:S2211-1247(23)00376-5 Tavakoli S, Garcia V, Gähwiler E, Adatto I, Rangan A, Messemer KA, Kakhki SA, Yang S, Chan VS, Manning ME, Fotowat H, Zhou Y, Wagers AJ, Zon LI. 2023. Transplantation-based screen identifies inducers of muscle progenitor cell engraftment across vertebrate species. Cell reports. 42(4):112365. Pubmed: 37018075 DOI:S2211-1247(23)00376-5 Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species.Published by Elsevier Inc. -
Yang JH, Hayano M, Griffin PT, Amorim JA, Bonkowski MS, Apostolides JK, Salfati EL, Blanchette M, Munding EM, Bhakta M, Chew YC, Guo W, Yang X, Maybury-Lewis S, Tian X, Ross JM, Coppotelli G, Meer MV, Rogers-Hammond R, Vera DL, Lu YR, Pippin JW, Creswell ML, Dou Z, Xu C, Mitchell SJ, Das A, O'Connell BL, Thakur S, Kane AE, Su Q, Mohri Y, Nishimura EK, Schaevitz L, Garg N, Balta AM, Rego MA, Gregory-Ksander M, Jakobs TC, Zhong L, Wakimoto H, El Andari J, Grimm D, Mostoslavsky R, Wagers AJ, Tsubota K, Bonasera SJ, Palmeira CM, Seidman JG, Seidman CE, Wolf NS, Kreiling JA, Sedivy JM, Murphy GF, Green RE, Garcia BA, Berger SL, Oberdoerffer P, Shankland SJ, Gladyshev VN, Ksander BR, Pfenning AR, Rajman LA, Sinclair DA. 2023. Loss of epigenetic information as a cause of mammalian aging. Cell. 186(2):305-326.e27. Pubmed: 36638792 DOI:S0092-8674(22)01570-7 Yang JH, Hayano M, Griffin PT, Amorim JA, Bonkowski MS, Apostolides JK, Salfati EL, Blanchette M, Munding EM, Bhakta M, Chew YC, Guo W, Yang X, Maybury-Lewis S, Tian X, Ross JM, Coppotelli G, Meer MV, Rogers-Hammond R, Vera DL, Lu YR, Pippin JW, Creswell ML, Dou Z, Xu C, Mitchell SJ, Das A, O'Connell BL, Thakur S, Kane AE, Su Q, Mohri Y, Nishimura EK, Schaevitz L, Garg N, Balta AM, Rego MA, Gregory-Ksander M, Jakobs TC, Zhong L, Wakimoto H, El Andari J, Grimm D, Mostoslavsky R, Wagers AJ, Tsubota K, Bonasera SJ, Palmeira CM, Seidman JG, Seidman CE, Wolf NS, Kreiling JA, Sedivy JM, Murphy GF, Green RE, Garcia BA, Berger SL, Oberdoerffer P, Shankland SJ, Gladyshev VN, Ksander BR, Pfenning AR, Rajman LA, Sinclair DA. 2023. Loss of epigenetic information as a cause of mammalian aging. Cell. 186(2):305-326.e27. Pubmed: 36638792 DOI:S0092-8674(22)01570-7 All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.Copyright © 2022 Elsevier Inc. All rights reserved. -
Florea M, Nicolaou F, Pacouret S, Zinn EM, Sanmiguel J, Andres-Mateos E, Unzu C, Wagers AJ, Vandenberghe LH. 2023. High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography. Molecular therapy. Methods & clinical development. 28:146-159. Pubmed: 36654797 DOI:10.1016/j.omtm.2022.12.009 Florea M, Nicolaou F, Pacouret S, Zinn EM, Sanmiguel J, Andres-Mateos E, Unzu C, Wagers AJ, Vandenberghe LH. 2023. High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography. Molecular therapy. Methods & clinical development. 28:146-159. Pubmed: 36654797 DOI:10.1016/j.omtm.2022.12.009 The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both and . Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%-80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes.© 2022 The Authors. -
Lian J, Walker RG, D'Amico A, Vujic A, Mills MJ, Messemer KA, Mendello KR, Goldstein JM, Leacock KA, Epp S, Stimpfl EV, Thompson TB, Wagers AJ, Lee RT. 2023. Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle. Life science alliance. 6(3). Pubmed: 36631218 DOI:10.26508/lsa.202201662 Lian J, Walker RG, D'Amico A, Vujic A, Mills MJ, Messemer KA, Mendello KR, Goldstein JM, Leacock KA, Epp S, Stimpfl EV, Thompson TB, Wagers AJ, Lee RT. 2023. Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle. Life science alliance. 6(3). Pubmed: 36631218 DOI:10.26508/lsa.202201662 Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF-β family proteins that interact with nearly identical signaling receptors and antagonists. However, GDF11 appears to activate SMAD2/3 more potently than GDF8 in vitro and in vivo. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced the activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of GDF8 to that of GDF11 yielded mice lacking GDF8, with GDF11 levels ∼50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with -deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify a specific requirement for GDF11 in early-stage skeletal development.© 2023 Lian et al. 2022
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Zinn E, Unzu C, Schmit PF, Turunen HT, Zabaleta N, Sanmiguel J, Fieldsend A, Bhatt U, Diop C, Merkel E, Gurrala R, Peacker B, Rios C, Messemer K, Santos J, Estelien R, Andres-Mateos E, Wagers AJ, Tipper C, Vandenberghe LH. 2022. Ancestral library identifies conserved reprogrammable liver motif on AAV capsid. Cell reports. Medicine. 3(11):100803. Pubmed: 36327973 DOI:10.1016/j.xcrm.2022.100803 Zinn E, Unzu C, Schmit PF, Turunen HT, Zabaleta N, Sanmiguel J, Fieldsend A, Bhatt U, Diop C, Merkel E, Gurrala R, Peacker B, Rios C, Messemer K, Santos J, Estelien R, Andres-Mateos E, Wagers AJ, Tipper C, Vandenberghe LH. 2022. Ancestral library identifies conserved reprogrammable liver motif on AAV capsid. Cell reports. Medicine. 3(11):100803. Pubmed: 36327973 DOI:10.1016/j.xcrm.2022.100803 Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved. -
Barutcu AR, Elizalde G, Gonzalez AE, Soni K, Rinn JL, Wagers AJ, Almada AE. 2022. Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells. Skeletal muscle. 12(1):20. Pubmed: 35971133 DOI:10.1186/s13395-022-00303-x Barutcu AR, Elizalde G, Gonzalez AE, Soni K, Rinn JL, Wagers AJ, Almada AE. 2022. Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells. Skeletal muscle. 12(1):20. Pubmed: 35971133 DOI:10.1186/s13395-022-00303-x Array© 2022. The Author(s). 2021
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Yagi M, Ji F, Charlton J, Cristea S, Messemer K, Horwitz N, Di Stefano B, Tsopoulidis N, Hoetker MS, Huebner AJ, Bar-Nur O, Almada AE, Yamamoto M, Patelunas A, Goldhamer DJ, Wagers AJ, Michor F, Meissner A, Sadreyev RI, Hochedlinger K. 2021. Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells. Genes & development. 35(17-18):1209-1228. Pubmed: 34413137 DOI:10.1101/gad.348678.121 Yagi M, Ji F, Charlton J, Cristea S, Messemer K, Horwitz N, Di Stefano B, Tsopoulidis N, Hoetker MS, Huebner AJ, Bar-Nur O, Almada AE, Yamamoto M, Patelunas A, Goldhamer DJ, Wagers AJ, Michor F, Meissner A, Sadreyev RI, Hochedlinger K. 2021. Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells. Genes & development. 35(17-18):1209-1228. Pubmed: 34413137 DOI:10.1101/gad.348678.121 The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7 iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.© 2021 Yagi et al.; Published by Cold Spring Harbor Laboratory Press. -
Regina C, Hamed E, Andrieux G, Angenendt S, Schneider M, Ku M, Follo M, Wachtel M, Ke E, Kikuchi K, Henssen AG, Schäfer BW, Boerries M, Wagers AJ, Keller C, Hettmer S. 2021. Negative correlation of single-cell expression with tumorigenicity in rhabdomyosarcoma. Life science alliance. 4(9). Pubmed: 34187933 DOI:10.26508/lsa.202001002 Regina C, Hamed E, Andrieux G, Angenendt S, Schneider M, Ku M, Follo M, Wachtel M, Ke E, Kikuchi K, Henssen AG, Schäfer BW, Boerries M, Wagers AJ, Keller C, Hettmer S. 2021. Negative correlation of single-cell expression with tumorigenicity in rhabdomyosarcoma. Life science alliance. 4(9). Pubmed: 34187933 DOI:10.26508/lsa.202001002 Rhabdomyosarcomas (RMS) are phenotypically and functionally heterogeneous. Both primary human RMS cultures and low-passage mouse RMS cell lines, which express the fusion oncoprotein Pax3:Foxo1 and lack the tumor suppressor (), exhibit marked heterogeneity in () expression at the single cell level. In mouse RMS cells, expression is directed by the promoter and coupled to YFP/P3F mouse RMS cells included 87% G0/G1 cells and reorganized their actin cytoskeleton to produce a cellular phenotype characterized by more efficient adhesion and migration. This translated into higher tumor-propagating cell frequencies of YFP/P3F compared with YFP/P3F cells. Both YFP/P3F and YFP/P3F cells gave rise to mixed clones in vitro, consistent with fluctuations in expression over time. Exposure to the anti-tropomyosin compound TR100 disrupted the cytoskeleton and reversed enhanced migration and adhesion of YFP/P3F RMS cells. Heterogeneous expression of at the single cell level may provide a critical advantage during tumor progression.© 2021 Regina et al. -
Almada AE, Horwitz N, Price FD, Gonzalez AE, Ko M, Bolukbasi OV, Messemer KA, Chen S, Sinha M, Rubin LL, Wagers AJ. 2021. FOS licenses early events in stem cell activation driving skeletal muscle regeneration. Cell reports. 34(4):108656. Pubmed: 33503437 DOI:S2211-1247(20)31645-4 Almada AE, Horwitz N, Price FD, Gonzalez AE, Ko M, Bolukbasi OV, Messemer KA, Chen S, Sinha M, Rubin LL, Wagers AJ. 2021. FOS licenses early events in stem cell activation driving skeletal muscle regeneration. Cell reports. 34(4):108656. Pubmed: 33503437 DOI:S2211-1247(20)31645-4 Muscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of "pro-regenerative" target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses.Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved. -
Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, Krunnfusz A, Tavakoli S, Widrick JJ, Messemer KA, Troiano EC, Moghadaszadeh B, Peacker BL, Leacock KA, Horwitz N, Beggs AH, Wagers AJ, Sabeti PC. 2021. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell. 184(19):4919-4938.e22. Pubmed: 34506722 DOI:S0092-8674(21)01002-3 Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, Krunnfusz A, Tavakoli S, Widrick JJ, Messemer KA, Troiano EC, Moghadaszadeh B, Peacker BL, Leacock KA, Horwitz N, Beggs AH, Wagers AJ, Sabeti PC. 2021. Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Cell. 184(19):4919-4938.e22. Pubmed: 34506722 DOI:S0092-8674(21)01002-3 Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. 2020
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Endo Y, Baldino K, Li B, Zhang Y, Sakthivel D, MacArthur M, Panayi AC, Kip P, Spencer DJ, Jasuja R, Bagchi D, Bhasin S, Nuutila K, Neppl RL, Wagers AJ, Sinha I. 2020. Loss of ARNT in skeletal muscle limits muscle regeneration in aging. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 34(12):16086-16104. Pubmed: 33064329 DOI:10.1096/fj.202000761RR Endo Y, Baldino K, Li B, Zhang Y, Sakthivel D, MacArthur M, Panayi AC, Kip P, Spencer DJ, Jasuja R, Bagchi D, Bhasin S, Nuutila K, Neppl RL, Wagers AJ, Sinha I. 2020. Loss of ARNT in skeletal muscle limits muscle regeneration in aging. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 34(12):16086-16104. Pubmed: 33064329 DOI:10.1096/fj.202000761RR The ability of skeletal muscle to regenerate declines significantly with aging. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT), a critical component of the hypoxia signaling pathway, was less abundant in skeletal muscle of old (23-25 months old) mice. This loss of ARNT was associated with decreased levels of Notch1 intracellular domain (N1ICD) and impaired regenerative response to injury in comparison to young (2-3 months old) mice. Knockdown of ARNT in a primary muscle cell line impaired differentiation in vitro. Skeletal muscle-specific ARNT deletion in young mice resulted in decreased levels of whole muscle N1ICD and limited muscle regeneration. Administration of a systemic hypoxia pathway activator (ML228), which simulates the actions of ARNT, rescued skeletal muscle regeneration in both old and ARNT-deleted mice. These results suggest that the loss of ARNT in skeletal muscle is partially responsible for diminished myogenic potential in aging and activation of hypoxia signaling holds promise for rescuing regenerative activity in old muscle.© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology. -
Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential. -
Würtemberger J, Tchessalova D, Regina C, Bauer C, Schneider M, Wagers AJ, Hettmer S. 2020. Growth inhibition associated with disruption of the actin cytoskeleton by Latrunculin A in rhabdomyosarcoma cells. PloS one. 15(9):e0238572. Pubmed: 32898143 DOI:10.1371/journal.pone.0238572 Würtemberger J, Tchessalova D, Regina C, Bauer C, Schneider M, Wagers AJ, Hettmer S. 2020. Growth inhibition associated with disruption of the actin cytoskeleton by Latrunculin A in rhabdomyosarcoma cells. PloS one. 15(9):e0238572. Pubmed: 32898143 DOI:10.1371/journal.pone.0238572 Functional genomic screening of KRAS-driven mouse sarcomas was previously employed to identify proliferation-relevant genes. Genes identified included Ubiquitin-conjugating enzyme E2 (Ube2c), Centromere Protein E (Cenpe), Hyaluronan Synthase 2 (Has2), and CAMP Responsive Element Binding Protein 3 Like 2 (Creb3l2). This study examines the expression and chemical inhibition of these candidate genes, identifying variable levels of protein expression and significant contributions to rhabdomyosarcoma (RMS) cell proliferation. Chemical treatment of human and murine RMS cell lines with bortezomib, UA62784, latrunculin A and sorafenib inhibited growth with approximate EC50 concentrations of 15-30nM for bortezomib, 25-80nM for UA62784 and 80-220nM for latrunculin A. The multi-kinase inhibitor sorafenib increased in vitro proliferation of 4 of 6 sarcoma cell lines tested. Latrunculin A was further associated with disruption of the actin cytoskeleton and reduced ERK1/2 phosphorylation. Together, this work advances opportunities for developing therapies to block progression of soft-tissue sarcomas and demonstrates that disruption of the actin cytoskeleton in sarcoma cells by latrunculin A is associated with a reduction in RMS cell growth. (167 words). -
Shah A, Dagdeviren S, Lewandowski JP, Schmider AB, Ricci-Blair EM, Natarajan N, Hundal H, Noh HL, Friedline RH, Vidoudez C, Kim JK, Wagers AJ, Soberman RJ, Lee RT. 2020. Thioredoxin Interacting Protein Is Required for a Chronic Energy-Rich Diet to Promote Intestinal Fructose Absorption. iScience. 23(9):101521. Pubmed: 32927265 DOI:10.1016/j.isci.2020.101521 Shah A, Dagdeviren S, Lewandowski JP, Schmider AB, Ricci-Blair EM, Natarajan N, Hundal H, Noh HL, Friedline RH, Vidoudez C, Kim JK, Wagers AJ, Soberman RJ, Lee RT. 2020. Thioredoxin Interacting Protein Is Required for a Chronic Energy-Rich Diet to Promote Intestinal Fructose Absorption. iScience. 23(9):101521. Pubmed: 32927265 DOI:10.1016/j.isci.2020.101521 Increased consumption of fats and added sugars has been associated with an increase in metabolic syndromes. Here we show that mice chronically fed an energy-rich diet (ERD) with high fat and moderate sucrose have enhanced the absorption of a gastrointestinal fructose load, and this required expression of the arrestin domain protein Txnip in the intestinal epithelial cells. ERD feeding induced gene and protein expression of Glut5, and this required the expression of Txnip. Furthermore, Txnip interacted with Rab11a, a small GTPase that facilitates the apical localization of Glut5. We also demonstrate that ERD promoted Txnip/Glut5 complexes in the apical intestinal epithelial cell. Our findings demonstrate that ERD facilitates fructose absorption through a Txnip-dependent mechanism in the intestinal epithelial cell, suggesting that increased fructose absorption could potentially provide a mechanism for worsening of metabolic syndromes in the setting of a chronic ERD.© 2020 The Author(s). -
Walker RG, Barrandon O, Poggioli T, Dagdeviren S, Carroll SH, Mills MJ, Mendello KR, Gomez Y, Loffredo FS, Pancoast JR, Macias-Trevino C, Marts C, LeClair KB, Noh HL, Kim T, Banks AS, Kim JK, Cohen DE, Wagers AJ, Melton DA, Lee RT. 2020. Exogenous GDF11, but not GDF8, reduces body weight and improves glucose homeostasis in mice. Scientific reports. 10(1):4561. Pubmed: 32165710 DOI:10.1038/s41598-020-61443-y Walker RG, Barrandon O, Poggioli T, Dagdeviren S, Carroll SH, Mills MJ, Mendello KR, Gomez Y, Loffredo FS, Pancoast JR, Macias-Trevino C, Marts C, LeClair KB, Noh HL, Kim T, Banks AS, Kim JK, Cohen DE, Wagers AJ, Melton DA, Lee RT. 2020. Exogenous GDF11, but not GDF8, reduces body weight and improves glucose homeostasis in mice. Scientific reports. 10(1):4561. Pubmed: 32165710 DOI:10.1038/s41598-020-61443-y Insulin resistance is associated with aging in mice and humans. We have previously shown that administration of recombinant GDF11 (rGDF11) to aged mice alters aging phenotypes in the brain, skeletal muscle, and heart. While the closely related protein GDF8 has a role in metabolism, limited data are available on the potential metabolic effects of GDF11 or GDF8 in aging. To determine the metabolic effects of these two ligands, we administered rGDF11 or rGDF8 protein to young or aged mice fed a standard chow diet, short-term high-fat diet (HFD), or long-term HFD. Under nearly all of these diet conditions, administration of exogenous rGDF11 reduced body weight by 3-17% and significantly improved glucose tolerance in aged mice fed a chow (~30% vs. saline) or HF (~50% vs. saline) diet and young mice fed a HFD (~30%). On the other hand, exogenous rGDF8 showed signifcantly lesser effect or no effect at all on glucose tolerance compared to rGDF11, consistent with data demonstrating that GFD11 is a more potent signaling ligand than GDF8. Collectively, our results show that administration of exogenous rGDF11, but not rGDF8, can reduce diet-induced weight gain and improve metabolic homeostasis. 2019
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Goldstein JM, Valido A, Lewandowski JP, Walker RG, Mills MJ, Messemer KA, Besseling P, Lee KH, Wattrus SJ, Cho M, Lee RT, Wagers AJ. 2019. Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus. Scientific reports. 9(1):18613. Pubmed: 31819086 DOI:10.1038/s41598-019-54766-y Goldstein JM, Valido A, Lewandowski JP, Walker RG, Mills MJ, Messemer KA, Besseling P, Lee KH, Wattrus SJ, Cho M, Lee RT, Wagers AJ. 2019. Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus. Scientific reports. 9(1):18613. Pubmed: 31819086 DOI:10.1038/s41598-019-54766-y Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11 allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts. -
Lewandowski JP, Lee JC, Hwang T, Sunwoo H, Goldstein JM, Groff AF, Chang NP, Mallard W, Williams A, Henao-Meija J, Flavell RA, Lee JT, Gerhardinger C, Wagers AJ, Rinn JL. 2019. The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis. Nature communications. 10(1):5137. Pubmed: 31723143 DOI:10.1038/s41467-019-12970-4 Lewandowski JP, Lee JC, Hwang T, Sunwoo H, Goldstein JM, Groff AF, Chang NP, Mallard W, Williams A, Henao-Meija J, Flavell RA, Lee JT, Gerhardinger C, Wagers AJ, Rinn JL. 2019. The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis. Nature communications. 10(1):5137. Pubmed: 31723143 DOI:10.1038/s41467-019-12970-4 RNA has been classically known to play central roles in biology, including maintaining telomeres, protein synthesis, and in sex chromosome compensation. While thousands of long noncoding RNAs (lncRNAs) have been identified, attributing RNA-based roles to lncRNA loci requires assessing whether phenotype(s) could be due to DNA regulatory elements, transcription, or the lncRNA. Here, we use the conserved X chromosome lncRNA locus Firre, as a model to discriminate between DNA- and RNA-mediated effects in vivo. We demonstrate that (i) Firre mutant mice have cell-specific hematopoietic phenotypes, and (ii) upon exposure to lipopolysaccharide, mice overexpressing Firre exhibit increased levels of pro-inflammatory cytokines and impaired survival. (iii) Deletion of Firre does not result in changes in local gene expression, but rather in changes on autosomes that can be rescued by expression of transgenic Firre RNA. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis. -
Yang VK, Rush JE, Bhasin S, Wagers AJ, Lee RT. 2019. Plasma growth differentiation factors 8 and 11 levels in cats with congestive heart failure secondary to hypertrophic cardiomyopathy. Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology. 25:41-51. Pubmed: 31568985 DOI:S1760-2734(18)30198-X Yang VK, Rush JE, Bhasin S, Wagers AJ, Lee RT. 2019. Plasma growth differentiation factors 8 and 11 levels in cats with congestive heart failure secondary to hypertrophic cardiomyopathy. Journal of veterinary cardiology : the official journal of the European Society of Veterinary Cardiology. 25:41-51. Pubmed: 31568985 DOI:S1760-2734(18)30198-X ArrayCopyright © 2019 Elsevier B.V. All rights reserved. -
Goldstein JM, Sengul H, Messemer KA, Fernández-Alfara M, Garbern JC, Kristl AC, Lee RT, Wagers AJ. 2019. Steady-state and regenerative hematopoiesis occurs normally in mice in the absence of GDF11. Blood. 134(20):1712-1716. Pubmed: 31530563 DOI:10.1182/blood.2019002066 Goldstein JM, Sengul H, Messemer KA, Fernández-Alfara M, Garbern JC, Kristl AC, Lee RT, Wagers AJ. 2019. Steady-state and regenerative hematopoiesis occurs normally in mice in the absence of GDF11. Blood. 134(20):1712-1716. Pubmed: 31530563 DOI:10.1182/blood.2019002066 Tightly regulated production of mature blood cells is essential for health and survival in vertebrates and dependent on discrete populations of blood-forming (hematopoietic) stem and progenitor cells. Prior studies suggested that inhibition of growth differentiation factor 11 (GDF11) through soluble activin receptor type II (ActRII) ligand traps or neutralizing antibodies promotes erythroid precursor cell maturation and red blood cell formation in contexts of homeostasis and anemia. As Gdf11 is expressed by mature hematopoietic cells, and erythroid precursor cell expression of Gdf11 has been implicated in regulating erythropoiesis, we hypothesized that genetic disruption of Gdf11 in blood cells might perturb normal hematopoiesis or recovery from hematopoietic insult. Contrary to these predictions, we found that deletion of Gdf11 in the hematopoietic lineage in mice does not alter erythropoiesis or erythroid precursor cell frequency under normal conditions or during hematopoietic recovery after irradiation and transplantation. In addition, although hematopoietic cell-derived Gdf11 may contribute to the pool of circulating GDF11 protein during adult homeostasis, loss of Gdf11 specifically in the blood system does not impair hematopoietic stem cell function or induce overt pathological consequences. Taken together, these results reveal that hematopoietic cell-derived Gdf11 is largely dispensable for native and transplant-induced blood formation.© 2019 by The American Society of Hematology. -
Garbern J, Kristl AC, Bassaneze V, Vujic A, Schoemaker H, Sereda R, Peng L, Ricci-Blair EM, Goldstein JM, Walker RG, Bhasin S, Wagers AJ, Lee RT. 2019. Analysis of Cre-mediated genetic deletion of in cardiomyocytes of young mice. American journal of physiology. Heart and circulatory physiology. 317(1):H201-H212. Pubmed: 31125255 DOI:10.1152/ajpheart.00615.2018 Garbern J, Kristl AC, Bassaneze V, Vujic A, Schoemaker H, Sereda R, Peng L, Ricci-Blair EM, Goldstein JM, Walker RG, Bhasin S, Wagers AJ, Lee RT. 2019. Analysis of Cre-mediated genetic deletion of in cardiomyocytes of young mice. American journal of physiology. Heart and circulatory physiology. 317(1):H201-H212. Pubmed: 31125255 DOI:10.1152/ajpheart.00615.2018 Administration of active growth differentiation factor 11 (GDF11) to aged mice can reduce cardiac hypertrophy, and low serum levels of GDF11 measured together with the related protein, myostatin (also known as GDF8), predict future morbidity and mortality in coronary heart patients. Using mice with a loxP-flanked ("floxed") allele of and -driven expression of Cre recombinase to delete in cardiomyocytes, we tested the hypothesis that cardiac-specific deficiency might lead to cardiac hypertrophy in young adulthood. We observed that targeted deletion of in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation when compared with control mice carrying only the or -floxed alleles, suggesting a possible etiology for dilated cardiomyopathy. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected model. First, whole heart expression did not decrease in -floxed mice, possibly because of upregulation of in noncardiomyocytes in the heart. Second, we observed Cre-associated toxicity, with lower body weights and increased global fibrosis, in Cre-only control male mice compared with flox-only controls, making it challenging to infer which changes in floxed mice were the result of Cre toxicity versus deletion of . Third, we observed differential expression of mRNA in Cre-only controls compared with the cardiomyocyte-specific knockout mice, also making comparison between these two groups difficult. Thus, targeted deletion in cardiomyocytes may lead to left ventricular dilation without hypertrophy, but alternative animal models are necessary to understand the mechanism for these findings. We observed that targeted deletion of growth differentiation factor 11 in cardiomyocytes does not cause cardiac hypertrophy but rather leads to left ventricular dilation compared with control mice carrying only the or growth differentiation factor 11-floxed alleles. However, the mechanism underlying this finding remains unclear because of multiple confounding effects associated with the selected mouse model. -
Carmody C, Ogawa-Wong AN, Martin C, Luongo C, Zuidwijk M, Sager B, Petersen T, Roginski Guetter A, Janssen R, Wu EY, Bogaards S, Neumann NM, Hau K, Marsili A, Boelen A, Silva JE, Dentice M, Salvatore D, Wagers AJ, Larsen PR, Simonides WS, Zavacki AM. 2019. A Global Loss of Dio2 Leads to Unexpected Changes in Function and Fiber Types of Slow Skeletal Muscle in Male Mice. Endocrinology. 160(5):1205-1222. Pubmed: 30951174 DOI:10.1210/en.2019-00088 Carmody C, Ogawa-Wong AN, Martin C, Luongo C, Zuidwijk M, Sager B, Petersen T, Roginski Guetter A, Janssen R, Wu EY, Bogaards S, Neumann NM, Hau K, Marsili A, Boelen A, Silva JE, Dentice M, Salvatore D, Wagers AJ, Larsen PR, Simonides WS, Zavacki AM. 2019. A Global Loss of Dio2 Leads to Unexpected Changes in Function and Fiber Types of Slow Skeletal Muscle in Male Mice. Endocrinology. 160(5):1205-1222. Pubmed: 30951174 DOI:10.1210/en.2019-00088 The type 2 iodothyronine-deiodinase (D2) enzyme converts T4 to T3, and mice deficient in this enzyme [D2 knockout (D2KO) mice] have decreased T3 derived from T4 in skeletal muscle despite normal circulating T3 levels. Because slow skeletal muscle is particularly susceptible to changes in T3 levels, we expected D2 inactivation to result in more pronounced slow-muscle characteristics in the soleus muscle, mirroring hypothyroidism. However, ex vivo studies of D2KO soleus revealed higher rates of twitch contraction and relaxation and reduced resistance to fatigue. Immunostaining of D2KO soleus showed that these properties were associated with changes in muscle fiber type composition, including a marked increase in the number of fast, glycolytic type IIB fibers. D2KO soleus muscle fibers had a larger cross-sectional area, and this correlated with increased myonuclear accretion in myotubes formed from D2KO skeletal muscle precursor cells differentiated in vitro. Consistent with our functional findings, D2KO soleus gene expression was markedly different from that in hypothyroid wild-type (WT) mice. Comparison of gene expression between euthyroid WT and D2KO mice indicated that PGC-1α, a T3-dependent regulator of slow muscle fiber type, was decreased by ∼50% in D2KO soleus. Disruption of Dio2 in the C2C12 myoblast cell line led to a significant decrease in PGC-1α expression and a faster muscle phenotype upon differentiation. These results indicate that D2 loss leads to significant changes in soleus contractile function and fiber type composition that are inconsistent with local hypothyroidism and suggest that reduced levels of PCG-1α may contribute to the observed phenotypical changes.Copyright © 2019 Endocrine Society. -
Burzyn D, Wagers A, Mathis D, Cerletti M. 2019. Methods of Isolation and Analysis of TREG Immune Infiltrates from Injured and Dystrophic Skeletal Muscle. Methods in molecular biology (Clifton, N.J.). 1899:229-237. Pubmed: 30649776 DOI:10.1007/978-1-4939-8938-6_16 Burzyn D, Wagers A, Mathis D, Cerletti M. 2019. Methods of Isolation and Analysis of TREG Immune Infiltrates from Injured and Dystrophic Skeletal Muscle. Methods in molecular biology (Clifton, N.J.). 1899:229-237. Pubmed: 30649776 DOI:10.1007/978-1-4939-8938-6_16 The immune infiltrate present in acutely injured or dystrophic skeletal muscle has been shown to play an important role in the process of muscle regeneration. Our work has described, for the first time, muscle regulatory T cells (Tregs), a unique population in phenotype and function capable of promoting skeletal muscle repair. Herein, we describe the methods we have optimized to study muscle Tregs, including their isolation from injured muscle, immuno-labeling for analysis/separation by flow cytometry, and measurement of their proliferation status. -
Yoshida H, Lareau CA, Ramirez RN, Rose SA, Maier B, Wroblewska A, Desland F, Chudnovskiy A, Mortha A, Dominguez C, Tellier J, Kim E, Dwyer D, Shinton S, Nabekura T, Qi Y, Yu B, Robinette M, Kim KW, Wagers A, Rhoads A, Nutt SL, Brown BD, Mostafavi S, Buenrostro JD, Benoist C. 2019. The cis-Regulatory Atlas of the Mouse Immune System. Cell. 176(4):897-912.e20. Pubmed: 30686579 DOI:S0092-8674(18)31650-7 Yoshida H, Lareau CA, Ramirez RN, Rose SA, Maier B, Wroblewska A, Desland F, Chudnovskiy A, Mortha A, Dominguez C, Tellier J, Kim E, Dwyer D, Shinton S, Nabekura T, Qi Y, Yu B, Robinette M, Kim KW, Wagers A, Rhoads A, Nutt SL, Brown BD, Mostafavi S, Buenrostro JD, Benoist C. 2019. The cis-Regulatory Atlas of the Mouse Immune System. Cell. 176(4):897-912.e20. Pubmed: 30686579 DOI:S0092-8674(18)31650-7 A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.Copyright © 2018 Elsevier Inc. All rights reserved. -
Goldstein JM, Tabebordbar M, Zhu K, Wang LD, Messemer KA, Peacker B, Ashrafi Kakhki S, Gonzalez-Celeiro M, Shwartz Y, Cheng JKW, Xiao R, Barungi T, Albright C, Hsu YC, Vandenberghe LH, Wagers AJ. 2019. In Situ Modification of Tissue Stem and Progenitor Cell Genomes. Cell reports. 27(4):1254-1264.e7. Pubmed: 31018138 DOI:S2211-1247(19)30452-8 Goldstein JM, Tabebordbar M, Zhu K, Wang LD, Messemer KA, Peacker B, Ashrafi Kakhki S, Gonzalez-Celeiro M, Shwartz Y, Cheng JKW, Xiao R, Barungi T, Albright C, Hsu YC, Vandenberghe LH, Wagers AJ. 2019. In Situ Modification of Tissue Stem and Progenitor Cell Genomes. Cell reports. 27(4):1254-1264.e7. Pubmed: 31018138 DOI:S2211-1247(19)30452-8 In vivo delivery of genome-modifying enzymes holds significant promise for therapeutic applications and functional genetic screening. Delivery to endogenous tissue stem cells, which provide an enduring source of cell replacement during homeostasis and regeneration, is of particular interest. Here, we use a sensitive Cre/lox fluorescent reporter system to test the efficiency of genome modification following in vivo transduction by adeno-associated viruses (AAVs) in tissue stem and progenitor cells. We combine immunophenotypic analyses with in vitro and in vivo assays of stem cell function to reveal effective targeting of skeletal muscle satellite cells, mesenchymal progenitors, hematopoietic stem cells, and dermal cell subsets using multiple AAV serotypes. Genome modification rates achieved through this system reached >60%, and modified cells retained key functional properties. This study establishes a powerful platform to genetically alter tissue progenitors within their physiological niche while preserving their native stem cell properties and regulatory interactions.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. 2018
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Goldstein JM, Wagers AJ. 2018. What's in a (Sub)strain?. Stem cell reports. 11(2):303-305. Pubmed: 30110622 DOI:S2213-6711(18)30316-3 Goldstein JM, Wagers AJ. 2018. What's in a (Sub)strain?. Stem cell reports. 11(2):303-305. Pubmed: 30110622 DOI:S2213-6711(18)30316-3 C57BL/6J and C57BL/6N inbred mice are widely, and often interchangeably, used for stem cell research; yet, these substrains harbor discrete genetic differences that can cause phenotypic disparities. In this issue of Stem Cell Reports, Morales-Hernández et al. identify one particular difference-disruption of Nicotinamide Nucleotide Transhydrogenase (Nnt)-that increases reactive oxygen exposure and impairs hematopoietic progenitor cell function in C57BL/6J, as compared to C57BL/6N, mice.Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved. -
Rao TN, Gupta MK, Softic S, Wang LD, Jang YC, Thomou T, Bezy O, Kulkarni RN, Kahn CR, Wagers AJ. 2018. Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors. The EMBO journal. 37(24). Pubmed: 30446598 DOI:10.15252/embj.2018100409 Rao TN, Gupta MK, Softic S, Wang LD, Jang YC, Thomou T, Bezy O, Kulkarni RN, Kahn CR, Wagers AJ. 2018. Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors. The EMBO journal. 37(24). Pubmed: 30446598 DOI:10.15252/embj.2018100409 A finely tuned balance of self-renewal, differentiation, proliferation, and survival governs the pool size and regenerative capacity of blood-forming hematopoietic stem and progenitor cells (HSPCs). Here, we report that protein kinase C delta (PKCδ) is a critical regulator of adult HSPC number and function that couples the proliferative and metabolic activities of HSPCs. PKCδ-deficient mice showed a pronounced increase in HSPC numbers, increased competence in reconstituting lethally irradiated recipients, enhanced long-term competitive advantage in serial transplantation studies, and an augmented HSPC recovery during stress. PKCδ-deficient HSPCs also showed accelerated proliferation and reduced apoptosis, but did not exhaust in serial transplant assays or induce leukemia. Using inducible knockout and transplantation models, we further found that PKCδ acts in a hematopoietic cell-intrinsic manner to restrict HSPC number and bone marrow regenerative function. Mechanistically, PKCδ regulates HSPC energy metabolism and coordinately governs multiple regulators within signaling pathways implicated in HSPC homeostasis. Together, these data identify PKCδ as a critical regulator of HSPC signaling and metabolism that acts to limit HSPC expansion in response to physiological and regenerative demands.© 2018 The Authors. Published under the terms of the CC BY 4.0 license. -
Teot LA, Schneider M, Thorner AR, Tian J, Chi YY, Ducar M, Lin L, Wlodarski M, Grier HE, Fletcher CDM, van Hummelen P, Skapek SX, Hawkins DS, Wagers AJ, Rodriguez-Galindo C, Hettmer S. 2018. Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group. Cancer. 124(9):1973-1981. Pubmed: 29461635 DOI:10.1002/cncr.31286 Teot LA, Schneider M, Thorner AR, Tian J, Chi YY, Ducar M, Lin L, Wlodarski M, Grier HE, Fletcher CDM, van Hummelen P, Skapek SX, Hawkins DS, Wagers AJ, Rodriguez-Galindo C, Hettmer S. 2018. Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group. Cancer. 124(9):1973-1981. Pubmed: 29461635 DOI:10.1002/cncr.31286 Array© 2018 American Cancer Society. -
Bar-Nur O, Gerli MFM, Di Stefano B, Almada AE, Galvin A, Coffey A, Huebner AJ, Feige P, Verheul C, Cheung P, Payzin-Dogru D, Paisant S, Anselmo A, Sadreyev RI, Ott HC, Tajbakhsh S, Rudnicki MA, Wagers AJ, Hochedlinger K. 2018. Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors. Stem cell reports. 10(5):1505-1521. Pubmed: 29742392 DOI:S2213-6711(18)30177-2 Bar-Nur O, Gerli MFM, Di Stefano B, Almada AE, Galvin A, Coffey A, Huebner AJ, Feige P, Verheul C, Cheung P, Payzin-Dogru D, Paisant S, Anselmo A, Sadreyev RI, Ott HC, Tajbakhsh S, Rudnicki MA, Wagers AJ, Hochedlinger K. 2018. Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors. Stem cell reports. 10(5):1505-1521. Pubmed: 29742392 DOI:S2213-6711(18)30177-2 Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7 cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved. 2017
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Walker RG, Czepnik M, Goebel EJ, McCoy JC, Vujic A, Cho M, Oh J, Aykul S, Walton KL, Schang G, Bernard DJ, Hinck AP, Harrison CA, Martinez-Hackert E, Wagers AJ, Lee RT, Thompson TB. 2017. Structural basis for potency differences between GDF8 and GDF11. BMC biology. 15(1):19. Pubmed: 28257634 DOI:10.1186/s12915-017-0350-1 Walker RG, Czepnik M, Goebel EJ, McCoy JC, Vujic A, Cho M, Oh J, Aykul S, Walton KL, Schang G, Bernard DJ, Hinck AP, Harrison CA, Martinez-Hackert E, Wagers AJ, Lee RT, Thompson TB. 2017. Structural basis for potency differences between GDF8 and GDF11. BMC biology. 15(1):19. Pubmed: 28257634 DOI:10.1186/s12915-017-0350-1 Array -
Sinha I, Sakthivel D, Olenchock BA, Kruse CR, Williams J, Varon DE, Smith JD, Madenci AL, Nuutila K, Wagers AJ. 2017. Prolyl Hydroxylase Domain-2 Inhibition Improves Skeletal Muscle Regeneration in a Male Murine Model of Obesity. Frontiers in endocrinology. 8:153. Pubmed: 28725215 DOI:10.3389/fendo.2017.00153 Sinha I, Sakthivel D, Olenchock BA, Kruse CR, Williams J, Varon DE, Smith JD, Madenci AL, Nuutila K, Wagers AJ. 2017. Prolyl Hydroxylase Domain-2 Inhibition Improves Skeletal Muscle Regeneration in a Male Murine Model of Obesity. Frontiers in endocrinology. 8:153. Pubmed: 28725215 DOI:10.3389/fendo.2017.00153 Obesity leads to a loss of muscle mass and impaired muscle regeneration. In obese individuals, pathologically elevated levels of prolyl hydroxylase domain enzyme 2 (PHD2) limit skeletal muscle hypoxia-inducible factor-1 alpha and vascular endothelial growth factor (VEGF) expression. Loss of local VEGF may further impair skeletal muscle regeneration. We hypothesized that PHD2 inhibition would restore vigorous muscle regeneration in a murine model of obesity. Adult (22-week-old) male mice were fed either a high-fat diet (HFD), with 60% of calories derived from fat, or a regular diet (RD), with 10% of calories derived from fat, for 16 weeks. On day 5 following cryoinjury to the tibialis anterior muscle, newly regenerated muscle fiber cross-sectional areas were significantly smaller in mice fed an HFD as compared to RD, indicating an impaired regenerative response. Cryoinjured gastrocnemius muscles of HFD mice also showed elevated PHD2 levels (twofold higher) and reduced VEGF levels (twofold lower) as compared to RD. Dimethyloxalylglycine, a cell permeable competitive inhibitor of PHD2, restored VEGF levels and significantly improved regenerating myofiber size in cryoinjured mice fed an HFD. We conclude that pathologically increased PHD2 in the obese state drives impairments in muscle regeneration, in part by blunting VEGF production. Inhibition of PHD2 over activity in the obese state normalizes VEGF levels and restores muscle regenerative potential. -
Kadoki M, Patil A, Thaiss CC, Brooks DJ, Pandey S, Deep D, Alvarez D, von Andrian UH, Wagers AJ, Nakai K, Mikkelsen TS, Soumillon M, Chevrier N. 2017. Organism-Level Analysis of Vaccination Reveals Networks of Protection across Tissues. Cell. 171(2):398-413.e21. Pubmed: 28942919 DOI:S0092-8674(17)30949-2 Kadoki M, Patil A, Thaiss CC, Brooks DJ, Pandey S, Deep D, Alvarez D, von Andrian UH, Wagers AJ, Nakai K, Mikkelsen TS, Soumillon M, Chevrier N. 2017. Organism-Level Analysis of Vaccination Reveals Networks of Protection across Tissues. Cell. 171(2):398-413.e21. Pubmed: 28942919 DOI:S0092-8674(17)30949-2 A fundamental challenge in immunology is to decipher the principles governing immune responses at the whole-organism scale. Here, using a comparative infection model, we observe immune signal propagation within and between organs to obtain a dynamic map of immune processes at the organism level. We uncover two inter-organ mechanisms of protective immunity mediated by soluble and cellular factors. First, analyzing ligand-receptor connectivity across tissues reveals that type I IFNs trigger a whole-body antiviral state, protecting the host within hours after skin vaccination. Second, combining parabiosis, single-cell analyses, and gene knockouts, we uncover a multi-organ web of tissue-resident memory T cells that functionally adapt to their environment to stop viral spread across the organism. These results have implications for manipulating tissue-resident memory T cells through vaccination and open up new lines of inquiry for the analysis of immune responses at the organism level.Copyright © 2017 Elsevier Inc. All rights reserved. 2016
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Oh J, Sinha I, Tan KY, Rosner B, Dreyfuss JM, Gjata O, Tran P, Shoelson SE, Wagers AJ. 2016. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging. 8(11):2871-2896. Pubmed: 27852976 DOI:10.18632/aging.101098 Oh J, Sinha I, Tan KY, Rosner B, Dreyfuss JM, Gjata O, Tran P, Shoelson SE, Wagers AJ. 2016. Age-associated NF-κB signaling in myofibers alters the satellite cell niche and re-strains muscle stem cell function. Aging. 8(11):2871-2896. Pubmed: 27852976 DOI:10.18632/aging.101098 Skeletal muscle is a highly regenerative tissue, but muscle repair potential is increasingly compromised with advancing age. In this study, we demonstrate that increased NF-κB activity in aged muscle fibers contributes to diminished myogenic potential of their associated satellite cells. We further examine the impact of genetic modulation of NF-κB signaling in muscle satellite cells or myofibers on recovery after damage. These studies reveal that NF-κB activity in differentiated myofibers is sufficient to drive dysfunction of muscle regenerative cells via cell-non-autonomous mechanisms. Inhibition of NF-κB, or its downstream target Phospholipase A2, in myofibers rescued muscle regenerative potential in aged muscle. Moreover, systemic administration of sodium salicylate, an FDA-approved NF-κB inhibitor, decreased inflammatory gene expression and improved repair in aged muscle. Together, these studies identify a unique NF-κB regulated, non-cell autonomous mechanism by which stem cell function is linked to lipid signaling and homeostasis, and provide important new targets to stimulate muscle repair in aged individuals. -
Dey D, Bagarova J, Hatsell SJ, Armstrong KA, Huang L, Ermann J, Vonner AJ, Shen Y, Mohedas AH, Lee A, Eekhoff EM, van Schie A, Demay MB, Keller C, Wagers AJ, Economides AN, Yu PB. 2016. Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic ossification. Science translational medicine. 8(366):366ra163. Pubmed: 27881824 Dey D, Bagarova J, Hatsell SJ, Armstrong KA, Huang L, Ermann J, Vonner AJ, Shen Y, Mohedas AH, Lee A, Eekhoff EM, van Schie A, Demay MB, Keller C, Wagers AJ, Economides AN, Yu PB. 2016. Two tissue-resident progenitor lineages drive distinct phenotypes of heterotopic ossification. Science translational medicine. 8(366):366ra163. Pubmed: 27881824 Fibrodysplasia ossificans progressiva (FOP), a congenital heterotopic ossification (HO) syndrome caused by gain-of-function mutations of bone morphogenetic protein (BMP) type I receptor ACVR1, manifests with progressive ossification of skeletal muscles, tendons, ligaments, and joints. In this disease, HO can occur in discrete flares, often triggered by injury or inflammation, or may progress incrementally without identified triggers. Mice harboring an Acvr1 knock-in allele recapitulate the phenotypic spectrum of FOP, including injury-responsive intramuscular HO and spontaneous articular, tendon, and ligament ossification. The cells that drive HO in these diverse tissues can be compartmentalized into two lineages: an Scx tendon-derived progenitor that mediates endochondral HO of ligaments and joints without exogenous injury, and a muscle-resident interstitial Mx1 population that mediates intramuscular, injury-dependent endochondral HO. Expression of Acvr1 in either lineage confers aberrant gain of BMP signaling and chondrogenic differentiation in response to activin A and gives rise to mutation-expressing hypertrophic chondrocytes in HO lesions. Compared to Acvr1, expression of the man-made, ligand-independent ACVR1 mutation accelerates and increases the penetrance of all observed phenotypes, but does not abrogate the need for antecedent injury in muscle HO, demonstrating the need for an injury factor in addition to enhanced BMP signaling. Both injury-dependent intramuscular and spontaneous ligament HO in Acvr1 knock-in mice were effectively controlled by the selective ACVR1 inhibitor LDN-212854. Thus, diverse phenotypes of HO found in FOP are rooted in cell-autonomous effects of dysregulated ACVR1 signaling in nonoverlapping tissue-resident progenitor pools that may be addressed by systemic therapy or by modulating injury-mediated factors involved in their local recruitment.Copyright © 2016, American Association for the Advancement of Science. -
Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. 2016. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science (New York, N.Y.). 351(6271):407-411. Pubmed: 26721686 DOI:10.1126/science.aad5177 Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran FA, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. 2016. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science (New York, N.Y.). 351(6271):407-411. Pubmed: 26721686 DOI:10.1126/science.aad5177 Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.Copyright © 2016, American Association for the Advancement of Science. -
Song L, Papaioannou G, Zhao H, Luderer HF, Miller C, Dall'Osso C, Nazarian RM, Wagers AJ, Demay MB. 2016. The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury. Endocrinology. 157(10):4066-4075. Pubmed: 27526034 Song L, Papaioannou G, Zhao H, Luderer HF, Miller C, Dall'Osso C, Nazarian RM, Wagers AJ, Demay MB. 2016. The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury. Endocrinology. 157(10):4066-4075. Pubmed: 27526034 Ligand-dependent actions of the vitamin D receptor (VDR) play a pleiotropic role in the regulation of innate and adaptive immunity. The liganded VDR is required for recruitment of macrophages during the inflammatory phase of cutaneous wound healing. Although the number of macrophages in the granulation tissue 2 days after wounding is markedly reduced in VDR knockout (KO) compared with wild-type mice, VDR ablation does not alter macrophage polarization. Parabiosis studies demonstrate that circulatory chimerism with wild-type mice is unable to rescue the macrophage defect in the wounds of VDR KO mice and reveal that wound macrophages are of local origin, regardless of VDR status. Wound cytokine analyses demonstrated a decrease in macrophage colony-stimulating factor (M-CSF) protein levels in VDR KO mice. Consistent with this, induction of M-CSF gene expression by TGFβ and 1,25-dihydroxyvitamin D was impaired in dermal fibroblasts isolated from VDR KO mice. Because M-CSF is important for macrophage self-renewal, studies were performed to evaluate the response of tissue resident macrophages to this cytokine. A decrease in M-CSF induced proliferation and cyclin D1 expression was observed in peritoneal resident macrophages isolated from VDR KO mice, suggesting an intrinsic macrophage abnormality. Consistent with this, wound-healing assays in mice with macrophage-specific VDR ablation demonstrate that a normal wound microenvironment cannot compensate for the absence of the VDR in macrophages and thus confirm a critical role for the macrophage VDR in the inflammatory response to injury. -
Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. 2016. A multifunctional AAV-CRISPR-Cas9 and its host response. Nature methods. 13(10):868-74. Pubmed: 27595405 DOI:10.1038/nmeth.3993 Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, Wagers AJ, Church GM. 2016. A multifunctional AAV-CRISPR-Cas9 and its host response. Nature methods. 13(10):868-74. Pubmed: 27595405 DOI:10.1038/nmeth.3993 CRISPR-Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV-CRISPR-Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics. -
Maesner CC, Almada AE, Wagers AJ. 2016. Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting. Skeletal muscle. 6:35. Pubmed: 27826411 DOI:10.1186/s13395-016-0106-6 Maesner CC, Almada AE, Wagers AJ. 2016. Established cell surface markers efficiently isolate highly overlapping populations of skeletal muscle satellite cells by fluorescence-activated cell sorting. Skeletal muscle. 6:35. Pubmed: 27826411 DOI:10.1186/s13395-016-0106-6 Array -
Poggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, Gonzalez E, Walker RG, Thompson TB, Wagers AJ, Fong YW, Lee RT. 2016. Circulating Growth Differentiation Factor 11/8 Levels Decline With Age. Circulation research. 118(1):29-37. Pubmed: 26489925 DOI:10.1161/CIRCRESAHA.115.307521 Poggioli T, Vujic A, Yang P, Macias-Trevino C, Uygur A, Loffredo FS, Pancoast JR, Cho M, Goldstein J, Tandias RM, Gonzalez E, Walker RG, Thompson TB, Wagers AJ, Fong YW, Lee RT. 2016. Circulating Growth Differentiation Factor 11/8 Levels Decline With Age. Circulation research. 118(1):29-37. Pubmed: 26489925 DOI:10.1161/CIRCRESAHA.115.307521 Array© 2015 American Heart Association, Inc. -
Katagiri S, Park K, Maeda Y, Rao TN, Khamaisi M, Li Q, Yokomizo H, Mima A, Lancerotto L, Wagers A, Orgill DP, King GL. 2016. Overexpressing IRS1 in Endothelial Cells Enhances Angioblast Differentiation and Wound Healing in Diabetes and Insulin Resistance. Diabetes. 65(9):2760-71. Pubmed: 27217486 DOI:10.2337/db15-1721 Katagiri S, Park K, Maeda Y, Rao TN, Khamaisi M, Li Q, Yokomizo H, Mima A, Lancerotto L, Wagers A, Orgill DP, King GL. 2016. Overexpressing IRS1 in Endothelial Cells Enhances Angioblast Differentiation and Wound Healing in Diabetes and Insulin Resistance. Diabetes. 65(9):2760-71. Pubmed: 27217486 DOI:10.2337/db15-1721 The effect of enhancing insulin's actions in endothelial cells (ECs) to improve angiogenesis and wound healing was studied in obesity and diabetes. Insulin receptor substrate 1 (IRS1) was overexpressed in ECs using the VE-cadherin promoter to create ECIRS1 TG mice, which elevated pAkt activation and expressions of vascular endothelial growth factor (VEGF), Flk1, and VE-cadherin in ECs and granulation tissues (GTs) of full-thickness wounds. Open wound and epithelialization rates and angiogenesis significantly improved in normal mice and high fat (HF) diet-induced diabetic mice with hyperinsulinemia in ECIRS1 TG versus wild type (WT), but not in insulin-deficient diabetic mice. Increased angioblasts and EC numbers in GT of ECIRS1 mice were due to proliferation in situ rather than uptake. GT in HF-fed diabetic mice exhibited parallel decreases in insulin and VEGF-induced pAkt and EC numbers by >50% without changes in angioblasts versus WT mice, which were improved in ECIRS1 TG mice on normal chow or HF diet. Thus, HF-induced diabetes impaired angiogenesis by inhibiting insulin signaling in GT to decrease the differentiation of angioblasts to EC, which was normalized by enhancing insulin's action targeted to EC, a potential target to improve wound healing in diabetes and obesity.© 2016 by the American Diabetes Association. -
Wang LD, Ficarro SB, Hutchinson JN, Csepanyi-Komi R, Nguyen PT, Wisniewski E, Sullivan J, Hofmann O, Ligeti E, Marto JA, Wagers AJ. 2016. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization. Blood. 128(11):1465-74. Pubmed: 27365422 DOI:10.1182/blood-2016-05-711424 Wang LD, Ficarro SB, Hutchinson JN, Csepanyi-Komi R, Nguyen PT, Wisniewski E, Sullivan J, Hofmann O, Ligeti E, Marto JA, Wagers AJ. 2016. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization. Blood. 128(11):1465-74. Pubmed: 27365422 DOI:10.1182/blood-2016-05-711424 Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization.© 2016 by The American Society of Hematology. -
Gage FH, Guarente LP, Wagers AJ. 2016. Aging and Rejuvenation: Insights from Rusty Gage, Leonard Guarente, and Amy Wagers. Trends in molecular medicine. 22(8):633-634. Pubmed: 27396512 DOI:S1471-4914(16)30073-9 Gage FH, Guarente LP, Wagers AJ. 2016. Aging and Rejuvenation: Insights from Rusty Gage, Leonard Guarente, and Amy Wagers. Trends in molecular medicine. 22(8):633-634. Pubmed: 27396512 DOI:S1471-4914(16)30073-9 -
Rowe RG, Wang LD, Coma S, Han A, Mathieu R, Pearson DS, Ross S, Sousa P, Nguyen PT, Rodriguez A, Wagers AJ, Daley GQ. 2016. Developmental regulation of myeloerythroid progenitor function by the Lin28b-let-7-Hmga2 axis. The Journal of experimental medicine. 213(8):1497-512. Pubmed: 27401346 DOI:10.1084/jem.20151912 Rowe RG, Wang LD, Coma S, Han A, Mathieu R, Pearson DS, Ross S, Sousa P, Nguyen PT, Rodriguez A, Wagers AJ, Daley GQ. 2016. Developmental regulation of myeloerythroid progenitor function by the Lin28b-let-7-Hmga2 axis. The Journal of experimental medicine. 213(8):1497-512. Pubmed: 27401346 DOI:10.1084/jem.20151912 For appropriate development, tissue and organ system morphogenesis and maturation must occur in synchrony with the overall developmental requirements of the host. Mistiming of such developmental events often results in disease. The hematopoietic system matures from the fetal state, characterized by robust erythrocytic output that supports prenatal growth in the hypoxic intrauterine environment, to the postnatal state wherein granulocytes predominate to provide innate immunity. Regulation of the developmental timing of these myeloerythroid states is not well understood. In this study, we find that expression of the heterochronic factor Lin28b decreases in common myeloid progenitors during hematopoietic maturation to adulthood in mice. This decrease in Lin28b coincides with accumulation of mature let-7 microRNAs, whose biogenesis is regulated by Lin28 proteins. We find that inhibition of let-7 in the adult hematopoietic system recapitulates fetal erythroid-dominant hematopoiesis. Conversely, deletion of Lin28b or ectopic activation of let-7 microRNAs in the fetal state induces a shift toward adult-like myeloid-dominant output. Furthermore, we identify Hmga2 as an effector of this genetic switch. These studies provide the first detailed analysis of the roles of endogenous Lin28b and let-7 in the timing of hematopoietic states during development.© 2016 Rowe et al. -
Khamaisi M, Katagiri S, Keenan H, Park K, Maeda Y, Li Q, Qi W, Thomou T, Eschuk D, Tellechea A, Veves A, Huang C, Orgill DP, Wagers A, King GL. 2016. PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts. The Journal of clinical investigation. 126(3):837-53. Pubmed: 26808499 DOI:82788 Khamaisi M, Katagiri S, Keenan H, Park K, Maeda Y, Li Q, Qi W, Thomou T, Eschuk D, Tellechea A, Veves A, Huang C, Orgill DP, Wagers A, King GL. 2016. PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts. The Journal of clinical investigation. 126(3):837-53. Pubmed: 26808499 DOI:82788 Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients. -
Kuswanto W, Burzyn D, Panduro M, Wang KK, Jang YC, Wagers AJ, Benoist C, Mathis D. 2016. Poor Repair of Skeletal Muscle in Aging Mice Reflects a Defect in Local, Interleukin-33-Dependent Accumulation of Regulatory T Cells. Immunity. 44(2):355-67. Pubmed: 26872699 DOI:S1074-7613(16)00034-0 Kuswanto W, Burzyn D, Panduro M, Wang KK, Jang YC, Wagers AJ, Benoist C, Mathis D. 2016. Poor Repair of Skeletal Muscle in Aging Mice Reflects a Defect in Local, Interleukin-33-Dependent Accumulation of Regulatory T Cells. Immunity. 44(2):355-67. Pubmed: 26872699 DOI:S1074-7613(16)00034-0 Normal repair of skeletal muscle requires local expansion of a special population of Foxp3(+)CD4(+) regulatory T (Treg) cells. Such cells failed to accumulate in acutely injured muscle of old mice, known to undergo ineffectual repair. This defect reflected reduced recruitment of Treg cells to injured muscle, as well as less proliferation and retention therein. Interleukin-33 (IL-33) regulated muscle Treg cell homeostasis in young mice, and its administration to old mice ameliorated their deficits in Treg cell accumulation and muscle regeneration. The major IL-33-expressing cells in skeletal muscle displayed a constellation of markers diagnostic of fibro/adipogenic progenitor cells and were often associated with neural structures, including nerve fibers, nerve bundles, and muscle spindles, which are stretch-sensitive mechanoreceptors important for proprioception. IL-33(+) cells were more frequent after muscle injury and were reduced in old mice. IL-33 is well situated to relay signals between the nervous and immune systems within the muscle context.Copyright © 2016 Elsevier Inc. All rights reserved. -
Olenchock BA, Moslehi J, Baik AH, Davidson SM, Williams J, Gibson WJ, Chakraborty AA, Pierce KA, Miller CM, Hanse EA, Kelekar A, Sullivan LB, Wagers AJ, Clish CB, Vander Heiden MG, Kaelin WG. 2016. EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection. Cell. 164(5):884-95. Pubmed: 26919427 DOI:S0092-8674(16)30072-1 Olenchock BA, Moslehi J, Baik AH, Davidson SM, Williams J, Gibson WJ, Chakraborty AA, Pierce KA, Miller CM, Hanse EA, Kelekar A, Sullivan LB, Wagers AJ, Clish CB, Vander Heiden MG, Kaelin WG. 2016. EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection. Cell. 164(5):884-95. Pubmed: 26919427 DOI:S0092-8674(16)30072-1 Ischemic preconditioning is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent, more prolonged, ischemic insult. In remote ischemic preconditioning (RIPC), ischemia to one organ protects others organs at a distance. We created mouse models to ask if inhibition of the alpha-ketoglutarate (αKG)-dependent dioxygenase Egln1, which senses oxygen and regulates the hypoxia-inducible factor (HIF) transcription factor, could suffice to mediate local and remote ischemic preconditioning. Using somatic gene deletion and a pharmacological inhibitor, we found that inhibiting Egln1 systemically or in skeletal muscles protects mice against myocardial ischemia-reperfusion (I/R) injury. Parabiosis experiments confirmed that RIPC in this latter model was mediated by a secreted factor. Egln1 loss causes accumulation of circulating αKG, which drives hepatic production and secretion of kynurenic acid (KYNA) that is necessary and sufficient to mediate cardiac ischemic protection in this setting.Copyright © 2016 Elsevier Inc. All rights reserved. -
Almada AE, Wagers AJ. 2016. Molecular circuitry of stem cell fate in skeletal muscle regeneration, ageing and disease. Nature reviews. Molecular cell biology. 17(5):267-79. Pubmed: 26956195 DOI:10.1038/nrm.2016.7 Almada AE, Wagers AJ. 2016. Molecular circuitry of stem cell fate in skeletal muscle regeneration, ageing and disease. Nature reviews. Molecular cell biology. 17(5):267-79. Pubmed: 26956195 DOI:10.1038/nrm.2016.7 Satellite cells are adult myogenic stem cells that repair damaged muscle. The enduring capacity for muscle regeneration requires efficient satellite cell expansion after injury, their differentiation to produce myoblasts that can reconstitute damaged fibres and their self-renewal to replenish the muscle stem cell pool for subsequent rounds of injury and repair. Emerging studies indicate that misregulation of satellite cell fate and function can contribute to age-associated muscle dysfunction and influence the severity of muscle diseases, including Duchenne muscular dystrophy (DMD). It has also become apparent that satellite cell fate during muscle regeneration and ageing, and in the context of DMD, is governed by an intricate network of intrinsic and extrinsic regulators. Targeted manipulation of this network may offer unique opportunities for muscle regenerative medicine. -
Walker RG, Poggioli T, Katsimpardi L, Buchanan SM, Oh J, Wattrus S, Heidecker B, Fong YW, Rubin LL, Ganz P, Thompson TB, Wagers AJ, Lee RT. 2016. Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation. Circulation research. 118(7):1125-41; discussion 1142. Pubmed: 27034275 DOI:10.1161/CIRCRESAHA.116.308391 Walker RG, Poggioli T, Katsimpardi L, Buchanan SM, Oh J, Wattrus S, Heidecker B, Fong YW, Rubin LL, Ganz P, Thompson TB, Wagers AJ, Lee RT. 2016. Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation. Circulation research. 118(7):1125-41; discussion 1142. Pubmed: 27034275 DOI:10.1161/CIRCRESAHA.116.308391 Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor β superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.© 2016 American Heart Association, Inc. -
Hettmer S, Lin MM, Tchessalova D, Tortorici SJ, Castiglioni A, Desai T, Mao J, McMahon AP, Wagers AJ. 2016. Hedgehog-driven myogenic tumors recapitulate skeletal muscle cellular heterogeneity. Experimental cell research. 340(1):43-52. Pubmed: 26460176 DOI:S0014-4827(15)30113-0 Hettmer S, Lin MM, Tchessalova D, Tortorici SJ, Castiglioni A, Desai T, Mao J, McMahon AP, Wagers AJ. 2016. Hedgehog-driven myogenic tumors recapitulate skeletal muscle cellular heterogeneity. Experimental cell research. 340(1):43-52. Pubmed: 26460176 DOI:S0014-4827(15)30113-0 Hedgehog (Hh) pathway activation in R26-SmoM2;CAGGS-CreER mice, which carry a tamoxifen-inducible activated Smoothened allele (SmoM2), results in numerous microscopic tumor foci in mouse skeletal muscle. These tumors exhibit a highly differentiated myogenic phenotype and resemble human fetal rhabdomyomas. This study sought to apply previously established strategies to isolate lineally distinct populations of normal mouse myofiber-associated cells in order to examine cellular heterogeneity in SmoM2 tumors. We demonstrate that established SmoM2 tumors are composed of cells expressing myogenic, adipocytic and hematopoietic lineage markers and differentiation capacity. SmoM2 tumors thus recapitulate the phenotypic and functional hetereogeneity observed in normal mouse skeletal muscle. SmoM2 tumors also contain an expanded population of PAX7+ and MyoD+ satellite-like cells with extremely low clonogenic activity. Selective activation of Hh signaling in freshly isolated muscle satellite cells enhanced terminal myogenic differentiation without stimulating proliferation. Our findings support the conclusion that SmoM2 tumors represent an aberrant skeletal muscle state and demonstrate that, similar to normal muscle, myogenic tumors contain functionally distinct cell subsets, including cells lacking myogenic differentiation potential.Copyright © 2015 Elsevier Inc. All rights reserved. -
Olenchock BA, Moslehi J, Baik AH, Davidson SM, Williams J, Gibson WJ, Chakraborty AA, Pierce KA, Miller CM, Hanse EA, Kelekar A, Sullivan LB, Wagers AJ, Clish CB, Vander Heiden MG, Kaelin WG. 2016. EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection. Cell. 165(2):497. Pubmed: 27058668 DOI:S0092-8674(16)30335-X Olenchock BA, Moslehi J, Baik AH, Davidson SM, Williams J, Gibson WJ, Chakraborty AA, Pierce KA, Miller CM, Hanse EA, Kelekar A, Sullivan LB, Wagers AJ, Clish CB, Vander Heiden MG, Kaelin WG. 2016. EGLN1 Inhibition and Rerouting of α-Ketoglutarate Suffice for Remote Ischemic Protection. Cell. 165(2):497. Pubmed: 27058668 DOI:S0092-8674(16)30335-X 2015
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Bhatt S, Gupta MK, Khamaisi M, Martinez R, Gritsenko MA, Wagner BK, Guye P, Busskamp V, Shirakawa J, Wu G, Liew CW, Clauss TR, Valdez I, El Ouaamari A, Dirice E, Takatani T, Keenan HA, Smith RD, Church G, Weiss R, Wagers AJ, Qian WJ, King GL, Kulkarni RN. 2015. Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes. Cell metabolism. 22(2):239-52. Pubmed: 26244933 DOI:S1550-4131(15)00344-7 Bhatt S, Gupta MK, Khamaisi M, Martinez R, Gritsenko MA, Wagner BK, Guye P, Busskamp V, Shirakawa J, Wu G, Liew CW, Clauss TR, Valdez I, El Ouaamari A, Dirice E, Takatani T, Keenan HA, Smith RD, Church G, Weiss R, Wagers AJ, Qian WJ, King GL, Kulkarni RN. 2015. Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes. Cell metabolism. 22(2):239-52. Pubmed: 26244933 DOI:S1550-4131(15)00344-7 The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. We propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.Copyright © 2015 Elsevier Inc. All rights reserved. -
Gupta MK, Teo AK, Rao TN, Bhatt S, Kleinridders A, Shirakawa J, Takatani T, Hu J, De Jesus DF, Windmueller R, Wagers AJ, Kulkarni RN. 2015. Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts. Stem cells translational medicine. 4(10):1101-8. Pubmed: 26253715 DOI:10.5966/sctm.2014-0217 Gupta MK, Teo AK, Rao TN, Bhatt S, Kleinridders A, Shirakawa J, Takatani T, Hu J, De Jesus DF, Windmueller R, Wagers AJ, Kulkarni RN. 2015. Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts. Stem cells translational medicine. 4(10):1101-8. Pubmed: 26253715 DOI:10.5966/sctm.2014-0217 Array©AlphaMed Press. -
Hettmer S, Bronson RT, Wagers AJ. 2015. Distinct malignant behaviors of mouse myogenic tumors induced by different oncogenetic lesions. Frontiers in oncology. 5:50. Pubmed: 25759794 DOI:10.3389/fonc.2015.00050 Hettmer S, Bronson RT, Wagers AJ. 2015. Distinct malignant behaviors of mouse myogenic tumors induced by different oncogenetic lesions. Frontiers in oncology. 5:50. Pubmed: 25759794 DOI:10.3389/fonc.2015.00050 Rhabdomyosarcomas (RMS) are heterogeneous cancers with myogenic differentiation features. The cytogenetic and mutational aberrations in RMS are diverse. This study examined differences in the malignant behavior of two genetically distinct and disease-relevant mouse myogenic tumor models. Kras; p1619(null) myogenic tumors, initiated by expression of oncogenic Kras in p16p19(null) mouse satellite cells, were metastatic to the lungs of the majority of tumor-bearing animals and repopulated tumors in seven of nine secondary recipients. In contrast, SmoM2 tumors, initiated by ubiquitous expression of a mutant Smoothened allele, did not metastasize and repopulated tumors in 2 of 18 recipients only. In summary, genetically distinct myogenic tumors in mice exhibit marked differences in malignant behavior. -
Kowalczyk MS, Tirosh I, Heckl D, Rao TN, Dixit A, Haas BJ, Schneider RK, Wagers AJ, Ebert BL, Regev A. 2015. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome research. 25(12):1860-72. Pubmed: 26430063 DOI:10.1101/gr.192237.115 Kowalczyk MS, Tirosh I, Heckl D, Rao TN, Dixit A, Haas BJ, Schneider RK, Wagers AJ, Ebert BL, Regev A. 2015. Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells. Genome research. 25(12):1860-72. Pubmed: 26430063 DOI:10.1101/gr.192237.115 Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs.© 2015 Kowalczyk et al.; Published by Cold Spring Harbor Laboratory Press. -
Hettmer S, Teot LA, Kozakewich H, Werger AM, Davies KJ, Fletcher CD, Grier HE, Rodriguez-Galindo C, Wagers AJ. 2015. Myogenic tumors in nevoid Basal cell carcinoma syndrome. Journal of pediatric hematology/oncology. 37(2):147-9. Pubmed: 24517962 DOI:10.1097/MPH.0000000000000115 Hettmer S, Teot LA, Kozakewich H, Werger AM, Davies KJ, Fletcher CD, Grier HE, Rodriguez-Galindo C, Wagers AJ. 2015. Myogenic tumors in nevoid Basal cell carcinoma syndrome. Journal of pediatric hematology/oncology. 37(2):147-9. Pubmed: 24517962 DOI:10.1097/MPH.0000000000000115 In mice, activated Hedgehog (Hh) signaling induces tumors with myogenic differentiation. In humans, hyperactive Hh signaling due to germline PATCHED1 (PTCH1) mutations has been linked to nevoid basal cell carcinoma syndrome (NBCCS). We report an embryonal rhabdomyosarcoma in a 16-month-old girl with NBCCS and review the literature on myogenic neoplasms in NBCCS, including 8 fetal rhabdomyomas and 3 rhabdomyosarcomas. Of note, 3 population studies, including 255 individuals with NBCCS aged 4 months to 87 years, did not identify any myogenic tumors. Thus, myogenic tumors in NBCCS are rare and include both rhabdomyosarcomas and fetal rhabdomyomas. -
Hettmer S, Schinzel AC, Tchessalova D, Schneider M, Parker CL, Bronson RT, Richards NG, Hahn WC, Wagers AJ. 2015. Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma. eLife. 4. Pubmed: 26499495 DOI:10.7554/eLife.09436 Hettmer S, Schinzel AC, Tchessalova D, Schneider M, Parker CL, Bronson RT, Richards NG, Hahn WC, Wagers AJ. 2015. Functional genomic screening reveals asparagine dependence as a metabolic vulnerability in sarcoma. eLife. 4. Pubmed: 26499495 DOI:10.7554/eLife.09436 Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value. -
Rao TN, Marks-Bluth J, Sullivan J, Gupta MK, Chandrakanthan V, Fitch SR, Ottersbach K, Jang YC, Piao X, Kulkarni RN, Serwold T, Pimanda JE, Wagers AJ. 2015. High-level Gpr56 expression is dispensable for the maintenance and function of hematopoietic stem and progenitor cells in mice. Stem cell research. 14(3):307-22. Pubmed: 25840412 DOI:S1873-5061(15)00028-8 Rao TN, Marks-Bluth J, Sullivan J, Gupta MK, Chandrakanthan V, Fitch SR, Ottersbach K, Jang YC, Piao X, Kulkarni RN, Serwold T, Pimanda JE, Wagers AJ. 2015. High-level Gpr56 expression is dispensable for the maintenance and function of hematopoietic stem and progenitor cells in mice. Stem cell research. 14(3):307-22. Pubmed: 25840412 DOI:S1873-5061(15)00028-8 Blood formation by hematopoietic stem cells (HSCs) is regulated by a still incompletely defined network of general and HSC-specific regulators. In this study, we analyzed the role of G-protein coupled receptor 56 (Gpr56) as a candidate HSC regulator based on its differential expression in quiescent relative to proliferating HSCs and its common targeting by core HSC regulators. Detailed expression analysis revealed that Gpr56 is abundantly expressed by HSPCs during definitive hematopoiesis in the embryo and in the adult bone marrow, but its levels are reduced substantially as HSPCs differentiate. However, despite enriched expression in HSPCs, Gpr56-deficiency did not impair HSPC maintenance or function during steady-state or myeloablative stress-induced hematopoiesis. Gpr56-deficient HSCs also responded normally to physiological and pharmacological mobilization signals, despite the reported role of this GPCR as a regulator of cell adhesion and migration in neuronal cells. Moreover, Gpr56-deficient bone marrow engrafted with equivalent efficiency as wild-type HSCs in primary recipients; however, their reconstituting ability was reduced when subjected to serial transplantation. These data indicate that although GPR56 is abundantly and selectively expressed by primitive HSPCs, its high level expression is largely dispensable for steady-state and regenerative hematopoiesis.Copyright © 2015. Published by Elsevier B.V. -
Artandi SE, Blau HM, de Haan G, Geiger H, Goodell MA, Jones L, Levine RL, Muñoz-Cánoves P, Rodewald HR, Wagers A, Wang ZQ, Yamashita Y. 2015. Stem Cells and Aging: What's Next?. Cell stem cell. 16(6):578-81. Pubmed: 26247067 Artandi SE, Blau HM, de Haan G, Geiger H, Goodell MA, Jones L, Levine RL, Muñoz-Cánoves P, Rodewald HR, Wagers A, Wang ZQ, Yamashita Y. 2015. Stem Cells and Aging: What's Next?. Cell stem cell. 16(6):578-81. Pubmed: 26247067 -
Castiglioni A, Corna G, Rigamonti E, Basso V, Vezzoli M, Monno A, Almada AE, Mondino A, Wagers AJ, Manfredi AA, Rovere-Querini P. 2015. FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration. PloS one. 10(6):e0128094. Pubmed: 26039259 DOI:10.1371/journal.pone.0128094 Castiglioni A, Corna G, Rigamonti E, Basso V, Vezzoli M, Monno A, Almada AE, Mondino A, Wagers AJ, Manfredi AA, Rovere-Querini P. 2015. FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration. PloS one. 10(6):e0128094. Pubmed: 26039259 DOI:10.1371/journal.pone.0128094 Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue. -
Reeves AZ, Spears WE, Du J, Tan KY, Wagers AJ, Lesser CF. 2015. Engineering Escherichia coli into a protein delivery system for mammalian cells. ACS synthetic biology. 4(5):644-54. Pubmed: 25853840 DOI:10.1021/acssynbio.5b00002 Reeves AZ, Spears WE, Du J, Tan KY, Wagers AJ, Lesser CF. 2015. Engineering Escherichia coli into a protein delivery system for mammalian cells. ACS synthetic biology. 4(5):644-54. Pubmed: 25853840 DOI:10.1021/acssynbio.5b00002 Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications. -
Wang LD, Rao TN, Rowe RG, Nguyen PT, Sullivan JL, Pearson DS, Doulatov S, Wu L, Lindsley RC, Zhu H, DeAngelo DJ, Daley GQ, Wagers AJ. 2015. The role of Lin28b in myeloid and mast cell differentiation and mast cell malignancy. Leukemia. 29(6):1320-30. Pubmed: 25655194 DOI:10.1038/leu.2015.19 Wang LD, Rao TN, Rowe RG, Nguyen PT, Sullivan JL, Pearson DS, Doulatov S, Wu L, Lindsley RC, Zhu H, DeAngelo DJ, Daley GQ, Wagers AJ. 2015. The role of Lin28b in myeloid and mast cell differentiation and mast cell malignancy. Leukemia. 29(6):1320-30. Pubmed: 25655194 DOI:10.1038/leu.2015.19 Mast cells (MCs) are critical components of the innate immune system and important for host defense, allergy, autoimmunity, tissue regeneration and tumor progression. Dysregulated MC development leads to systemic mastocytosis (SM), a clinically variable but often devastating family of hematologic disorders. Here we report that induced expression of Lin28, a heterochronic gene and pluripotency factor implicated in driving a fetal hematopoietic program, caused MC accumulation in adult mice in target organs such as the skin and peritoneal cavity. In vitro assays revealed a skewing of myeloid commitment in LIN28B-expressing hematopoietic progenitors, with increased levels of LIN28B in common myeloid and basophil-MC progenitors altering gene expression patterns to favor cell fate choices that enhanced MC specification. In addition, LIN28B-induced MCs appeared phenotypically and functionally immature, and in vitro assays suggested a slowing of MC terminal differentiation in the context of LIN28B upregulation. Finally, interrogation of human MC leukemia samples revealed upregulation of LIN28B in abnormal MCs from patients with SM. This work identifies Lin28 as a novel regulator of innate immune function and a new protein of interest in MC disease. -
Kolodin D, van Panhuys N, Li C, Magnuson AM, Cipolletta D, Miller CM, Wagers A, Germain RN, Benoist C, Mathis D. 2015. Antigen- and cytokine-driven accumulation of regulatory T cells in visceral adipose tissue of lean mice. Cell metabolism. 21(4):543-57. Pubmed: 25863247 DOI:S1550-4131(15)00108-4 Kolodin D, van Panhuys N, Li C, Magnuson AM, Cipolletta D, Miller CM, Wagers A, Germain RN, Benoist C, Mathis D. 2015. Antigen- and cytokine-driven accumulation of regulatory T cells in visceral adipose tissue of lean mice. Cell metabolism. 21(4):543-57. Pubmed: 25863247 DOI:S1550-4131(15)00108-4 A unique population of Foxp3(+)CD4(+) regulatory T (Treg) cells, with a distinct transcriptome and antigen-receptor repertoire, resides in visceral adipose tissue (VAT) of lean individuals. These cells regulate local inflammation and both local and systemic metabolic indices. Here we focus on expansion of the VAT Treg compartment in aging lean mice-assessing these cells' phenotypic conversion from conventional CD4(+) T cells, influx from lymphoid organs, and local population dynamics. Our findings establish that the VAT Treg compartment is seeded from thymocytes generated during the first weeks of life and expands beyond 10 weeks of age due to indolent proliferation, of certain clones in particular, coupled with enhanced survival. Accumulation of VAT Tregs depends on the antigen(s) presented by MHC class-II molecules and soluble mediators, notably interleukin(IL)-33. Addressing such factors therapeutically promises novel approaches for harnessing Tregs to stem the growing epidemic of obesity and consequent metabolic abnormalities.Copyright © 2015 Elsevier Inc. All rights reserved. -
Kim MJ, Miller CM, Shadrach JL, Wagers AJ, Serwold T. 2015. Young, proliferative thymic epithelial cells engraft and function in aging thymuses. Journal of immunology (Baltimore, Md. : 1950). 194(10):4784-95. Pubmed: 25870244 DOI:10.4049/jimmunol.1403158 Kim MJ, Miller CM, Shadrach JL, Wagers AJ, Serwold T. 2015. Young, proliferative thymic epithelial cells engraft and function in aging thymuses. Journal of immunology (Baltimore, Md. : 1950). 194(10):4784-95. Pubmed: 25870244 DOI:10.4049/jimmunol.1403158 The thymus reaches its maximum size early in life and then begins to shrink, producing fewer T cells with increasing age. This thymic decline is thought to contribute to age-related T cell lymphopenias and hinder T cell recovery after bone marrow transplantation. Although several cellular and molecular processes have been implicated in age-related thymic involution, their relative contributions are not known. Using heterochronic parabiosis, we observe that young circulating factors are not sufficient to drive regeneration of the aged thymus. In contrast, we find that resupplying young, engraftable thymic epithelial cells (TECs) to a middle-aged or defective thymus leads to thymic growth and increased T cell production. Intrathymic transplantation and in vitro colony-forming assays reveal that the engraftment and proliferative capacities of TECs diminish early in life, whereas the receptivity of the thymus to TEC engraftment remains relatively constant with age. These results support a model in which thymic growth and subsequent involution are driven by cell-intrinsic changes in the proliferative capacity of TECs, and further show that young TECs can engraft and directly drive the growth of involuted thymuses.Copyright © 2015 by The American Association of Immunologists, Inc. 2014
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Castiglioni A, Hettmer S, Lynes MD, Rao TN, Tchessalova D, Sinha I, Lee BT, Tseng YH, Wagers AJ. 2014. Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle. Stem cell reports. 2(1):92-106. Pubmed: 24678452 DOI:10.1016/j.stemcr.2013.12.006 Castiglioni A, Hettmer S, Lynes MD, Rao TN, Tchessalova D, Sinha I, Lee BT, Tseng YH, Wagers AJ. 2014. Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle. Stem cell reports. 2(1):92-106. Pubmed: 24678452 DOI:10.1016/j.stemcr.2013.12.006 Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells. These CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(+) fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7(+) CD45(-)CD11b(-)GlyA(-)CD31(-)CD34(-)CD56(int)ITGA7(hi) hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis. -
Kikuchi K, Hettmer S, Aslam MI, Michalek JE, Laub W, Wilky BA, Loeb DM, Rubin BP, Wagers AJ, Keller C. 2014. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma. PLoS genetics. 10(1):e1004107. Pubmed: 24453992 DOI:10.1371/journal.pgen.1004107 Kikuchi K, Hettmer S, Aslam MI, Michalek JE, Laub W, Wilky BA, Loeb DM, Rubin BP, Wagers AJ, Keller C. 2014. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma. PLoS genetics. 10(1):e1004107. Pubmed: 24453992 DOI:10.1371/journal.pgen.1004107 Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells. -
Abraham J, Nuñez-Álvarez Y, Hettmer S, Carrió E, Chen HI, Nishijo K, Huang ET, Prajapati SI, Walker RL, Davis S, Rebeles J, Wiebush H, McCleish AT, Hampton ST, Bjornson CR, Brack AS, Wagers AJ, Rando TA, Capecchi MR, Marini FC, Ehler BR, Zarzabal LA, Goros MW, Michalek JE, Meltzer PS, Langenau DM, LeGallo RD, Mansoor A, Chen Y, Suelves M, Rubin BP, Keller C. 2014. Lineage of origin in rhabdomyosarcoma informs pharmacological response. Genes & development. 28(14):1578-91. Pubmed: 25030697 DOI:10.1101/gad.238733.114 Abraham J, Nuñez-Álvarez Y, Hettmer S, Carrió E, Chen HI, Nishijo K, Huang ET, Prajapati SI, Walker RL, Davis S, Rebeles J, Wiebush H, McCleish AT, Hampton ST, Bjornson CR, Brack AS, Wagers AJ, Rando TA, Capecchi MR, Marini FC, Ehler BR, Zarzabal LA, Goros MW, Michalek JE, Meltzer PS, Langenau DM, LeGallo RD, Mansoor A, Chen Y, Suelves M, Rubin BP, Keller C. 2014. Lineage of origin in rhabdomyosarcoma informs pharmacological response. Genes & development. 28(14):1578-91. Pubmed: 25030697 DOI:10.1101/gad.238733.114 Lineage or cell of origin of cancers is often unknown and thus is not a consideration in therapeutic approaches. Alveolar rhabdomyosarcoma (aRMS) is an aggressive childhood cancer for which the cell of origin remains debated. We used conditional genetic mouse models of aRMS to activate the pathognomonic Pax3:Foxo1 fusion oncogene and inactivate p53 in several stages of prenatal and postnatal muscle development. We reveal that lineage of origin significantly influences tumor histomorphology and sensitivity to targeted therapeutics. Furthermore, we uncovered differential transcriptional regulation of the Pax3:Foxo1 locus by tumor lineage of origin, which led us to identify the histone deacetylase inhibitor entinostat as a pharmacological agent for the potential conversion of Pax3:Foxo1-positive aRMS to a state akin to fusion-negative RMS through direct transcriptional suppression of Pax3:Foxo1.© 2014 Abraham et al.; Published by Cold Spring Harbor Laboratory Press. -
Hung CM, Calejman CM, Sanchez-Gurmaches J, Li H, Clish CB, Hettmer S, Wagers AJ, Guertin DA. 2014. Rictor/mTORC2 loss in the Myf5 lineage reprograms brown fat metabolism and protects mice against obesity and metabolic disease. Cell reports. 8(1):256-71. Pubmed: 25001283 DOI:S2211-1247(14)00457-4 Hung CM, Calejman CM, Sanchez-Gurmaches J, Li H, Clish CB, Hettmer S, Wagers AJ, Guertin DA. 2014. Rictor/mTORC2 loss in the Myf5 lineage reprograms brown fat metabolism and protects mice against obesity and metabolic disease. Cell reports. 8(1):256-71. Pubmed: 25001283 DOI:S2211-1247(14)00457-4 The in vivo functions of mechanistic target of rapamycin complex 2 (mTORC2) and the signaling mechanisms that control brown adipose tissue (BAT) fuel utilization and activity are not well understood. Here, by conditionally deleting Rictor in the Myf5 lineage, we provide in vivo evidence that mTORC2 is dispensable for skeletal muscle development and regeneration but essential for BAT growth. Furthermore, deleting Rictor in Myf5 precursors shifts BAT metabolism to a more oxidative and less lipogenic state and protects mice from obesity and metabolic disease at thermoneutrality. We additionally find that Rictor is required for brown adipocyte differentiation in vitro and that the mechanism specifically requires AKT1 hydrophobic motif phosphorylation but is independent of pan-AKT signaling and is rescued with BMP7. Our findings provide insights into the signaling circuitry that regulates brown adipocytes and could have important implications for developing therapies aimed at increasing energy expenditure as a means to combat human obesity.Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved. -
Sinha I, Sinha-Hikim AP, Wagers AJ, Sinha-Hikim I. 2014. Testosterone is essential for skeletal muscle growth in aged mice in a heterochronic parabiosis model. Cell and tissue research. 357(3):815-21. Pubmed: 24859218 DOI:10.1007/s00441-014-1900-2 Sinha I, Sinha-Hikim AP, Wagers AJ, Sinha-Hikim I. 2014. Testosterone is essential for skeletal muscle growth in aged mice in a heterochronic parabiosis model. Cell and tissue research. 357(3):815-21. Pubmed: 24859218 DOI:10.1007/s00441-014-1900-2 As humans age, they lose both muscle mass and strength (sarcopenia). Testosterone, a circulating hormone, progressively declines in aging and is associated with loss of muscle mass and strength. The surgical joining of a young and old mouse (heterochronic parabiosis) activates Notch signaling and restores muscle regenerative potential in aged mice. We hypothesize that testosterone is at least one of the factors required for the improvement seen in muscles in old mice in heterochronic parabiosis with young mice. To test this hypothesis, we established the following heterochronic parabioses between young (Y; 5 months old) and old (O; 22-23 months old) C57BL6 male mice: (1) Y:O; (2) castrated Y:O (ØY:O); (3) castrated + testosterone-treated Y:O (ØY + T:O). A group of normal young mice received empty implants, and old mice were used as controls. Parabiotic pairings were maintained for 4 weeks prior to analysis. Serum testosterone levels were three-fold higher in young than in old mice. The ØY + T:O pairing demonstrated significantly elevated levels of serum testosterone and an improvement in gastrocnemius muscle weight, muscle ultrastructure, muscle fiber cross-sectional area, and Notch-1 expression in old mice. These changes were not present in aged mice in the ØY:O pairing. These data indicate that testosterone has a critical role in mediating the improved muscle mass and ultrastructure seen in an experimental model of heterochronic parabiosis. -
Sinha M, Jang YC, Oh J, Khong D, Wu EY, Manohar R, Miller C, Regalado SG, Loffredo FS, Pancoast JR, Hirshman MF, Lebowitz J, Shadrach JL, Cerletti M, Kim MJ, Serwold T, Goodyear LJ, Rosner B, Lee RT, Wagers AJ. 2014. Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science (New York, N.Y.). 344(6184):649-52. Pubmed: 24797481 DOI:10.1126/science.1251152 Sinha M, Jang YC, Oh J, Khong D, Wu EY, Manohar R, Miller C, Regalado SG, Loffredo FS, Pancoast JR, Hirshman MF, Lebowitz J, Shadrach JL, Cerletti M, Kim MJ, Serwold T, Goodyear LJ, Rosner B, Lee RT, Wagers AJ. 2014. Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science (New York, N.Y.). 344(6184):649-52. Pubmed: 24797481 DOI:10.1126/science.1251152 Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral "rejuvenating" factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction. -
Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. 2014. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science (New York, N.Y.). 344(6184):630-4. Pubmed: 24797482 DOI:10.1126/science.1251141 Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. 2014. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science (New York, N.Y.). 344(6184):630-4. Pubmed: 24797482 DOI:10.1126/science.1251141 In the adult central nervous system, the vasculature of the neurogenic niche regulates neural stem cell behavior by providing circulating and secreted factors. Age-related decline of neurogenesis and cognitive function is associated with reduced blood flow and decreased numbers of neural stem cells. Therefore, restoring the functionality of the niche should counteract some of the negative effects of aging. We show that factors found in young blood induce vascular remodeling, culminating in increased neurogenesis and improved olfactory discrimination in aging mice. Further, we show that GDF11 alone can improve the cerebral vasculature and enhance neurogenesis. The identification of factors that slow the age-dependent deterioration of the neurogenic niche in mice may constitute the basis for new methods of treating age-related neurodegenerative and neurovascular diseases. -
Lee JH, Bhang DH, Beede A, Huang TL, Stripp BR, Bloch KD, Wagers AJ, Tseng YH, Ryeom S, Kim CF. 2014. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis. Cell. 156(3):440-55. Pubmed: 24485453 DOI:S0092-8674(14)00009-9 Lee JH, Bhang DH, Beede A, Huang TL, Stripp BR, Bloch KD, Wagers AJ, Tseng YH, Ryeom S, Kim CF. 2014. Lung stem cell differentiation in mice directed by endothelial cells via a BMP4-NFATc1-thrombospondin-1 axis. Cell. 156(3):440-55. Pubmed: 24485453 DOI:S0092-8674(14)00009-9 Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal 3D cocultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell cocultures. Gain- and loss-of-function experiments showed that BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1 null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide tools to understand the mechanisms of respiratory diseases at the single-cell level.Copyright © 2014 Elsevier Inc. All rights reserved. -
Hettmer S, Archer NM, Somers GR, Novokmet A, Wagers AJ, Diller L, Rodriguez-Galindo C, Teot LA, Malkin D. 2014. Anaplastic rhabdomyosarcoma in TP53 germline mutation carriers. Cancer. 120(7):1068-75. Pubmed: 24382691 DOI:10.1002/cncr.28507 Hettmer S, Archer NM, Somers GR, Novokmet A, Wagers AJ, Diller L, Rodriguez-Galindo C, Teot LA, Malkin D. 2014. Anaplastic rhabdomyosarcoma in TP53 germline mutation carriers. Cancer. 120(7):1068-75. Pubmed: 24382691 DOI:10.1002/cncr.28507 Array© 2013 American Cancer Society. -
Hettmer S, Li Z, Billin AN, Barr FG, Cornelison DD, Ehrlich AR, Guttridge DC, Hayes-Jordan A, Helman LJ, Houghton PJ, Khan J, Langenau DM, Linardic CM, Pal R, Partridge TA, Pavlath GK, Rota R, Schäfer BW, Shipley J, Stillman B, Wexler LH, Wagers AJ, Keller C. 2014. Rhabdomyosarcoma: current challenges and their implications for developing therapies. Cold Spring Harbor perspectives in medicine. 4(11):a025650. Pubmed: 25368019 DOI:10.1101/cshperspect.a025650 Hettmer S, Li Z, Billin AN, Barr FG, Cornelison DD, Ehrlich AR, Guttridge DC, Hayes-Jordan A, Helman LJ, Houghton PJ, Khan J, Langenau DM, Linardic CM, Pal R, Partridge TA, Pavlath GK, Rota R, Schäfer BW, Shipley J, Stillman B, Wexler LH, Wagers AJ, Keller C. 2014. Rhabdomyosarcoma: current challenges and their implications for developing therapies. Cold Spring Harbor perspectives in medicine. 4(11):a025650. Pubmed: 25368019 DOI:10.1101/cshperspect.a025650 Rhabdomyosarcoma (RMS) represents a rare, heterogeneous group of mesodermal malignancies with skeletal muscle differentiation. One major subgroup of RMS tumors (so-called "fusion-positive" tumors) carries exclusive chromosomal translocations that join the DNA-binding domain of the PAX3 or PAX7 gene to the transactivation domain of the FOXO1 (previously known as FKHR) gene. Fusion-negative RMS represents a heterogeneous spectrum of tumors with frequent RAS pathway activation. Overtly metastatic disease at diagnosis is more frequently found in individuals with fusion-positive than in those with fusion-negative tumors. RMS is the most common pediatric soft-tissue sarcoma, and approximately 60% of all children and adolescents diagnosed with RMS are cured by currently available multimodal therapies. However, a curative outcome is achieved in <30% of high-risk individuals with RMS, including all those diagnosed as adults, those diagnosed with fusion-positive tumors during childhood (including metastatic and nonmetastatic tumors), and those diagnosed with metastatic disease during childhood (including fusion-positive and fusion-negative tumors). This white paper outlines current challenges in RMS research and their implications for developing more effective therapies. Urgent clinical problems include local control, systemic disease, need for improved risk stratification, and characterization of differences in disease course in children and adults. Biological challenges include definition of the cellular functions of PAX-FOXO1 fusion proteins, clarification of disease heterogeneity, elucidation of the cellular origins of RMS, delineation of the tumor microenvironment, and identification of means for rational selection and testing of new combination therapies. To streamline future therapeutic developments, it will be critical to improve access to fresh tumor tissue for research purposes, consider alternative trial designs to optimize early clinical testing of candidate drugs, coalesce advocacy efforts to garner public and industry support, and facilitate collaborative efforts between academia and industry.Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved. -
Wagers AJ. 2014. How stem cells get "turned on". The EMBO journal. 33(23):2743-4. Pubmed: 25344560 DOI:10.15252/embj.201490272 Wagers AJ. 2014. How stem cells get "turned on". The EMBO journal. 33(23):2743-4. Pubmed: 25344560 DOI:10.15252/embj.201490272 Recent research began to link autophagic processes to the functional integrity of certain stem cells. A novel study published in this issue of reports on autophagic flux as crucial checkpoint to meet the energy demands during muscle stem cell activation. -
Mostafavi S, Ortiz-Lopez A, Bogue MA, Hattori K, Pop C, Koller D, Mathis D, Benoist C. 2014. Variation and genetic control of gene expression in primary immunocytes across inbred mouse strains. Journal of immunology (Baltimore, Md. : 1950). 193(9):4485-96. Pubmed: 25267973 DOI:10.4049/jimmunol.1401280 Mostafavi S, Ortiz-Lopez A, Bogue MA, Hattori K, Pop C, Koller D, Mathis D, Benoist C. 2014. Variation and genetic control of gene expression in primary immunocytes across inbred mouse strains. Journal of immunology (Baltimore, Md. : 1950). 193(9):4485-96. Pubmed: 25267973 DOI:10.4049/jimmunol.1401280 To determine the breadth and underpinning of changes in immunocyte gene expression due to genetic variation in mice, we performed, as part of the Immunological Genome Project, gene expression profiling for CD4(+) T cells and neutrophils purified from 39 inbred strains of the Mouse Phenome Database. Considering both cell types, a large number of transcripts showed significant variation across the inbred strains, with 22% of the transcriptome varying by 2-fold or more. These included 119 loci with apparent complete loss of function, where the corresponding transcript was not expressed in some of the strains, representing a useful resource of "natural knockouts." We identified 1222 cis-expression quantitative trait loci (cis-eQTL) that control some of this variation. Most (60%) cis-eQTLs were shared between T cells and neutrophils, but a significant portion uniquely impacted one of the cell types, suggesting cell type-specific regulatory mechanisms. Using a conditional regression algorithm, we predicted regulatory interactions between transcription factors and potential targets, and we demonstrated that these predictions overlap with regulatory interactions inferred from transcriptional changes during immunocyte differentiation. Finally, comparison of these and parallel data from CD4(+) T cells of healthy humans demonstrated intriguing similarities in variability of a gene's expression: the most variable genes tended to be the same in both species, and there was an overlap in genes subject to strong cis-acting genetic variants. We speculate that this "conservation of variation" reflects a differential constraint on intraspecies variation in expression levels of different genes, either through lower pressure for some genes, or by favoring variability for others.Copyright © 2014 by The American Association of Immunologists, Inc. -
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. -
Oh J, Lee YD, Wagers AJ. 2014. Stem cell aging: mechanisms, regulators and therapeutic opportunities. Nature medicine. 20(8):870-80. Pubmed: 25100532 DOI:10.1038/nm.3651 Oh J, Lee YD, Wagers AJ. 2014. Stem cell aging: mechanisms, regulators and therapeutic opportunities. Nature medicine. 20(8):870-80. Pubmed: 25100532 DOI:10.1038/nm.3651 Aging tissues experience a progressive decline in homeostatic and regenerative capacities, which has been attributed to degenerative changes in tissue-specific stem cells, stem cell niches and systemic cues that regulate stem cell activity. Understanding the molecular pathways involved in this age-dependent deterioration of stem cell function will be critical for developing new therapies for diseases of aging that target the specific causes of age-related functional decline. Here we explore key molecular pathways that are commonly perturbed as tissues and stem cells age and degenerate. We further consider experimental evidence both supporting and refuting the notion that modulation of these pathways per se can reverse aging phenotypes. Finally, we ask whether stem cell aging establishes an epigenetic 'memory' that is indelibly written or one that can be reset. -
Tremblay AM, Missiaglia E, Galli GG, Hettmer S, Urcia R, Carrara M, Judson RN, Thway K, Nadal G, Selfe JL, Murray G, Calogero RA, De Bari C, Zammit PS, Delorenzi M, Wagers AJ, Shipley J, Wackerhage H, Camargo FD. 2014. The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation. Cancer cell. 26(2):273-87. Pubmed: 25087979 DOI:S1535-6108(14)00230-X Tremblay AM, Missiaglia E, Galli GG, Hettmer S, Urcia R, Carrara M, Judson RN, Thway K, Nadal G, Selfe JL, Murray G, Calogero RA, De Bari C, Zammit PS, Delorenzi M, Wagers AJ, Shipley J, Wackerhage H, Camargo FD. 2014. The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation. Cancer cell. 26(2):273-87. Pubmed: 25087979 DOI:S1535-6108(14)00230-X The role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy.Copyright © 2014 Elsevier Inc. All rights reserved. -
Painter MW, Brosius Lutz A, Cheng YC, Latremoliere A, Duong K, Miller CM, Posada S, Cobos EJ, Zhang AX, Wagers AJ, Havton LA, Barres B, Omura T, Woolf CJ. 2014. Diminished Schwann cell repair responses underlie age-associated impaired axonal regeneration. Neuron. 83(2):331-343. Pubmed: 25033179 DOI:S0896-6273(14)00538-8 Painter MW, Brosius Lutz A, Cheng YC, Latremoliere A, Duong K, Miller CM, Posada S, Cobos EJ, Zhang AX, Wagers AJ, Havton LA, Barres B, Omura T, Woolf CJ. 2014. Diminished Schwann cell repair responses underlie age-associated impaired axonal regeneration. Neuron. 83(2):331-343. Pubmed: 25033179 DOI:S0896-6273(14)00538-8 The regenerative capacity of the peripheral nervous system declines with age. Why this occurs, however, is unknown. We demonstrate that 24-month-old mice exhibit an impairment of functional recovery after nerve injury compared to 2-month-old animals. We find no difference in the intrinsic growth capacity between aged and young sensory neurons in vitro or in their ability to activate growth-associated transcriptional programs after injury. Instead, using age-mismatched nerve transplants in vivo, we show that the extent of functional recovery depends on the age of the nerve graft, and not the age of the host. Molecular interrogation of the sciatic nerve reveals that aged Schwann cells (SCs) fail to rapidly activate a transcriptional repair program after injury. Functionally, aged SCs exhibit impaired dedifferentiation, myelin clearance, and macrophage recruitment. These results suggest that the age-associated decline in axonal regeneration results from diminished Schwann cell plasticity, leading to slower myelin clearance.Copyright © 2014 Elsevier Inc. All rights reserved. 2013
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Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, van Wijngaarden P, Wagers AJ, Williams A, Franklin RJM, Ffrench-Constant C. 2013. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nature neuroscience. 16(9):1211-1218. Pubmed: 23872599 DOI:10.1038/nn.3469 Miron VE, Boyd A, Zhao JW, Yuen TJ, Ruckh JM, Shadrach JL, van Wijngaarden P, Wagers AJ, Williams A, Franklin RJM, Ffrench-Constant C. 2013. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nature neuroscience. 16(9):1211-1218. Pubmed: 23872599 DOI:10.1038/nn.3469 The lack of therapies for progressive multiple sclerosis highlights the need to understand the regenerative process of remyelination that can follow CNS demyelination. This involves an innate immune response consisting of microglia and macrophages, which can be polarized to distinct functional phenotypes: pro-inflammatory (M1) and anti-inflammatory or immunoregulatory (M2). We found that a switch from an M1- to an M2-dominant response occurred in microglia and peripherally derived macrophages as remyelination started. Oligodendrocyte differentiation was enhanced in vitro with M2 cell conditioned media and impaired in vivo following intra-lesional M2 cell depletion. M2 cell densities were increased in lesions of aged mice in which remyelination was enhanced by parabiotic coupling to a younger mouse and in multiple sclerosis lesions that normally show remyelination. Blocking M2 cell-derived activin-A inhibited oligodendrocyte differentiation during remyelination in cerebellar slice cultures. Thus, our results indicate that M2 cell polarization is essential for efficient remyelination and identify activin-A as a therapeutic target for CNS regeneration. -
Hettmer S, Teot LA, van Hummelen P, MacConaill L, Bronson RT, Dall'Osso C, Mao J, McMahon AP, Gruber PJ, Grier HE, Rodriguez-Galindo C, Fletcher CD, Wagers AJ. 2013. Mutations in Hedgehog pathway genes in fetal rhabdomyomas. The Journal of pathology. 231(1):44-52. Pubmed: 23780909 DOI:10.1002/path.4229 Hettmer S, Teot LA, van Hummelen P, MacConaill L, Bronson RT, Dall'Osso C, Mao J, McMahon AP, Gruber PJ, Grier HE, Rodriguez-Galindo C, Fletcher CD, Wagers AJ. 2013. Mutations in Hedgehog pathway genes in fetal rhabdomyomas. The Journal of pathology. 231(1):44-52. Pubmed: 23780909 DOI:10.1002/path.4229 Ligand-independent, constitutive activation of Hedgehog signalling in mice expressing a mutant, activated SmoM2 allele results in the development of multifocal, highly differentiated tumours that express myogenic markers (including desmin, actin, MyoD and myogenin). The histopathology of these tumours, commonly classified as rhabdomyosarcomas, more closely resembles human fetal rhabdomyoma (FRM), a benign tumour that can be difficult to distinguish from highly differentiated rhabdomyosarcomas. We evaluated the spectrum of Hedgehog (HH) pathway gene mutations in a cohort of human FRM tumours by targeted Illumina sequencing and fluorescence in situ hybridization testing for PTCH1. Our studies identified functionally relevant aberrations at the PTCH1 locus in three of five FRM tumours surveyed, including a PTCH1 frameshift mutation in one tumour and homozygous deletions of PTCH1 in two tumours. These data suggest that activated Hedgehog signalling contributes to the biology of human FRM.Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. -
Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Später D, Xu H, Tabebordbar M, Gorbatov R, Sena B, Nahrendorf M, Briscoe DM, Li RA, Wagers AJ, Rossi DJ, Pu WT, Chien KR. 2013. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nature biotechnology. 31(10):898-907. Pubmed: 24013197 DOI:10.1038/nbt.2682 Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Später D, Xu H, Tabebordbar M, Gorbatov R, Sena B, Nahrendorf M, Briscoe DM, Li RA, Wagers AJ, Rossi DJ, Pu WT, Chien KR. 2013. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nature biotechnology. 31(10):898-907. Pubmed: 24013197 DOI:10.1038/nbt.2682 In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair. -
Teo AK, Wagers AJ, Kulkarni RN. 2013. New opportunities: harnessing induced pluripotency for discovery in diabetes and metabolism. Cell metabolism. 18(6):775-91. Pubmed: 24035588 DOI:S1550-4131(13)00336-7 Teo AK, Wagers AJ, Kulkarni RN. 2013. New opportunities: harnessing induced pluripotency for discovery in diabetes and metabolism. Cell metabolism. 18(6):775-91. Pubmed: 24035588 DOI:S1550-4131(13)00336-7 The landmark discovery of induced pluripotent stem cells (iPSCs) by Shinya Yamanaka has transformed regenerative biology. Previously, insights into the pathogenesis of chronic human diseases have been hindered by the inaccessibility of patient samples. However, scientists are now able to convert patient fibroblasts into iPSCs and differentiate them into disease-relevant cell types. This ability opens new avenues for investigating disease pathogenesis and designing novel treatments. In this review, we highlight the uses of human iPSCs to uncover the underlying causes and pathological consequences of diabetes and metabolic syndromes, multifactorial diseases whose etiologies have been difficult to unravel using traditional methodologies.Copyright © 2013 Elsevier Inc. All rights reserved. -
Xu C, Tabebordbar M, Iovino S, Ciarlo C, Liu J, Castiglioni A, Price E, Liu M, Barton ER, Kahn CR, Wagers AJ, Zon LI. 2013. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell. 155(4):909-921. Pubmed: 24209627 DOI:10.1016/j.cell.2013.10.023 Xu C, Tabebordbar M, Iovino S, Ciarlo C, Liu J, Castiglioni A, Price E, Liu M, Barton ER, Kahn CR, Wagers AJ, Zon LI. 2013. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell. 155(4):909-921. Pubmed: 24209627 DOI:10.1016/j.cell.2013.10.023 Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors, and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle.Copyright © 2013 Elsevier Inc. All rights reserved. -
Aslam MI, Hettmer S, Abraham J, Latocha D, Soundararajan A, Huang ET, Goros MW, Michalek JE, Wang S, Mansoor A, Druker BJ, Wagers AJ, Tyner JW, Keller C. 2013. Dynamic and nuclear expression of PDGFRα and IGF-1R in alveolar Rhabdomyosarcoma. Molecular cancer research : MCR. 11(11):1303-13. Pubmed: 23928059 DOI:10.1158/1541-7786.MCR-12-0598 Aslam MI, Hettmer S, Abraham J, Latocha D, Soundararajan A, Huang ET, Goros MW, Michalek JE, Wang S, Mansoor A, Druker BJ, Wagers AJ, Tyner JW, Keller C. 2013. Dynamic and nuclear expression of PDGFRα and IGF-1R in alveolar Rhabdomyosarcoma. Molecular cancer research : MCR. 11(11):1303-13. Pubmed: 23928059 DOI:10.1158/1541-7786.MCR-12-0598 Array©2013 AACR. -
Gazit R, Garrison BS, Rao TN, Shay T, Costello J, Ericson J, Kim F, Collins JJ, Regev A, Wagers AJ, Rossi DJ. 2013. Transcriptome analysis identifies regulators of hematopoietic stem and progenitor cells. Stem cell reports. 1(3):266-80. Pubmed: 24319662 DOI:10.1016/j.stemcr.2013.07.004 Gazit R, Garrison BS, Rao TN, Shay T, Costello J, Ericson J, Kim F, Collins JJ, Regev A, Wagers AJ, Rossi DJ. 2013. Transcriptome analysis identifies regulators of hematopoietic stem and progenitor cells. Stem cell reports. 1(3):266-80. Pubmed: 24319662 DOI:10.1016/j.stemcr.2013.07.004 Hematopoietic stem cells (HSCs) maintain blood homeostasis and are the functional units of bone marrow transplantation. To improve the molecular understanding of HSCs and their proximal progenitors, we performed transcriptome analysis within the context of the ImmGen Consortium data set. Gene sets that define steady-state and mobilized HSCs, as well as hematopoietic stem and progenitor cells (HSPCs), were determined. Genes involved in transcriptional regulation, including a group of putative transcriptional repressors, were identified in multipotent progenitors and HSCs. Proximal promoter analyses combined with ImmGen module analysis identified candidate regulators of HSCs. Enforced expression of one predicted regulator, Hlf, in diverse HSPC subsets led to extensive self-renewal activity ex vivo. These analyses reveal unique insights into the mechanisms that control the core properties of HSPCs. -
Tabebordbar M, Wang ET, Wagers AJ. 2013. Skeletal muscle degenerative diseases and strategies for therapeutic muscle repair. Annual review of pathology. 8:441-75. Pubmed: 23121053 DOI:10.1146/annurev-pathol-011811-132450 Tabebordbar M, Wang ET, Wagers AJ. 2013. Skeletal muscle degenerative diseases and strategies for therapeutic muscle repair. Annual review of pathology. 8:441-75. Pubmed: 23121053 DOI:10.1146/annurev-pathol-011811-132450 Skeletal muscle is a highly specialized, postmitotic tissue that must withstand chronic mechanical and physiological stress throughout life to maintain proper contractile function. Muscle damage or disease leads to progressive weakness and disability, and manifests in more than 100 different human disorders. Current therapies to treat muscle degenerative diseases are limited mostly to the amelioration of symptoms, although promising new therapeutic directions are emerging. In this review, we discuss the pathological basis for the most common muscle degenerative diseases and highlight new and encouraging experimental and clinical opportunities to prevent or reverse these afflictions. -
Cohen NR, Brennan PJ, Shay T, Watts GF, Brigl M, Kang J, Brenner MB. 2013. Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells. Nature immunology. 14(1):90-9. Pubmed: 23202270 DOI:10.1038/ni.2490 Cohen NR, Brennan PJ, Shay T, Watts GF, Brigl M, Kang J, Brenner MB. 2013. Shared and distinct transcriptional programs underlie the hybrid nature of iNKT cells. Nature immunology. 14(1):90-9. Pubmed: 23202270 DOI:10.1038/ni.2490 Invariant natural killer T cells (iNKT cells) are innate-like T lymphocytes that act as critical regulators of the immune response. To better characterize this population, we profiled gene expression in iNKT cells during ontogeny and in peripheral subsets as part of the Immunological Genome Project. High-resolution comparative transcriptional analyses defined developmental and subset-specific programs of gene expression by iNKT cells. In addition, we found that iNKT cells shared an extensive transcriptional program with NK cells, similar in magnitude to that shared with major histocompatibility complex (MHC)-restricted T cells. Notably, the program shared by NK cells and iNKT cells also operated constitutively in γδ T cells and in adaptive T cells after activation. Together our findings highlight a core effector program regulated distinctly in innate and adaptive lymphocytes. -
Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, Sinha M, Dall'Osso C, Khong D, Shadrach JL, Miller CM, Singer BS, Stewart A, Psychogios N, Gerszten RE, Hartigan AJ, Kim MJ, Serwold T, Wagers AJ, Lee RT. 2013. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 153(4):828-39. Pubmed: 23663781 DOI:S0092-8674(13)00456-X Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, Sinha M, Dall'Osso C, Khong D, Shadrach JL, Miller CM, Singer BS, Stewart A, Psychogios N, Gerszten RE, Hartigan AJ, Kim MJ, Serwold T, Wagers AJ, Lee RT. 2013. Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell. 153(4):828-39. Pubmed: 23663781 DOI:S0092-8674(13)00456-X The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-β superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.Copyright © 2013 Elsevier Inc. All rights reserved. -
Best JA, Blair DA, Knell J, Yang E, Mayya V, Doedens A, Dustin ML, Goldrath AW. 2013. Transcriptional insights into the CD8(+) T cell response to infection and memory T cell formation. Nature immunology. 14(4):404-12. Pubmed: 23396170 DOI:10.1038/ni.2536 Best JA, Blair DA, Knell J, Yang E, Mayya V, Doedens A, Dustin ML, Goldrath AW. 2013. Transcriptional insights into the CD8(+) T cell response to infection and memory T cell formation. Nature immunology. 14(4):404-12. Pubmed: 23396170 DOI:10.1038/ni.2536 After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity. -
Nabors LK, Wang LD, Wagers AJ, Kansas GS. 2013. Overlapping roles for endothelial selectins in murine hematopoietic stem/progenitor cell homing to bone marrow. Experimental hematology. 41(7):588-96. Pubmed: 23499761 DOI:S0301-472X(13)00067-2 Nabors LK, Wang LD, Wagers AJ, Kansas GS. 2013. Overlapping roles for endothelial selectins in murine hematopoietic stem/progenitor cell homing to bone marrow. Experimental hematology. 41(7):588-96. Pubmed: 23499761 DOI:S0301-472X(13)00067-2 Selectins are carbohydrate-binding adhesion molecules that are critically involved in leukocyte recognition of endothelium. The endothelial selectins have been implicated in homing of hematopoietic stem and progenitor cells (HSPCs) to the bone marrow (BM) during bone marrow transplant (BMT), but the precise roles of individual selectins in this process have never been defined. BMT of lethally irradiated mice lacking both endothelial selectins (E/P KO) with limiting numbers of wild type BM cells rescued significantly fewer E/P KO than WT recipients, but higher numbers of transplanted WT cells rescued E/P KOs in a dose-dependent fashion. Short-term homing assays confirmed a substantial defect in HSPC homing to BM in E/P KO mice. In contrast, BMT of E-selectin null or P-selectin null mice at limiting cell number uniformly rescued greater than 95% of the transplanted animals. Consistent with these functional results, flow cytometric analysis revealed both E-selectin ligands and P-selectin ligands on distinct subsets of HSPC. These results demonstrate overlapping functions for the endothelial selectins in HSPC homing to BM in the setting of BMT, and define a novel aspect of HSPC heterogeneity linked to selectin ligand expression.Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved. -
Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T, Wagers AJ, Hsiao EC, Passegué E. 2013. Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell stem cell. 13(3):285-99. Pubmed: 23850243 DOI:S1934-5909(13)00267-1 Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T, Wagers AJ, Hsiao EC, Passegué E. 2013. Myeloproliferative neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche. Cell stem cell. 13(3):285-99. Pubmed: 23850243 DOI:S1934-5909(13)00267-1 Multipotent stromal cells (MSCs) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis, favors leukemic stem cell (LSC) function, and contributes to BM fibrosis. We show that leukemic myeloid cells stimulate MSCs to overproduce functionally altered OBCs, which accumulate in the BM cavity as inflammatory myelofibrotic cells. We identify roles for thrombopoietin, CCL3, and direct cell-cell interactions in driving OBC expansion, and for changes in TGF-β, Notch, and inflammatory signaling in OBC remodeling. MPN-expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of MPN-affected patients and prevention of myelofibrosis.Copyright © 2013 Elsevier Inc. All rights reserved. -
Mingueneau M, Kreslavsky T, Gray D, Heng T, Cruse R, Ericson J, Bendall S, Spitzer MH, Nolan GP, Kobayashi K, von Boehmer H, Mathis D, Benoist C, Best AJ, Knell J, Goldrath A, Joic V, Koller D, Shay T, Regev A, Cohen N, Brennan P, Brenner M, Kim F, Nageswara Rao T, Wagers A, Heng T, Ericson J, Rothamel K, Ortiz-Lopez A, Mathis D, Benoist C, Bezman NA, Sun JC, Min-Oo G, Kim CC, Lanier LL, Miller J, Brown B, Merad M, Gautier EL, Jakubzick C, Randolph GJ, Monach P, Blair DA, Dustin ML, Shinton SA, Hardy RR, Laidlaw D, Collins J, Gazit R, Rossi DJ, Malhotra N, Sylvia K, Kang J, Kreslavsky T, Fletcher A, Elpek K, Bellemare-Pelletier A, Malhotra D, Turley S. 2013. The transcriptional landscape of αβ T cell differentiation. Nature immunology. 14(6):619-32. Pubmed: 23644507 DOI:10.1038/ni.2590 Mingueneau M, Kreslavsky T, Gray D, Heng T, Cruse R, Ericson J, Bendall S, Spitzer MH, Nolan GP, Kobayashi K, von Boehmer H, Mathis D, Benoist C, Best AJ, Knell J, Goldrath A, Joic V, Koller D, Shay T, Regev A, Cohen N, Brennan P, Brenner M, Kim F, Nageswara Rao T, Wagers A, Heng T, Ericson J, Rothamel K, Ortiz-Lopez A, Mathis D, Benoist C, Bezman NA, Sun JC, Min-Oo G, Kim CC, Lanier LL, Miller J, Brown B, Merad M, Gautier EL, Jakubzick C, Randolph GJ, Monach P, Blair DA, Dustin ML, Shinton SA, Hardy RR, Laidlaw D, Collins J, Gazit R, Rossi DJ, Malhotra N, Sylvia K, Kang J, Kreslavsky T, Fletcher A, Elpek K, Bellemare-Pelletier A, Malhotra D, Turley S. 2013. The transcriptional landscape of αβ T cell differentiation. Nature immunology. 14(6):619-32. Pubmed: 23644507 DOI:10.1038/ni.2590 The differentiation of αβT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation. -
Jojic V, Shay T, Sylvia K, Zuk O, Sun X, Kang J, Regev A, Koller D, Best AJ, Knell J, Goldrath A, Joic V, Koller D, Shay T, Regev A, Cohen N, Brennan P, Brenner M, Kim F, Rao TN, Wagers A, Heng T, Ericson J, Rothamel K, Ortiz-Lopez A, Mathis D, Benoist C, Bezman NA, Sun JC, Min-Oo G, Kim CC, Lanier LL, Miller J, Brown B, Merad M, Gautier EL, Jakubzick C, Randolph GJ, Monach P, Blair DA, Dustin ML, Shinton SA, Hardy RR, Laidlaw D, Collins J, Gazit R, Rossi DJ, Malhotra N, Sylvia K, Kang J, Kreslavsky T, Fletcher A, Elpek K, Bellemarte-Pelletier A, Malhotra D, Turley S. 2013. Identification of transcriptional regulators in the mouse immune system. Nature immunology. 14(6):633-43. Pubmed: 23624555 DOI:10.1038/ni.2587 Jojic V, Shay T, Sylvia K, Zuk O, Sun X, Kang J, Regev A, Koller D, Best AJ, Knell J, Goldrath A, Joic V, Koller D, Shay T, Regev A, Cohen N, Brennan P, Brenner M, Kim F, Rao TN, Wagers A, Heng T, Ericson J, Rothamel K, Ortiz-Lopez A, Mathis D, Benoist C, Bezman NA, Sun JC, Min-Oo G, Kim CC, Lanier LL, Miller J, Brown B, Merad M, Gautier EL, Jakubzick C, Randolph GJ, Monach P, Blair DA, Dustin ML, Shinton SA, Hardy RR, Laidlaw D, Collins J, Gazit R, Rossi DJ, Malhotra N, Sylvia K, Kang J, Kreslavsky T, Fletcher A, Elpek K, Bellemarte-Pelletier A, Malhotra D, Turley S. 2013. Identification of transcriptional regulators in the mouse immune system. Nature immunology. 14(6):633-43. Pubmed: 23624555 DOI:10.1038/ni.2587 The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans. -
Le X, Pugach EK, Hettmer S, Storer NY, Liu J, Wills AA, DiBiase A, Chen EY, Ignatius MS, Poss KD, Wagers AJ, Langenau DM, Zon LI. 2013. A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development. Development (Cambridge, England). 140(11):2354-64. Pubmed: 23615277 DOI:10.1242/dev.088427 Le X, Pugach EK, Hettmer S, Storer NY, Liu J, Wills AA, DiBiase A, Chen EY, Ignatius MS, Poss KD, Wagers AJ, Langenau DM, Zon LI. 2013. A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development. Development (Cambridge, England). 140(11):2354-64. Pubmed: 23615277 DOI:10.1242/dev.088427 The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth. -
Burzyn D, Kuswanto W, Kolodin D, Shadrach JL, Cerletti M, Jang Y, Sefik E, Tan TG, Wagers AJ, Benoist C, Mathis D. 2013. A special population of regulatory T cells potentiates muscle repair. Cell. 155(6):1282-95. Pubmed: 24315098 DOI:S0092-8674(13)01413-X Burzyn D, Kuswanto W, Kolodin D, Shadrach JL, Cerletti M, Jang Y, Sefik E, Tan TG, Wagers AJ, Benoist C, Mathis D. 2013. A special population of regulatory T cells potentiates muscle repair. Cell. 155(6):1282-95. Pubmed: 24315098 DOI:S0092-8674(13)01413-X Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.Copyright © 2013 Elsevier Inc. All rights reserved. -
El Ouaamari A, Kawamori D, Dirice E, Liew CW, Shadrach JL, Hu J, Katsuta H, Hollister-Lock J, Qian WJ, Wagers AJ, Kulkarni RN. 2013. Liver-derived systemic factors drive β cell hyperplasia in insulin-resistant states. Cell reports. 3(2):401-10. Pubmed: 23375376 DOI:S2211-1247(13)00014-4 El Ouaamari A, Kawamori D, Dirice E, Liew CW, Shadrach JL, Hu J, Katsuta H, Hollister-Lock J, Qian WJ, Wagers AJ, Kulkarni RN. 2013. Liver-derived systemic factors drive β cell hyperplasia in insulin-resistant states. Cell reports. 3(2):401-10. Pubmed: 23375376 DOI:S2211-1247(13)00014-4 Integrative organ crosstalk regulates key aspects of energy homeostasis, and its dysregulation may underlie metabolic disorders such as obesity and diabetes. To test the hypothesis that crosstalk between the liver and pancreatic islets modulates β cell growth in response to insulin resistance, we used the liver-specific insulin receptor knockout (LIRKO) mouse, a unique model that exhibits dramatic islet hyperplasia. Using complementary in vivo parabiosis and transplantation assays, as well as in vitro islet culture approaches, we demonstrate that humoral, nonneural, non-cell-autonomous factor(s) induces β cell proliferation in LIRKO mice. Furthermore, we report that a hepatocyte-derived factor(s) stimulates mouse and human β cell proliferation in ex vivo assays, independent of ambient glucose and insulin levels. These data implicate the liver as a critical source of β cell growth factor(s) in insulin-resistant states.Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved. 2012
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Gibney BC, Chamoto K, Lee GS, Simpson DC, Miele LF, Tsuda A, Konerding MA, Wagers A, Mentzer SJ. 2012. Cross-circulation and cell distribution kinetics in parabiotic mice. Journal of cellular physiology. 227(2):821-8. Pubmed: 21503883 DOI:10.1002/jcp.22796 Gibney BC, Chamoto K, Lee GS, Simpson DC, Miele LF, Tsuda A, Konerding MA, Wagers A, Mentzer SJ. 2012. Cross-circulation and cell distribution kinetics in parabiotic mice. Journal of cellular physiology. 227(2):821-8. Pubmed: 21503883 DOI:10.1002/jcp.22796 Blood-borne nucleated cells participate not only in inflammation, but in tissue repair and regeneration. Because progenitor and stem cell populations have a low concentration in the blood, the circulation kinetics and tissue distribution of these cells is largely unknown. An important approach to tracking cell lineage is the use of fluorescent tracers and parabiotic models of cross-circulation. Here, we investigated the cross-circulation and cell distribution kinetics of C57/B6 GFP(+)/wild-type parabionts. Flow cytometry analysis of the peripheral blood after parabiosis demonstrated no evidence for a "parabiotic barrier" based on cell size or surface characterstics; all peripheral blood cell subpopulations in this study reached equilibrium within 14 days. Whole blood fluorescence analysis indicated that the mean exchange flow rate was 16 µl/h or 0.66% of the circulating blood volume per hour. Studies of peripheral lymphoid organs indicated differential cell distribution kinetics. Some subpopulations, such as CD8(+) and CD11c(+), equilibrated in both lymph nodes and spleen indicating a residence time <28 days; in contrast, other lymphocyte subpopulations, such as B220(+) and CD4(+) cells, had not yet reached equilibrium at 28 days. We conclude that parabiosis can provide important insights into defining tissue distribution, residence times, and recirculating pools using fluorochrome markers of cell lineage.Copyright © 2011 Wiley Periodicals, Inc. -
Jiang X, Clark RA, Liu L, Wagers AJ, Fuhlbrigge RC, Kupper TS. 2012. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity. Nature. 483(7388):227-31. Pubmed: 22388819 DOI:10.1038/nature10851 Jiang X, Clark RA, Liu L, Wagers AJ, Fuhlbrigge RC, Kupper TS. 2012. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity. Nature. 483(7388):227-31. Pubmed: 22388819 DOI:10.1038/nature10851 Protective T-cell memory has long been thought to reside in blood and lymph nodes, but recently the concept of immune memory in peripheral tissues mediated by resident memory T (T(RM)) cells has been proposed. Here we show in mice that localized vaccinia virus (VACV) skin infection generates long-lived non-recirculating CD8(+) skin T(RM) cells that reside within the entire skin. These skin T(RM) cells are potent effector cells, and are superior to circulating central memory T (T(CM)) cells at providing rapid long-term protection against cutaneous re-infection. We find that CD8(+) T cells are rapidly recruited to skin after acute VACV infection. CD8(+) T-cell recruitment to skin is independent of CD4(+) T cells and interferon-γ, but requires the expression of E- and P-selectin ligands by CD8(+) T cells. Using parabiotic mice, we further show that circulating CD8(+) T(CM) and CD8(+) skin T(RM) cells are both generated after skin infection; however, CD8(+) T(CM) cells recirculate between blood and lymph nodes whereas T(RM) cells remain in the skin. Cutaneous CD8(+) T(RM) cells produce effector cytokines and persist for at least 6 months after infection. Mice with CD8(+) skin T(RM) cells rapidly cleared a subsequent re-infection with VACV whereas mice with circulating T(CM) but no skin T(RM) cells showed greatly impaired viral clearance, indicating that T(RM) cells provide superior protection. Finally, we show that T(RM) cells generated as a result of localized VACV skin infection reside not only in the site of infection, but also populate the entire skin surface and remain present for many months. Repeated re-infections lead to progressive accumulation of highly protective T(RM) cells in non-involved skin. These findings have important implications for our understanding of protective immune memory at epithelial interfaces with the environment, and suggest novel strategies for vaccines that protect against tissue tropic organisms. -
Wagers AJ. 2012. The stem cell niche in regenerative medicine. Cell stem cell. 10(4):362-9. Pubmed: 22482502 DOI:10.1016/j.stem.2012.02.018 Wagers AJ. 2012. The stem cell niche in regenerative medicine. Cell stem cell. 10(4):362-9. Pubmed: 22482502 DOI:10.1016/j.stem.2012.02.018 Stem cells are fundamental units for achieving regenerative therapies, which leads naturally to a theoretical and experimental focus on these cells for therapeutic screening and intervention. A growing body of data in many tissue systems indicates that stem cell function is critically influenced by extrinsic signals derived from the microenvironment, or "niche." In this vein, the stem cell niche represents a significant, and largely untapped, entry point for therapeutic modulation of stem cell behavior. This Perspective will discuss how the niche influences stem cells in homeostasis, in the progression of degenerative and malignant diseases, and in therapeutic strategies for tissue repair.Copyright © 2012 Elsevier Inc. All rights reserved. -
Cerletti M, Jang YC, Finley LW, Haigis MC, Wagers AJ. 2012. Short-term calorie restriction enhances skeletal muscle stem cell function. Cell stem cell. 10(5):515-9. Pubmed: 22560075 DOI:10.1016/j.stem.2012.04.002 Cerletti M, Jang YC, Finley LW, Haigis MC, Wagers AJ. 2012. Short-term calorie restriction enhances skeletal muscle stem cell function. Cell stem cell. 10(5):515-9. Pubmed: 22560075 DOI:10.1016/j.stem.2012.04.002 Calorie restriction (CR) extends life span and ameliorates age-related pathologies in most species studied, yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle after transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.Copyright © 2012 Elsevier Inc. All rights reserved. -
Gottumukkala RV, Lv H, Cornivelli L, Wagers AJ, Kwong RY, Bronson R, Stewart GC, Schulze PC, Chutkow W, Wolpert HA, Lee RT, Lipes MA. 2012. Myocardial infarction triggers chronic cardiac autoimmunity in type 1 diabetes. Science translational medicine. 4(138):138ra80. Pubmed: 22700956 DOI:10.1126/scitranslmed.3003551 Gottumukkala RV, Lv H, Cornivelli L, Wagers AJ, Kwong RY, Bronson R, Stewart GC, Schulze PC, Chutkow W, Wolpert HA, Lee RT, Lipes MA. 2012. Myocardial infarction triggers chronic cardiac autoimmunity in type 1 diabetes. Science translational medicine. 4(138):138ra80. Pubmed: 22700956 DOI:10.1126/scitranslmed.3003551 Patients with type 1 diabetes (T1D) suffer excessive morbidity and mortality after myocardial infarction (MI) that is not fully explained by the metabolic effects of diabetes. Acute MI is known to trigger a profound innate inflammatory response with influx of mononuclear cells and production of proinflammatory cytokines that are crucial for cardiac repair. We hypothesized that these same pathways might exert "adjuvant effects" and induce pathological responses in autoimmune-prone T1D hosts. Here, we show that experimental MI in nonobese diabetic mice, but not in control C57BL/6 mice, results in a severe post-infarction autoimmune (PIA) syndrome characterized by destructive lymphocytic infiltrates in the myocardium, infarct expansion, sustained cardiac autoantibody production, and T helper type 1 effector cell responses against cardiac (α-)myosin. PIA was prevented by inducing tolerance to α-myosin, demonstrating that immune responses to cardiac myosin are essential for this disease process. Extending these findings to humans, we developed a panel of immunoassays for cardiac autoantibody detection and found autoantibody positivity in 83% post-MI T1D patients. We further identified shared cardiac myosin autoantibody signatures between post-MI T1D patients and nondiabetic patients with myocarditis, which were absent in post-MI type 2 diabetic patients, and confirmed the presence of myocarditis in T1D by cardiac magnetic resonance imaging techniques. These data provide experimental and clinical evidence for a distinct post-MI autoimmune syndrome in T1D. Our findings suggest that PIA may contribute to worsened post-MI outcomes in T1D and highlight a role for antigen-specific immunointervention to selectively block this pathway. -
Liu J, Hettmer S, Milsom MD, Hofmann I, Hua F, Miller C, Bronson RT, Wagers AJ. 2012. Induction of histiocytic sarcoma in mouse skeletal muscle. PloS one. 7(8):e44044. Pubmed: 22952867 DOI:10.1371/journal.pone.0044044 Liu J, Hettmer S, Milsom MD, Hofmann I, Hua F, Miller C, Bronson RT, Wagers AJ. 2012. Induction of histiocytic sarcoma in mouse skeletal muscle. PloS one. 7(8):e44044. Pubmed: 22952867 DOI:10.1371/journal.pone.0044044 Myeloid sarcomas are extramedullary accumulations of immature myeloid cells that may present with or without evidence of pathologic involvement of the bone marrow or peripheral blood, and often coincide with or precede a diagnosis of acute myeloid leukemia (AML). A dearth of experimental models has hampered the study of myeloid sarcomas and led us to establish a new system in which tumor induction can be evaluated in an easily accessible non-hematopoietic tissue compartment. Using ex-vivo transduction of oncogenic Kras(G12V) into p16/p19(-/-) bone marrow cells, we generated transplantable leukemia-initiating cells that rapidly induced tumor formation in the skeletal muscle of immunocompromised NOD.SCID mice. In this model, murine histiocytic sarcomas, equivalent to human myeloid sarcomas, emerged at the injection site 30-50 days after cell implantation and consisted of tightly packed monotypic cells that were CD48+, CD47+ and Mac1+, with low or absent expression of other hematopoietic lineage markers. Tumor cells also infiltrated the bone marrow, spleen and other non-hematopoietic organs of tumor-bearing animals, leading to systemic illness (leukemia) within two weeks of tumor detection. P16/p19(-/-); Kras(G12V) myeloid sarcomas were multi-clonal, with dominant clones selected during secondary transplantation. The systemic leukemic phenotypes exhibited by histiocytic sarcoma-bearing mice were nearly identical to those of animals in which leukemia was introduced by intravenous transplantation of the same donor cells. Moreover, murine histiocytic sarcoma could be similarly induced by intramuscular injection of MLL-AF9 leukemia cells. This study establishes a novel, transplantable model of murine histiocytic/myeloid sarcoma that recapitulates the natural progression of these malignancies to systemic disease and indicates a cell autonomous leukemogenic mechanism. -
Hayhurst M, Wagner AK, Cerletti M, Wagers AJ, Rubin LL. 2012. A cell-autonomous defect in skeletal muscle satellite cells expressing low levels of survival of motor neuron protein. Developmental biology. 368(2):323-34. Pubmed: 22705478 DOI:10.1016/j.ydbio.2012.05.037 Hayhurst M, Wagner AK, Cerletti M, Wagers AJ, Rubin LL. 2012. A cell-autonomous defect in skeletal muscle satellite cells expressing low levels of survival of motor neuron protein. Developmental biology. 368(2):323-34. Pubmed: 22705478 DOI:10.1016/j.ydbio.2012.05.037 Mutations in the Survival of Motor Neuron (SMN) gene underlie the development of spinal muscular atrophy (SMA), which currently represents the leading genetic cause of mortality in infants and toddlers. SMA is characterized by degeneration of spinal cord motor neurons and muscle atrophy. Although SMA is often considered to be a motor neuron disease, accumulating evidence suggests that muscle cells themselves may be affected by low levels of SMN. Here, we examine satellite cells, tissue-resident stem cells that play an essential role in the growth and repair of skeletal muscle, isolated from a severe SMA mouse model (Smn(-/-); SMN2(+/+)). We found similar numbers of satellite cells in the muscles of SMA and wild-type (Smn(+/+); SMN2(+/+)) mice at postnatal day 2 (P2), and, when isolated from skeletal muscle using cell surface marker expression, these cells showed comparable survival and proliferative potential. However, SMA satellite cells differentiate abnormally, revealed by the premature expression of muscle differentiation markers, and, especially, by a reduced efficiency in forming myotubes. These phenotypes suggest a critical role of SMN protein in the intrinsic regulation of muscle differentiation and suggest that abnormal muscle development contributes to the manifestation of SMA symptoms.Copyright © 2012 Elsevier Inc. All rights reserved. -
Ruckh JM, Zhao JW, Shadrach JL, van Wijngaarden P, Rao TN, Wagers AJ, Franklin RJ. 2012. Rejuvenation of regeneration in the aging central nervous system. Cell stem cell. 10(1):96-103. Pubmed: 22226359 DOI:10.1016/j.stem.2011.11.019 Ruckh JM, Zhao JW, Shadrach JL, van Wijngaarden P, Rao TN, Wagers AJ, Franklin RJ. 2012. Rejuvenation of regeneration in the aging central nervous system. Cell stem cell. 10(1):96-103. Pubmed: 22226359 DOI:10.1016/j.stem.2011.11.019 Remyelination is a regenerative process in the central nervous system (CNS) that produces new myelin sheaths from adult stem cells. The decline in remyelination that occurs with advancing age poses a significant barrier to therapy in the CNS, particularly for long-term demyelinating diseases such as multiple sclerosis (MS). Here we show that remyelination of experimentally induced demyelination is enhanced in old mice exposed to a youthful systemic milieu through heterochronic parabiosis. Restored remyelination in old animals involves recruitment to the repairing lesions of blood-derived monocytes from the young parabiotic partner, and preventing this recruitment partially inhibits rejuvenation of remyelination. These data suggest that enhanced remyelinating activity requires both youthful monocytes and other factors, and that remyelination-enhancing therapies targeting endogenous cells can be effective throughout life.Copyright © 2012 Elsevier Inc. All rights reserved. 2011
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Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, Shadrach JL, Cerletti M, McDougall LE, Giorgadze N, Tchkonia T, Schrier D, Falb D, Kirkland JL, Wagers AJ, Tseng YH. 2011. Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proceedings of the National Academy of Sciences of the United States of America. 108(1):143-8. Pubmed: 21173238 DOI:10.1073/pnas.1010929108 Schulz TJ, Huang TL, Tran TT, Zhang H, Townsend KL, Shadrach JL, Cerletti M, McDougall LE, Giorgadze N, Tchkonia T, Schrier D, Falb D, Kirkland JL, Wagers AJ, Tseng YH. 2011. Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat. Proceedings of the National Academy of Sciences of the United States of America. 108(1):143-8. Pubmed: 21173238 DOI:10.1073/pnas.1010929108 Brown fat is specialized for energy expenditure and has therefore been proposed to function as a defense against obesity. Despite recent advances in delineating the transcriptional regulation of brown adipocyte differentiation, cellular lineage specification and developmental cues specifying brown-fat cell fate remain poorly understood. In this study, we identify and isolate a subpopulation of adipogenic progenitors (Sca-1(+)/CD45(-)/Mac1(-); referred to as Sca-1(+) progenitor cells, ScaPCs) residing in murine brown fat, white fat, and skeletal muscle. ScaPCs derived from different tissues possess unique molecular expression signatures and adipogenic capacities. Importantly, although the ScaPCs from interscapular brown adipose tissue (BAT) are constitutively committed brown-fat progenitors, Sca-1(+) cells from skeletal muscle and subcutaneous white fat are highly inducible to differentiate into brown-like adipocytes upon stimulation with bone morphogenetic protein 7 (BMP7). Consistent with these findings, human preadipocytes isolated from subcutaneous white fat also exhibit the greatest inducible capacity to become brown adipocytes compared with cells isolated from mesenteric or omental white fat. When muscle-resident ScaPCs are re-engrafted into skeletal muscle of syngeneic mice, BMP7-treated ScaPCs efficiently develop into adipose tissue with brown fat-specific characteristics. Importantly, ScaPCs from obesity-resistant mice exhibit markedly higher thermogenic capacity compared with cells isolated from obesity-prone mice. These data establish the molecular characteristics of tissue-resident adipose progenitors and demonstrate a dynamic interplay between these progenitors and inductive signals that act in concert to specify brown adipocyte development. -
Hettmer S, Liu J, Miller CM, Lindsay MC, Sparks CA, Guertin DA, Bronson RT, Langenau DM, Wagers AJ. 2011. Sarcomas induced in discrete subsets of prospectively isolated skeletal muscle cells. Proceedings of the National Academy of Sciences of the United States of America. 108(50):20002-7. Pubmed: 22135462 DOI:10.1073/pnas.1111733108 Hettmer S, Liu J, Miller CM, Lindsay MC, Sparks CA, Guertin DA, Bronson RT, Langenau DM, Wagers AJ. 2011. Sarcomas induced in discrete subsets of prospectively isolated skeletal muscle cells. Proceedings of the National Academy of Sciences of the United States of America. 108(50):20002-7. Pubmed: 22135462 DOI:10.1073/pnas.1111733108 Soft-tissue sarcomas are heterogeneous cancers that can present with tissue-specific differentiation markers. To examine the cellular basis for this histopathological variation and to identify sarcoma-relevant molecular pathways, we generated a chimeric mouse model in which sarcoma-associated genetic lesions can be introduced into discrete, muscle-resident myogenic and mesenchymal cell lineages. Expression of Kirsten rat sarcoma viral oncogene [Kras(G12V)] and disruption of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16p19) in prospectively isolated satellite cells gave rise to pleomorphic rhabdomyosarcomas (MyoD-, Myogenin- and Desmin-positive), whereas introduction of the same oncogenetic hits in nonmyogenic progenitors induced pleomorphic sarcomas lacking myogenic features. Transcriptional profiling demonstrated that myogenic and nonmyogenic Kras; p16p19(null) sarcomas recapitulate gene-expression signatures of human rhabdomyosarcomas and identified a cluster of genes that is concordantly up-regulated in both mouse and human sarcomas. This cluster includes genes associated with Ras and mechanistic target of rapamycin (mTOR) signaling, a finding consistent with activation of the Ras and mTOR pathways both in Kras; p16p19(null) sarcomas and in 26-50% of human rhabdomyosarcomas surveyed. Moreover, chemical inhibition of Ras or mTOR signaling arrested the growth of mouse Kras; p16p19(null) sarcomas and of human rhabdomyosarcoma cells in vitro and in vivo. Taken together, these data demonstrate the critical importance of lineage commitment within the tumor cell-of-origin in determining sarcoma histotype and introduce an experimental platform for rapid dissection of sarcoma-relevant cellular and molecular events. -
Woo M, Isganaitis E, Cerletti M, Fitzpatrick C, Wagers AJ, Jimenez-Chillaron J, Patti ME. 2011. Early life nutrition modulates muscle stem cell number: implications for muscle mass and repair. Stem cells and development. 20(10):1763-9. Pubmed: 21247245 DOI:10.1089/scd.2010.0349 Woo M, Isganaitis E, Cerletti M, Fitzpatrick C, Wagers AJ, Jimenez-Chillaron J, Patti ME. 2011. Early life nutrition modulates muscle stem cell number: implications for muscle mass and repair. Stem cells and development. 20(10):1763-9. Pubmed: 21247245 DOI:10.1089/scd.2010.0349 Suboptimal nutrition during prenatal and early postnatal development is associated with increased risk for type 2 diabetes during adult life. A hallmark of such diabetes risk is altered body composition, including reduced lean mass and increased adiposity. Since stem cell number and activity are important determinants of muscle mass, modulation of perinatal nutrition could alter stem cell number/function, potentially mediating developmentally programmed reductions in muscle mass. Skeletal muscle precursors (SMP) were purified from muscle of mice subjected to prenatal undernutrition and/or early postnatal high-fat diet (HFD)--experimental models that are both associated with obesity and diabetes risk. SMP number was determined by flow cytometry, proliferative capacity measured in vitro, and regenerative capacity of these cells determined in vivo after muscle freeze injury. Prenatally undernutrition (UN) mice showed significantly reduced SMP frequencies [Control (C) 4.8% ± 0.3% (% live cells) vs. UN 3.2% ± 0.4%, P=0.015] at 6 weeks; proliferative capacity was unaltered. Reduced SMP in UN was associated with 32% decrease in regeneration after injury (C 16% ± 3% of injured area vs. UN 11% ± 2%; P<0.0001). SMP frequency was also reduced in HFD-fed mice (chow 6.4% ± 0.6% vs. HFD 4.7% ± 0.4%, P=0.03), and associated with 44% decreased regeneration (chow 16% ± 2.7% vs. HFD 9% ± 2.2%; P<0.0001). Prenatal undernutrition was additive with postnatal HFD. Thus, both prenatal undernutrition and postnatal overnutrition reduce myogenic stem cell frequency and function, indicating that developmentally established differences in muscle-resident stem cell populations may provoke reductions in muscle mass and repair and contribute to diabetes risk. -
Shadrach JL, Wagers AJ. 2011. Stem cells for skeletal muscle repair. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 366(1575):2297-306. Pubmed: 21727135 DOI:10.1098/rstb.2011.0027 Shadrach JL, Wagers AJ. 2011. Stem cells for skeletal muscle repair. Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 366(1575):2297-306. Pubmed: 21727135 DOI:10.1098/rstb.2011.0027 Skeletal muscle is a highly specialized tissue composed of non-dividing, multi-nucleated muscle fibres that contract to generate force in a controlled and directed manner. Skeletal muscle is formed during embryogenesis from a subset of muscle precursor cells, which generate both differentiated muscle fibres and specialized muscle-forming stem cells known as satellite cells. Satellite cells remain associated with muscle fibres after birth and are responsible for muscle growth and repair throughout life. Failure in satellite cell function can lead to delayed, impaired or failed recovery after muscle injury, and such failures become increasingly prominent in cases of progressive muscle disease and in old age. Recent progress in the isolation of muscle satellite cells and elucidation of the cellular and molecular mediators controlling their activity indicate that these cells represent promising therapeutic targets. Such satellite cell-based therapies may involve either direct cell replacement or development of drugs that enhance endogenous muscle repair mechanisms. Here, we discuss recent breakthroughs in understanding both the cell intrinsic and extrinsic regulators that determine the formation and function of muscle satellite cells, as well as promising paths forward to realizing their full therapeutic potential. -
Wang LD, Wagers AJ. 2011. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nature reviews. Molecular cell biology. 12(10):643-55. Pubmed: 21886187 DOI:10.1038/nrm3184 Wang LD, Wagers AJ. 2011. Dynamic niches in the origination and differentiation of haematopoietic stem cells. Nature reviews. Molecular cell biology. 12(10):643-55. Pubmed: 21886187 DOI:10.1038/nrm3184 Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic 'niche', or microenvironment. Recent discoveries, aided in part by technological advances in in vivo imaging, have engendered a new appreciation for the dynamic nature of the niche, identifying novel cellular and acellular niche components and uncovering fluctuations in the relative importance of these components over time. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche. -
Jang YC, Sinha M, Cerletti M, Dall'Osso C, Wagers AJ. 2011. Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function. Cold Spring Harbor symposia on quantitative biology. 76:101-11. Pubmed: 21960527 DOI:10.1101/sqb.2011.76.010652 Jang YC, Sinha M, Cerletti M, Dall'Osso C, Wagers AJ. 2011. Skeletal muscle stem cells: effects of aging and metabolism on muscle regenerative function. Cold Spring Harbor symposia on quantitative biology. 76:101-11. Pubmed: 21960527 DOI:10.1101/sqb.2011.76.010652 Homeostatic and regenerative replacement of skeletal muscle fibers requires the activity of a dedicated pool of myogenic stem cells, called satellite cells, that are activated by muscle injury and act as a renewable source of muscle-forming cells throughout adult life. Satellite cell function is controlled by both intrinsic and extrinsic regulatory cues, whose integration determines the success of muscle regenerative responses. Pathological deregulation of satellite cell function through perturbation of these signaling pathways appears to play an important role in age-dependent deterioration of muscle function and in muscle dystrophic disease. The regenerative activity of skeletal muscle also appears to be tightly linked to metabolism, and alterations in metabolic state can directly influence the activity of these tissue-specific stem cells. Here, we review recent and emerging insights into the molecular and biochemical signals that control satellite cell function and discuss these in the context of muscle degenerative diseases such as dystrophy and sarcopenia. Novel discoveries from this ongoing work bring new opportunities to enhance or restore muscle repair and are likely to facilitate satellite cell transplantation in clinical applications. -
Tan KY, Eminli S, Hettmer S, Hochedlinger K, Wagers AJ. 2011. Efficient generation of iPS cells from skeletal muscle stem cells. PloS one. 6(10):e26406. Pubmed: 22028872 DOI:10.1371/journal.pone.0026406 Tan KY, Eminli S, Hettmer S, Hochedlinger K, Wagers AJ. 2011. Efficient generation of iPS cells from skeletal muscle stem cells. PloS one. 6(10):e26406. Pubmed: 22028872 DOI:10.1371/journal.pone.0026406 Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that "epigenetic memory" significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. 2010
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Forsberg EC, Passegué E, Prohaska SS, Wagers AJ, Koeva M, Stuart JM, Weissman IL. 2010. Molecular signatures of quiescent, mobilized and leukemia-initiating hematopoietic stem cells. PloS one. 5(1):e8785. Pubmed: 20098702 DOI:10.1371/journal.pone.0008785 Forsberg EC, Passegué E, Prohaska SS, Wagers AJ, Koeva M, Stuart JM, Weissman IL. 2010. Molecular signatures of quiescent, mobilized and leukemia-initiating hematopoietic stem cells. PloS one. 5(1):e8785. Pubmed: 20098702 DOI:10.1371/journal.pone.0008785 Hematopoietic stem cells (HSC) are rare, multipotent cells capable of generating all specialized cells of the blood system. Appropriate regulation of HSC quiescence is thought to be crucial to maintain their lifelong function; however, the molecular pathways controlling stem cell quiescence remain poorly characterized. Likewise, the molecular events driving leukemogenesis remain elusive. In this study, we compare the gene expression profiles of steady-state bone marrow HSC to non-self-renewing multipotent progenitors; to HSC treated with mobilizing drugs that expand the HSC pool and induce egress from the marrow; and to leukemic HSC in a mouse model of chronic myelogenous leukemia. By intersecting the resulting lists of differentially regulated genes we identify a subset of molecules that are downregulated in all three circumstances, and thus may be particularly important for the maintenance and function of normal, quiescent HSC. These results identify potential key regulators of HSC and give insights into the clinically important processes of HSC mobilization for transplantation and leukemic development from cancer stem cells. -
Rask-Madsen C, Li Q, Freund B, Feather D, Abramov R, Wu IH, Chen K, Yamamoto-Hiraoka J, Goldenbogen J, Sotiropoulos KB, Clermont A, Geraldes P, Dall'Osso C, Wagers AJ, Huang PL, Rekhter M, Scalia R, Kahn CR, King GL. 2010. Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice. Cell metabolism. 11(5):379-89. Pubmed: 20444418 DOI:10.1016/j.cmet.2010.03.013 Rask-Madsen C, Li Q, Freund B, Feather D, Abramov R, Wu IH, Chen K, Yamamoto-Hiraoka J, Goldenbogen J, Sotiropoulos KB, Clermont A, Geraldes P, Dall'Osso C, Wagers AJ, Huang PL, Rekhter M, Scalia R, Kahn CR, King GL. 2010. Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice. Cell metabolism. 11(5):379-89. Pubmed: 20444418 DOI:10.1016/j.cmet.2010.03.013 To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls but reduced to below control values by a VCAM-1-blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications. -
Hettmer S, Wagers AJ. 2010. Muscling in: Uncovering the origins of rhabdomyosarcoma. Nature medicine. 16(2):171-3. Pubmed: 20134473 DOI:10.1038/nm0210-171 Hettmer S, Wagers AJ. 2010. Muscling in: Uncovering the origins of rhabdomyosarcoma. Nature medicine. 16(2):171-3. Pubmed: 20134473 DOI:10.1038/nm0210-171 -
Mayack SR, Shadrach JL, Kim FS, Wagers AJ. 2010. Systemic signals regulate ageing and rejuvenation of blood stem cell niches. Nature. 463(7280):495-500. Pubmed: 20110993 DOI:10.1038/nature08749 Mayack SR, Shadrach JL, Kim FS, Wagers AJ. 2010. Systemic signals regulate ageing and rejuvenation of blood stem cell niches. Nature. 463(7280):495-500. Pubmed: 20110993 DOI:10.1038/nature08749 Ageing in multicellular organisms typically involves a progressive decline in cell replacement and repair processes, resulting in several physiological deficiencies, including inefficient muscle repair, reduced bone mass, and dysregulation of blood formation (haematopoiesis). Although defects in tissue-resident stem cells clearly contribute to these phenotypes, it is unclear to what extent they reflect stem cell intrinsic alterations or age-related changes in the stem cell supportive microenvironment, or niche. Here, using complementary in vivo and in vitro heterochronic models, we show that age-associated changes in stem cell supportive niche cells deregulate normal haematopoiesis by causing haematopoietic stem cell dysfunction. Furthermore, we find that age-dependent defects in niche cells are systemically regulated and can be reversed by exposure to a young circulation or by neutralization of the conserved longevity regulator, insulin-like growth factor-1, in the marrow microenvironment. Together, these results show a new and critical role for local and systemic factors in signalling age-related haematopoietic decline, and highlight a new model in which blood-borne factors in aged animals act through local niche cells to induce age-dependent disruption of stem cell function. -
Schemionek M, Elling C, Steidl U, Bäumer N, Hamilton A, Spieker T, Göthert JR, Stehling M, Wagers A, Huettner CS, Tenen DG, Tickenbrock L, Berdel WE, Serve H, Holyoake TL, Müller-Tidow C, Koschmieder S. 2010. BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells. Blood. 115(16):3185-95. Pubmed: 20053753 DOI:10.1182/blood-2009-04-215376 Schemionek M, Elling C, Steidl U, Bäumer N, Hamilton A, Spieker T, Göthert JR, Stehling M, Wagers A, Huettner CS, Tenen DG, Tickenbrock L, Berdel WE, Serve H, Holyoake TL, Müller-Tidow C, Koschmieder S. 2010. BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells. Blood. 115(16):3185-95. Pubmed: 20053753 DOI:10.1182/blood-2009-04-215376 In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL(+) Lin(-)Sca-1(+)c-kit(+) (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin(-)Sca-1(-)c-kit(+)), nor mature granulocytes (CD11b(+)Gr-1(+)), nor potential stem cell niche cells (CD45(-)Ter119(-)) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL(+) LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity. -
Liu J, Sato C, Cerletti M, Wagers A. 2010. Notch signaling in the regulation of stem cell self-renewal and differentiation. Current topics in developmental biology. 92:367-409. Pubmed: 20816402 DOI:10.1016/S0070-2153(10)92012-7 Liu J, Sato C, Cerletti M, Wagers A. 2010. Notch signaling in the regulation of stem cell self-renewal and differentiation. Current topics in developmental biology. 92:367-409. Pubmed: 20816402 DOI:10.1016/S0070-2153(10)92012-7 Stem cells are rare and unique precursor cells that participate in the building and rebuilding of tissues and organs during embryogenesis, postnatal growth, and injury repair. Stem cells are distinctively endowed with the ability to both self-renew and differentiate, such that they can replenish the stem cell pool while continuing to produce the differentiated daughter cells that are essential for tissue function. Stem cell self-renewal/differentiation decisions must be carefully controlled during organogenesis, tissue homeostasis, and regeneration, as failure in stem cell maintenance or activation can lead to progressive tissue wasting, while unchecked self-renewal is a hallmark of many cancers. Here, we review evidence implicating the Notch signaling pathway, an evolutionarily conserved cell fate determinant with widespread roles in a variety of tissues and organisms, as a crucial regulator of stem cell behavior. As discussed below, this pathway plays varied and critical roles at multiple stages of organismal development, in lineage-specific differentiation of pluripotent embryonic stem cells, and in controlling stem cell numbers and activity in the context of age-related tissue degeneration, injury-induced tissue repair, and malignancy.Copyright 2010 Elsevier Inc. All rights reserved. -
Wagers A. 2010. Delivering on a promise. Interviewed by Kristie Nybo. BioTechniques. 49(2):545. Pubmed: 20945585 Wagers A. 2010. Delivering on a promise. Interviewed by Kristie Nybo. BioTechniques. 49(2):545. Pubmed: 20945585 -
Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, Apostolou E, Stadtfeld M, Li Y, Shioda T, Natesan S, Wagers AJ, Melnick A, Evans T, Hochedlinger K. 2010. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nature biotechnology. 28(8):848-55. Pubmed: 20644536 DOI:10.1038/nbt.1667 Polo JM, Liu S, Figueroa ME, Kulalert W, Eminli S, Tan KY, Apostolou E, Stadtfeld M, Li Y, Shioda T, Natesan S, Wagers AJ, Melnick A, Evans T, Hochedlinger K. 2010. Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nature biotechnology. 28(8):848-55. Pubmed: 20644536 DOI:10.1038/nbt.1667 Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages. -
Young AP, Wagers AJ. 2010. Pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors. Journal of cell science. 123(Pt 15):2632-9. Pubmed: 20605921 DOI:10.1242/jcs.061606 Young AP, Wagers AJ. 2010. Pax3 induces differentiation of juvenile skeletal muscle stem cells without transcriptional upregulation of canonical myogenic regulatory factors. Journal of cell science. 123(Pt 15):2632-9. Pubmed: 20605921 DOI:10.1242/jcs.061606 Pax3 is an essential myogenic regulator of fetal and embryonic development, but its role in postnatal myogenesis remains a topic of debate. We show that constitutive expression of Pax3 in postnatal, juvenile mouse skeletal muscle stem cells, a subset of the heterogeneous satellite cell pool highly enriched for myogenic activity, potently induces differentiation. This differentiation-promoting activity stands in contrast to the differentiation-inhibiting effects of Pax3 in the commonly used mouse myoblast cell line C2C12. Pax3 mRNA levels in distinct muscles correlate with the rate of myogenic differentiation of their muscle stem cells. Although Pax3 controls embryonic myogenesis through regulation of the canonical myogenic regulatory factors (MRFs) Myf-5, MyoD, myogenin and Mrf4, we find that in postnatal muscle stem cells, ectopic Pax3 expression fails to induce expression of any of these factors. Unexpectedly, overexpression of neither Myf-5 nor myogenin is sufficient to induce differentiation of juvenile stem cells; and knockdown of Myf-5, rather than inhibiting differentiation, promotes it. Taken together, our results suggest that there are distinct myogenic regulatory pathways that control the embryonic development, juvenile myogenesis and adult regeneration of skeletal myofibers. -
Taylor PL, Barker RA, Blume KG, Cattaneo E, Colman A, Deng H, Edgar H, Fox IJ, Gerstle C, Goldstein LS, High KA, Lyall A, Parkman R, Pitossi FJ, Prentice ED, Rooke HM, Sipp DA, Srivastava A, Stayn S, Steinberg GK, Wagers AJ, Weissman IL. 2010. Patients beware: commercialized stem cell treatments on the web. Cell stem cell. 7(1):43-9. Pubmed: 20621049 DOI:10.1016/j.stem.2010.06.001 Taylor PL, Barker RA, Blume KG, Cattaneo E, Colman A, Deng H, Edgar H, Fox IJ, Gerstle C, Goldstein LS, High KA, Lyall A, Parkman R, Pitossi FJ, Prentice ED, Rooke HM, Sipp DA, Srivastava A, Stayn S, Steinberg GK, Wagers AJ, Weissman IL. 2010. Patients beware: commercialized stem cell treatments on the web. Cell stem cell. 7(1):43-9. Pubmed: 20621049 DOI:10.1016/j.stem.2010.06.001 A report by the International Society for Stem Cell Research (ISSCR)'s Task Force on Unproven Stem Cell Treatments outlines development of resources for patients, their families, and physicians seeking information on stem cell treatments.Copyright (c) 2010 Elsevier Inc. All rights reserved. -
Song G, Nguyen DT, Pietramaggiori G, Scherer S, Chen B, Zhan Q, Ogawa R, Yannas IV, Wagers AJ, Orgill DP, Murphy GF. 2010. Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 18(4):426-32. Pubmed: 20546556 DOI:10.1111/j.1524-475X.2010.00595.x Song G, Nguyen DT, Pietramaggiori G, Scherer S, Chen B, Zhan Q, Ogawa R, Yannas IV, Wagers AJ, Orgill DP, Murphy GF. 2010. Use of the parabiotic model in studies of cutaneous wound healing to define the participation of circulating cells. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 18(4):426-32. Pubmed: 20546556 DOI:10.1111/j.1524-475X.2010.00595.x Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP- parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses. -
Conboy MJ, Cerletti M, Wagers AJ, Conboy IM. 2010. Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells. Methods in molecular biology (Clifton, N.J.). 621:165-73. Pubmed: 20405366 DOI:10.1007/978-1-60761-063-2_11 Conboy MJ, Cerletti M, Wagers AJ, Conboy IM. 2010. Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells. Methods in molecular biology (Clifton, N.J.). 621:165-73. Pubmed: 20405366 DOI:10.1007/978-1-60761-063-2_11 We describe the immunostaining methods we commonly use to detect the more robust cell markers identifying the various cell populations obtained by the enzymatic and or mechanical dissociation of muscle satellite cells: CD34, m-Cadherin, and Pax7, self-renewing muscle stem cells expressing CXCR4 and beta1-integrin, populations of proliferative myogenic progenitor cells expressing, Pax3, Pax7, Myf5, MyoD1, and desmin, differentiating myoblasts expressing myogenin and eMHC, and the CD45 expressing leukocyte lineage cells that infiltrate injured and regenerating skeletal muscle. 2009
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Pietramaggiori G, Scherer SS, Alperovich M, Chen B, Orgill DP, Wagers AJ. 2009. Improved cutaneous healing in diabetic mice exposed to healthy peripheral circulation. The Journal of investigative dermatology. 129(9):2265-74. Pubmed: 19295612 DOI:10.1038/jid.2009.60 Pietramaggiori G, Scherer SS, Alperovich M, Chen B, Orgill DP, Wagers AJ. 2009. Improved cutaneous healing in diabetic mice exposed to healthy peripheral circulation. The Journal of investigative dermatology. 129(9):2265-74. Pubmed: 19295612 DOI:10.1038/jid.2009.60 Impaired repair of skin defects is a major complication of diabetes; yet, the pathophysiology of diabetic (db) wound healing remains largely opaque. Here, we investigate the role of humoral factors in modulating db wound repair by generating chimeric animals through parabiotic joining of wild-type (wt) and diabetic (db/db) mice. This strategy allows wounds on healing-deficient db/db mice to be exposed to factors derived from the wt circulation at physiologically appropriate concentrations. When compared with db controls, chimeric db/db animals showed significantly improved healing of full-thickness, cutaneous wounds, with enhanced granulation tissue formation, angiogenesis, cell proliferation, and collagen deposition. Glycemic control was unaffected by parabiosis; however, the distribution of circulating leukocytes, altered in db controls, normalized in db-chimeras. Both wt and db cells were recruited from circulation into db wounds, but wt cells never exceeded 20% of total cells. Improved angiogenesis persisted in db-chimeras separated 24 hours after wounding, suggesting the existence of long-term normalizing factors. This study establishes a new model for studying db wound healing, and shows a key role for circulating factors in normalizing wound repair in diabetes. -
Kumar D, Shadrach JL, Wagers AJ, Lassar AB. 2009. Id3 is a direct transcriptional target of Pax7 in quiescent satellite cells. Molecular biology of the cell. 20(14):3170-7. Pubmed: 19458195 Kumar D, Shadrach JL, Wagers AJ, Lassar AB. 2009. Id3 is a direct transcriptional target of Pax7 in quiescent satellite cells. Molecular biology of the cell. 20(14):3170-7. Pubmed: 19458195 Pax7 is a key regulator of skeletal muscle stem cells and is required along with Pax3 to generate skeletal muscle precursors. We have identified a collection of genes induced by either Pax3 or Pax7 in C2C12 muscle cells. Two notable Pax3/7 targets are the inhibitory helix-loop-helix (HLH) proteins inhibitor of DNA binding (Id) 2 and Id3, both of which are coordinately expressed with Pax7 in quiescent satellite cells and are induced in quiescent C2C12 myogenic cells after ectopic expression of either Pax3 or Pax7. Ectopic Pax7 activates expression of a luciferase reporter driven by the Id3 promoter, and maximal induction of this reporter requires a conserved Pax7 binding site located upstream of the Id3 gene. Chromatin immunoprecipitation indicated that Pax7 is bound upstream of the Id3 promoter in quiescent satellite cells. In addition, short hairpin RNA-mediated knockdown of Pax7 expression in cultured satellite cells coordinately decreased both Id2 and Id3 expression. Together, these findings indicate that Id3 is a direct transcriptional target for Pax7 in quiescent satellite cells, and they suggest that Pax7 acts to block premature differentiation of quiescent satellite cells by inducing the expression of Id2 and Id3, which in turn may act to block either the precocious induction of myogenic basic (b)HLH proteins, the activity of myogenic bHLH proteins, or both. -
Guibal FC, Alberich-Jorda M, Hirai H, Ebralidze A, Levantini E, Di Ruscio A, Zhang P, Santana-Lemos BA, Neuberg D, Wagers AJ, Rego EM, Tenen DG. 2009. Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia. Blood. 114(27):5415-25. Pubmed: 19797526 DOI:10.1182/blood-2008-10-182071 Guibal FC, Alberich-Jorda M, Hirai H, Ebralidze A, Levantini E, Di Ruscio A, Zhang P, Santana-Lemos BA, Neuberg D, Wagers AJ, Rego EM, Tenen DG. 2009. Identification of a myeloid committed progenitor as the cancer-initiating cell in acute promyelocytic leukemia. Blood. 114(27):5415-25. Pubmed: 19797526 DOI:10.1182/blood-2008-10-182071 Acute promyelocytic leukemia (APL) is characterized by a block in differentiation and accumulation of promyelocytes in the bone marrow and blood. The majority of APL patients harbor the t(15:17) translocation leading to expression of the fusion protein promyelocytic-retinoic acid receptor alpha. Treatment with retinoic acid leads to degradation of promyelocytic-retinoic acid receptor alpha protein and disappearance of leukemic cells; however, 30% of APL patients relapse after treatment. One potential mechanism for relapse is the persistence of cancer "stem" cells in hematopoietic organs after treatment. Using a novel sorting strategy we developed to isolate murine myeloid cells at distinct stages of differentiation, we identified a population of committed myeloid cells (CD34(+), c-kit(+), FcgammaRIII/II(+), Gr1(int)) that accumulates in the spleen and bone marrow in a murine model of APL. We observed that these cells are capable of efficiently generating leukemia in recipient mice, demonstrating that this population represents the APL cancer-initiating cell. These cells down-regulate the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) possibly through a methylation-dependent mechanism, indicating that C/EBPalpha deregulation contributes to transformation of APL cancer-initiating cells. Our findings provide further understanding of the biology of APL by demonstrating that a committed transformed progenitor can initiate and propagate the disease. 2008
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Jakubzick C, Tacke F, Ginhoux F, Wagers AJ, van Rooijen N, Mack M, Merad M, Randolph GJ. 2008. Blood monocyte subsets differentially give rise to CD103+ and CD103- pulmonary dendritic cell populations. Journal of immunology (Baltimore, Md. : 1950). 180(5):3019-27. Pubmed: 18292524 Jakubzick C, Tacke F, Ginhoux F, Wagers AJ, van Rooijen N, Mack M, Merad M, Randolph GJ. 2008. Blood monocyte subsets differentially give rise to CD103+ and CD103- pulmonary dendritic cell populations. Journal of immunology (Baltimore, Md. : 1950). 180(5):3019-27. Pubmed: 18292524 There are two major myeloid pulmonary dendritic cell (DC) populations: CD103+ DCs and CD11bhigh DCs. In this study, we investigated in detail the origins of both myeloid DC pools using multiple experimental approaches. We show that, in resting lung, Ly-6ChighCCR2high monocytes repopulated CD103+ DCs using a CCR2-dependent mechanism, and these DCs preferentially retained residual CCR2 in the lung, whereas, conversely, Ly-6ClowCCR2low monocytes repopulated CD11bhigh DCs. CX3CR1 was required to generate normal numbers of pulmonary CD11bhigh DCs, possibly because Ly-6Clow monocytes in the circulation, which normally express high levels of CX3CR1, failed to express bcl-2 and may have diminished survival in the circulation in the absence of CX3CR1. Overall, these data demonstrate that the two circulating subsets of monocytes give rise to distinct tissue DC populations. -
Min IM, Pietramaggiori G, Kim FS, Passegué E, Stevenson KE, Wagers AJ. 2008. The transcription factor EGR1 controls both the proliferation and localization of hematopoietic stem cells. Cell stem cell. 2(4):380-91. Pubmed: 18397757 DOI:10.1016/j.stem.2008.01.015 Min IM, Pietramaggiori G, Kim FS, Passegué E, Stevenson KE, Wagers AJ. 2008. The transcription factor EGR1 controls both the proliferation and localization of hematopoietic stem cells. Cell stem cell. 2(4):380-91. Pubmed: 18397757 DOI:10.1016/j.stem.2008.01.015 EGR1 is a member of the immediate early response transcription factor family and functions in cell growth, development, and stress responses in many tissues. Here we report an additional role for EGR1 in regulating homeostasis of hematopoietic stem cells (HSCs). HSCs normally express Egr1 at high levels, but dramatically downregulate its expression when induced to divide and migrate. Consistent with this finding, mice lacking Egr1 exhibit significant increases in steady-state levels of dividing HSCs in the bone marrow (BM), and a striking spontaneous mobilization of HSCs into the peripheral blood. These data identify EGR1 as a transcriptional regulator of stem cell migration that normally functions to promote HSC quiescence and retention in the niche. The ability of this single factor to regulate both proliferation and mobilization of HSCs suggests that EGR1 commands a genetic program that coordinates stem cell division and migration to maintain appropriate HSC number and function. -
Cao YA, Wagers AJ, Karsunky H, Zhao H, Reeves R, Wong RJ, Stevenson DK, Weissman IL, Contag CH. 2008. Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors. Blood. 112(12):4494-502. Pubmed: 18509090 DOI:10.1182/blood-2007-12-127621 Cao YA, Wagers AJ, Karsunky H, Zhao H, Reeves R, Wong RJ, Stevenson DK, Weissman IL, Contag CH. 2008. Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors. Blood. 112(12):4494-502. Pubmed: 18509090 DOI:10.1182/blood-2007-12-127621 An effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1(+/-)) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1(+/-) HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1(+/-) HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1(+/-) HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1(+/-) HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell-based therapies. -
Cerletti M, Jurga S, Witczak CA, Hirshman MF, Shadrach JL, Goodyear LJ, Wagers AJ. 2008. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell. 134(1):37-47. Pubmed: 18614009 DOI:10.1016/j.cell.2008.05.049 Cerletti M, Jurga S, Witczak CA, Hirshman MF, Shadrach JL, Goodyear LJ, Wagers AJ. 2008. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell. 134(1):37-47. Pubmed: 18614009 DOI:10.1016/j.cell.2008.05.049 Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease. -
Laird DJ, von Andrian UH, Wagers AJ. 2008. Stem cell trafficking in tissue development, growth, and disease. Cell. 132(4):612-30. Pubmed: 18295579 DOI:10.1016/j.cell.2008.01.041 Laird DJ, von Andrian UH, Wagers AJ. 2008. Stem cell trafficking in tissue development, growth, and disease. Cell. 132(4):612-30. Pubmed: 18295579 DOI:10.1016/j.cell.2008.01.041 Regulated movement of stem cells is critical for organogenesis during development and for homeostasis and repair in adulthood. Here we analyze the biological significance and molecular mechanisms underlying stem cell trafficking in the generation of the germline, and the generation and regeneration of blood and muscle. Comparison across organisms and lineages reveals remarkable conservation as well as specialization in homing and migration mechanisms used by mature leukocytes, adult and fetal stem cells, and cancer stem cells. In vivo trafficking underpins the successful therapeutic application of hematopoietic stem cells for bone-marrow transplant, and further elucidation of homing and migration pathways in other systems will enable broader application of stem cells for targeted cell therapy and drug delivery. -
Wagers AJ. 2008. Wnt not, waste not. Cell stem cell. 2(1):6-7. Pubmed: 18371413 DOI:10.1016/j.stem.2007.12.005 Wagers AJ. 2008. Wnt not, waste not. Cell stem cell. 2(1):6-7. Pubmed: 18371413 DOI:10.1016/j.stem.2007.12.005 The molecular signals that regulate the regenerative function of satellite cells in the skeletal muscle remain largely obscure. In this issue of Cell Stem Cell, Brack et al. (2008) report that direct molecular crosstalk between stem cell self-renewal and differentiation pathways determines the timing and efficiency of muscle repair. -
Mayack SR, Wagers AJ. 2008. Osteolineage niche cells initiate hematopoietic stem cell mobilization. Blood. 112(3):519-31. Pubmed: 18456874 DOI:10.1182/blood-2008-01-133710 Mayack SR, Wagers AJ. 2008. Osteolineage niche cells initiate hematopoietic stem cell mobilization. Blood. 112(3):519-31. Pubmed: 18456874 DOI:10.1182/blood-2008-01-133710 Recent studies have implicated bone-lining osteoblasts as important regulators of hematopoietic stem cell (HSC) self-renewal and differentiation; however, because much of the evidence supporting this notion derives from indirect in vivo experiments, which are unavoidably complicated by the presence of other cell types within the complex bone marrow milieu, the sufficiency of osteoblasts in modulating HSC activity has remained controversial. To address this, we prospectively isolated mouse osteoblasts, using a novel flow cytometry-based approach, and directly tested their activity as HSC niche cells and their role in cyclophosphamide/granulocyte colony-stimulating factor (G-CSF)-induced HSC proliferation and mobilization. We found that osteoblasts expand rapidly after cyclophosphamide/G-CSF treatment and exhibit phenotypic and functional changes that directly influence HSC proliferation and maintenance of reconstituting potential. Effects of mobilization on osteoblast number and function depend on the function of ataxia telangiectasia mutated (ATM), the product of the Atm gene, demonstrating a new role for ATM in stem cell niche activity. These studies demonstrate that signals from osteoblasts can directly initiate and modulate HSC proliferation in the context of mobilization. This work also establishes that direct interaction with osteolineage niche cells, in the absence of additional environmental inputs, is sufficient to modulate stem cell activity. -
Breault DT, Min IM, Carlone DL, Farilla LG, Ambruzs DM, Henderson DE, Algra S, Montgomery RK, Wagers AJ, Hole N. 2008. Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells. Proceedings of the National Academy of Sciences of the United States of America. 105(30):10420-5. Pubmed: 18650388 DOI:10.1073/pnas.0804800105 Breault DT, Min IM, Carlone DL, Farilla LG, Ambruzs DM, Henderson DE, Algra S, Montgomery RK, Wagers AJ, Hole N. 2008. Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells. Proceedings of the National Academy of Sciences of the United States of America. 105(30):10420-5. Pubmed: 18650388 DOI:10.1073/pnas.0804800105 Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of "stemness" have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP(+) BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues. -
Jones DL, Wagers AJ. 2008. No place like home: anatomy and function of the stem cell niche. Nature reviews. Molecular cell biology. 9(1):11-21. Pubmed: 18097443 Jones DL, Wagers AJ. 2008. No place like home: anatomy and function of the stem cell niche. Nature reviews. Molecular cell biology. 9(1):11-21. Pubmed: 18097443 Stem cells are rare cells that are uniquely capable of both reproducing themselves (self-renewing) and generating the differentiated cell types that are needed to carry out specialized functions in the body. Stem cell behaviour, in particular the balance between self-renewal and differentiation, is ultimately controlled by the integration of intrinsic factors with extrinsic cues supplied by the surrounding microenvironment, known as the stem cell niche. The identification and characterization of niches within tissues has revealed an intriguing conservation of many components, although the mechanisms that regulate how niches are established, maintained and modified to support specific tissue stem cell functions are just beginning to be uncovered. -
Cerletti M, Shadrach JL, Jurga S, Sherwood R, Wagers AJ. 2008. Regulation and function of skeletal muscle stem cells. Cold Spring Harbor symposia on quantitative biology. 73:317-22. Pubmed: 19204065 DOI:10.1101/sqb.2008.73.054 Cerletti M, Shadrach JL, Jurga S, Sherwood R, Wagers AJ. 2008. Regulation and function of skeletal muscle stem cells. Cold Spring Harbor symposia on quantitative biology. 73:317-22. Pubmed: 19204065 DOI:10.1101/sqb.2008.73.054 Skeletal muscle satellite cells, which reside beneath the basal lamina of mature muscle fibers, function as myogenic precursors and are required for normal muscle growth and repair. Satellite cells share a common anatomical localization, yet they exhibit substantial phenotypic and functional heterogeneity. Recent efforts in the field of adult myogenesis have been aimed at dissecting this heterogeneity and reveal the presence of discrete cell lineages within the muscle that function independently and interactively to maintain muscle homeostasis and to determine the outcome of muscle damage. Normal developmental regulation of the frequency and function of these distinct tissue precursors, and pathological deregulation of their activity, may have an important role in age- and disease-dependent loss of muscle regenerative activity. 2007
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Massberg S, Schaerli P, Knezevic-Maramica I, Köllnberger M, Tubo N, Moseman EA, Huff IV, Junt T, Wagers AJ, Mazo IB, von Andrian UH. 2007. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues. Cell. 131(5):994-1008. Pubmed: 18045540 Massberg S, Schaerli P, Knezevic-Maramica I, Köllnberger M, Tubo N, Moseman EA, Huff IV, Junt T, Wagers AJ, Mazo IB, von Andrian UH. 2007. Immunosurveillance by hematopoietic progenitor cells trafficking through blood, lymph, and peripheral tissues. Cell. 131(5):994-1008. Pubmed: 18045540 Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on sphingosine-1-phosphate receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals. -
Monach PA, Verschoor A, Jacobs JP, Carroll MC, Wagers AJ, Benoist C, Mathis D. 2007. Circulating C3 is necessary and sufficient for induction of autoantibody-mediated arthritis in a mouse model. Arthritis and rheumatism. 56(9):2968-74. Pubmed: 17763447 Monach PA, Verschoor A, Jacobs JP, Carroll MC, Wagers AJ, Benoist C, Mathis D. 2007. Circulating C3 is necessary and sufficient for induction of autoantibody-mediated arthritis in a mouse model. Arthritis and rheumatism. 56(9):2968-74. Pubmed: 17763447 Array 2006
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Sherwood RI, Wagers AJ. 2006. Harnessing the potential of myogenic satellite cells. Trends in molecular medicine. 12(5):189-92. Pubmed: 16595190 Sherwood RI, Wagers AJ. 2006. Harnessing the potential of myogenic satellite cells. Trends in molecular medicine. 12(5):189-92. Pubmed: 16595190 Adult skeletal muscle has remarkable regenerative potential, which is mainly attributable to a small population of undifferentiated skeletal muscle precursors called satellite cells. These cells reside underneath the basal lamina of skeletal myofibers and can be activated to proliferate, differentiate and fuse to form new muscle tissue. Satellite cells have long been considered promising mediators of therapeutic muscle regeneration. However, in practice, the regenerative function of such cells, which in many cases have been derived or expanded by ex vivo cultures, can be surprisingly low. A recent study from Montarras and colleagues has provided new insights into the requirements for efficient muscle engraftment from purified muscle satellite cells, suggesting possible strategies to enhance their therapeutic potential. -
Passegué E, Wagers AJ. 2006. Regulating quiescence: new insights into hematopoietic stem cell biology. Developmental cell. 10(4):415-7. Pubmed: 16580989 Passegué E, Wagers AJ. 2006. Regulating quiescence: new insights into hematopoietic stem cell biology. Developmental cell. 10(4):415-7. Pubmed: 16580989 Blood-forming hematopoietic stem cells (HSCs) ensure production of all mature blood cells during homeostatic and regenerative hematopoiesis. Proliferation, cell cycle regulation, and quiescence are key processes involved in this function, and in a recent issue of Cancer Cell, show that HSC quiescence is actively regulated by specific molecular mechanisms that appear to distinguish normal HSC maintenance from HSC responses to hematologic injury. -
Eggan K, Jurga S, Gosden R, Min IM, Wagers AJ. 2006. Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature. 441(7097):1109-14. Pubmed: 16799565 Eggan K, Jurga S, Gosden R, Min IM, Wagers AJ. 2006. Ovulated oocytes in adult mice derive from non-circulating germ cells. Nature. 441(7097):1109-14. Pubmed: 16799565 Decades of research in reproductive biology have led to the generally accepted belief that in female mammals, all surviving germ cells enter meiosis at the end of fetal development and as a result, the postnatal ovary harbours a limited supply of oocytes that cannot be replenished or regenerated if lost to injury or disease. However, recent reports have challenged this view, suggesting instead that oocyte production is maintained through continual seeding of the ovary by circulating, bone-marrow-derived germ cells. To test directly the physiological relevance of circulating cells for female fertility, we established transplantation and parabiotic mouse models to assess the capacity of circulating bone marrow cells to generate ovulated oocytes, both in the steady state and after induced damage. Our studies showed no evidence that bone marrow cells, or any other normally circulating cells, contribute to the formation of mature, ovulated oocytes. Instead, cells that travelled to the ovary through the bloodstream exhibited properties characteristic of committed blood leukocytes. -
Santarelli JG, Udani V, Yung YC, Cheshier S, Wagers A, Brekken RA, Weissman I, Tse V. 2006. Incorporation of bone marrow-derived Flk-1-expressing CD34+ cells in the endothelium of tumor vessels in the mouse brain. Neurosurgery. 59(2):374-82; discussion 374-82. Pubmed: 16883178 Santarelli JG, Udani V, Yung YC, Cheshier S, Wagers A, Brekken RA, Weissman I, Tse V. 2006. Incorporation of bone marrow-derived Flk-1-expressing CD34+ cells in the endothelium of tumor vessels in the mouse brain. Neurosurgery. 59(2):374-82; discussion 374-82. Pubmed: 16883178 Array -
Wagers AJ, Weissman IL. 2006. Differential expression of alpha2 integrin separates long-term and short-term reconstituting Lin-/loThy1.1(lo)c-kit+ Sca-1+ hematopoietic stem cells. Stem cells (Dayton, Ohio). 24(4):1087-94. Pubmed: 16373693 Wagers AJ, Weissman IL. 2006. Differential expression of alpha2 integrin separates long-term and short-term reconstituting Lin-/loThy1.1(lo)c-kit+ Sca-1+ hematopoietic stem cells. Stem cells (Dayton, Ohio). 24(4):1087-94. Pubmed: 16373693 Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin- Thy1.1(lo)c-kit+ Sca-1+ subset of mouse bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically and perhaps functionally. We previously identified alpha2-integrin (CD49b) as a surface marker with heterogeneous expression on Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ stem cells. To determine whether differences in alpha2 expression were indicative of differences in stem cell function, we purified alpha2- and alpha2hi stem cells by fluorescence-activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both alpha2- and alpha2hi cells could give rise to mature lymphoid and myeloid cells after transplantation into lethally irradiated congenic recipients. However, alpha2hi cells supported hematopoiesis for only a short time (<4 weeks), whereas alpha2- cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that alpha2- cells represent a more primitive population than do alpha2hi cells. Consistent with this idea, alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells exhibited an approximately sixfold decreased frequency of spleen colony-forming units (day 12) versus alpha2hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells included both alpha2- and alpha2hi stem cells of donor origin, indicating that alpha2hi cells are likely lineal descendents of alpha2- cells. Interestingly, alpha2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of alpha2 selectively marks a subset of primitive hematopoietic cells which retains multilineage reconstitution potential but exhibits reduced self-renewal capacity. -
Bogunovic M, Ginhoux F, Wagers A, Loubeau M, Isola LM, Lubrano L, Najfeld V, Phelps RG, Grosskreutz C, Scigliano E, Frenette PS, Merad M. 2006. Identification of a radio-resistant and cycling dermal dendritic cell population in mice and men. The Journal of experimental medicine. 203(12):2627-38. Pubmed: 17116734 Bogunovic M, Ginhoux F, Wagers A, Loubeau M, Isola LM, Lubrano L, Najfeld V, Phelps RG, Grosskreutz C, Scigliano E, Frenette PS, Merad M. 2006. Identification of a radio-resistant and cycling dermal dendritic cell population in mice and men. The Journal of experimental medicine. 203(12):2627-38. Pubmed: 17116734 In this study, we explored dermal dendritic cell (DC) homeostasis in mice and humans both in the steady state and after hematopoietic cell transplantation. We discovered that dermal DCs proliferate in situ in mice and human quiescent dermis. In parabiotic mice with separate organs but shared blood circulation, the majority of dermal DCs failed to be replaced by circulating precursors for >6 mo. In lethally irradiated mice injected with donor congenic bone marrow (BM) cells, a subset of recipient DCs remained in the dermis and proliferated locally throughout life. Consistent with these findings, a large proportion of recipient dermal DCs remained in patients' skin after allogeneic hematopoietic cell transplantation, despite complete donor BM chimerism. Collectively, our results oppose the traditional view that DCs are nondividing terminally differentiated cells maintained by circulating precursors and support the new paradigm that tissue DCs have local proliferative properties that control their homeostasis in the steady state. Given the role of residual host tissue DCs in transplant immune reactions, these results suggest that dermal DC homeostasis may contribute to the development of cutaneous graft-versus-host disease in clinical transplantation. 2005
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Passegué E, Wagers AJ, Giuriato S, Anderson WC, Weissman IL. 2005. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. The Journal of experimental medicine. 202(11):1599-611. Pubmed: 16330818 Passegué E, Wagers AJ, Giuriato S, Anderson WC, Weissman IL. 2005. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. The Journal of experimental medicine. 202(11):1599-611. Pubmed: 16330818 Knowledge of the molecular networks controlling the proliferation and fate of hematopoietic stem cells (HSC) is essential to understand their function in maintaining blood cell production during normal hematopoiesis and upon clinical transplantation. Using highly purified stem and progenitor cell populations, we define the proliferation index and status of the cell cycle machinery at discrete stages of hematopoietic differentiation and during cytokine-mediated HSC mobilization. We identify distinct sets of cell cycle proteins that specifically associate with differentiation, self-renewal, and maintenance of quiescence in HSC and progenitor cells. Moreover, we describe a striking inequality of function among in vivo cycling and quiescent HSC by demonstrating that their long-term engraftment potential resides predominantly in the G(0) fraction. These data provide a direct link between HSC proliferation and function and identify discrete molecular targets in regulating HSC cell fate decisions that could have implications for both the therapeutic use of HSC and the understanding of leukemic transformation. -
Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. 2005. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature. 433(7027):760-4. Pubmed: 15716955 Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA. 2005. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature. 433(7027):760-4. Pubmed: 15716955 The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-alpha and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-alpha complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age. -
Udani VM, Santarelli JG, Yung YC, Wagers AJ, Cheshier SH, Weissman IL, Tse V. 2005. Hematopoietic stem cells give rise to perivascular endothelial-like cells during brain tumor angiogenesis. Stem cells and development. 14(5):478-86. Pubmed: 16305333 Udani VM, Santarelli JG, Yung YC, Wagers AJ, Cheshier SH, Weissman IL, Tse V. 2005. Hematopoietic stem cells give rise to perivascular endothelial-like cells during brain tumor angiogenesis. Stem cells and development. 14(5):478-86. Pubmed: 16305333 Bone marrow (BM) cells have recently been shown to give rise to skeletal, hepatic, cardiac, neural, and vascular endothelial tissues. However, it has been shown that this is the result of cell fusion rather than transdifferentiation of hematopoietic stem cells (HSC). For this study, we established a mouse model of brain tumor growth to investigate the differentiation potential of HSC into endothelial cells during brain tumor-induced angiogenesis. Nontransgenic (GFP(neg)) recipient mice were lethally irradiated, and their hematopoietic cells were subsequently repopulated by transplantation of a single green fluorescent protein (GFP)-expressing HSC. Rat glioma (RT-2/RAG) cells were then injected into the striatum of the chimeric mice 6-8 weeks post-transplantation. The animals were sacrificed 3-9 days after tumor implantation, and the mobilization, temporal-spatial distribution, and lineage-specific marker expression profile of the GFP(+) cells within the growing tumor were analyzed. We saw that GFP(+) cells gave rise to elongated, CD34(+)/Flk-1(+) cells that incorporated into the endothelium of tumor blood vessels. However, all GFP(+) cells were also CD45(+), and the presence of CD45 on the HSC-derived endothelial-like cells supports the hypothesis that the hematopoietic cells were recruited into the tumor milieu. The fact that we failed to demonstrate the expression of von Willebrand factor in these cells argues against a true endothelial identity. Nevertheless, the recruitment of HSC-derived endothelial-like cells was an extremely rare event in normal brain parenchyma, and, thus, the permissive influence afforded by the growing tumor appeared to enhance the perivascular tropism and acquisition of an endothelial phenotypes by a population of HSC-derived cells. -
Wagers AJ, Conboy IM. 2005. Cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell. 122(5):659-67. Pubmed: 16143100 Wagers AJ, Conboy IM. 2005. Cellular and molecular signatures of muscle regeneration: current concepts and controversies in adult myogenesis. Cell. 122(5):659-67. Pubmed: 16143100 Adult skeletal muscle generates force in a controlled and directed manner through the contraction of highly specialized, postmitotic, multinucleated myofibers. Life-long muscle function relies on maintenance and regeneration of myofibers through a highly regulated process beginning with activation of normally quiescent muscle precursor cells and proceeding with formation of proliferating progenitors that fuse to generate differentiated myofibers. In this review, we describe the historical basis and current evidence for the identification of satellite cells as adult muscle stem cells, critically evaluate contributions of other cells to adult myogenesis, and summarize existing data regarding the origins, genetic markers, and molecular regulation of satellite cells in normal, diseased, and aged muscle. -
Santarelli JG, Udani V, Yung CY, Cheshier S, Wagers A, Brekken RA, Weissman I, Tse V. 2005. Preuss Resident Research Award: bone marrow-derived Flk-1-expressing CD34+ cells contribute to the endothelium of tumor vessels in mouse brain. Clinical neurosurgery. 52:384-8. Pubmed: 16626098 Santarelli JG, Udani V, Yung CY, Cheshier S, Wagers A, Brekken RA, Weissman I, Tse V. 2005. Preuss Resident Research Award: bone marrow-derived Flk-1-expressing CD34+ cells contribute to the endothelium of tumor vessels in mouse brain. Clinical neurosurgery. 52:384-8. Pubmed: 16626098 -
Wagers AJ. 2005. Stem cell grand SLAM. Cell. 121(7):967-70. Pubmed: 15989946 Wagers AJ. 2005. Stem cell grand SLAM. Cell. 121(7):967-70. Pubmed: 15989946 Stem cells in both embryonic and adult tissues are defined by their unique ability to balance self-renewal and differentiation such that mature cells necessary for tissue function can be generated and replaced without depletion of the stem cell pool. In this issue of Cell, report a major step forward for studying the mechanisms and regulation of such stem cell fate decisions in the blood-forming (hematopoietic) system by providing a simple and broadly applicable method to isolate these cells and to visualize them in their normal environment. -
Massengale M, Wagers AJ, Vogel H, Weissman IL. 2005. Hematopoietic cells maintain hematopoietic fates upon entering the brain. The Journal of experimental medicine. 201(10):1579-89. Pubmed: 15897275 Massengale M, Wagers AJ, Vogel H, Weissman IL. 2005. Hematopoietic cells maintain hematopoietic fates upon entering the brain. The Journal of experimental medicine. 201(10):1579-89. Pubmed: 15897275 Several studies have reported that bone marrow (BM) cells may give rise to neurons and astrocytes in vitro and in vivo. To further test this hypothesis, we analyzed for incorporation of neural cell types expressing donor markers in normal or injured brains of irradiated mice reconstituted with whole BM or single, purified c-kit(+)Thy1.1(lo)Lin(-)Sca-1(+) (KTLS) hematopoietic stem cells (HSCs), and of unirradiated parabionts with surgically anastomosed vasculature. Each model showed low-level parenchymal engraftment of donor-marker(+) cells with 96-100% immunoreactivity for panhematopoietic (CD45) or microglial (Iba1 or Mac1) lineage markers in all cases studied. Other than one arborizing structure in the olfactory bulb of one BM-transplanted animal, possibly representing a neuronal or glial cell process, we found no donor-marker-expressing astrocytes or non-Purkinje neurons among >10,000 donor-marker(+) cells from 21 animals. These data strongly suggest that HSCs and their progeny maintain lineage fidelity in the brain and do not adopt neural cell fates with any measurable frequency. -
Surdez D, Kunz B, Wagers AJ, Weissman IL, Terskikh AV. 2005. Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice. Stem cells (Dayton, Ohio). 23(10):1617-25. Pubmed: 16091556 Surdez D, Kunz B, Wagers AJ, Weissman IL, Terskikh AV. 2005. Simple and efficient isolation of hematopoietic stem cells from H2K-zFP transgenic mice. Stem cells (Dayton, Ohio). 23(10):1617-25. Pubmed: 16091556 We have generated a transgenic mouse line that allows for simple and highly efficient enrichment for mouse hematopoietic stem cells (HSCs). The transgene expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs express exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2K-zFP mice. Up to 90% of zFP(bright) cells are also c-kit(high), Sca-1(high), Lin(neg), Flk-2(neg), which is a bona fide phenotype for long-term HSCs. Double-sorted zFP(bright) HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2K-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs without using fluorescence-conjugated monoclonal antibodies. This approach will greatly facilitate gene transfer, including short interfering RNA for gene knockdown, into HSCs and, consequently, into all other hematopoietic lineages. -
Rossi DJ, Bryder D, Zahn JM, Ahlenius H, Sonu R, Wagers AJ, Weissman IL. 2005. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proceedings of the National Academy of Sciences of the United States of America. 102(26):9194-9. Pubmed: 15967997 Rossi DJ, Bryder D, Zahn JM, Ahlenius H, Sonu R, Wagers AJ, Weissman IL. 2005. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proceedings of the National Academy of Sciences of the United States of America. 102(26):9194-9. Pubmed: 15967997 Loss of immune function and an increased incidence of myeloid leukemia are two of the most clinically significant consequences of aging of the hematopoietic system. To better understand the mechanisms underlying hematopoietic aging, we evaluated the cell intrinsic functional and molecular properties of highly purified long-term hematopoietic stem cells (LT-HSCs) from young and old mice. We found that LT-HSC aging was accompanied by cell autonomous changes, including increased stem cell self-renewal, differential capacity to generate committed myeloid and lymphoid progenitors, and diminished lymphoid potential. Expression profiling revealed that LT-HSC aging was accompanied by the systemic down-regulation of genes mediating lymphoid specification and function and up-regulation of genes involved in specifying myeloid fate and function. Moreover, LT-HSCs from old mice expressed elevated levels of many genes involved in leukemic transformation. These data support a model in which age-dependent alterations in gene expression at the stem cell level presage downstream developmental potential and thereby contribute to age-dependent immune decline, and perhaps also to the increased incidence of leukemia in the elderly. 2004
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Cao YA, Wagers AJ, Beilhack A, Dusich J, Bachmann MH, Negrin RS, Weissman IL, Contag CH. 2004. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proceedings of the National Academy of Sciences of the United States of America. 101(1):221-6. Pubmed: 14688412 Cao YA, Wagers AJ, Beilhack A, Dusich J, Bachmann MH, Negrin RS, Weissman IL, Contag CH. 2004. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proceedings of the National Academy of Sciences of the United States of America. 101(1):221-6. Pubmed: 14688412 To reveal the early events and dynamics of hematopoietic reconstitution in living animals in real-time, we used bioluminescence imaging to monitor engraftment from single luciferase-labeled hematopoietic stem cells (HSC) in irradiated recipients. Transplanted HSC generated discrete foci in the spleen and bone marrow (BM), at a frequency that correlated with BM compartment size. Initially detected foci could expand locally, seed other sites in BM or spleen, and/or recede with different kinetics. These studies reveal dynamic and variable patterns of engraftment from highly purified HSC and indicate that the final overall contribution of individual HSC to hematopoietic chimerism does not depend on the specific site of initial engraftment and expansion. -
Yung YC, Cheshier S, Santarelli JG, Huang Z, Wagers A, Weissman I, Tse V. 2004. Incorporation of naive bone marrow derived cells into the vascular architecture of brain tumor. Microcirculation (New York, N.Y. : 1994). 11(8):699-708. Pubmed: 15726837 Yung YC, Cheshier S, Santarelli JG, Huang Z, Wagers A, Weissman I, Tse V. 2004. Incorporation of naive bone marrow derived cells into the vascular architecture of brain tumor. Microcirculation (New York, N.Y. : 1994). 11(8):699-708. Pubmed: 15726837 Array -
Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL, Wagers AJ. 2004. Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell. 119(4):543-54. Pubmed: 15537543 Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL, Wagers AJ. 2004. Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell. 119(4):543-54. Pubmed: 15537543 Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors. -
Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. 2004. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 428(6983):668-73. Pubmed: 15034594 Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. 2004. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature. 428(6983):668-73. Pubmed: 15034594 Under conditions of tissue injury, myocardial replication and regeneration have been reported. A growing number of investigators have implicated adult bone marrow (BM) in this process, suggesting that marrow serves as a reservoir for cardiac precursor cells. It remains unclear which BM cell(s) can contribute to myocardium, and whether they do so by transdifferentiation or cell fusion. Here, we studied the ability of c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells to regenerate myocardium in an infarct model. Cells were isolated from transgenic mice expressing green fluorescent protein (GFP) and injected directly into ischaemic myocardium of wild-type mice. Abundant GFP+ cells were detected in the myocardium after 10 days, but by 30 days, few cells were detectable. These GFP+ cells did not express cardiac tissue-specific markers, but rather, most of them expressed the haematopoietic marker CD45 and myeloid marker Gr-1. We also studied the role of circulating cells in the repair of ischaemic myocardium using GFP+-GFP- parabiotic mice. Again, we found no evidence of myocardial regeneration from blood-borne partner-derived cells. Our data suggest that even in the microenvironment of the injured heart, c-kit-enriched BM cells, Lin- c-kit+ BM cells and c-kit+ Thy1.1(lo) Lin- Sca-1+ long-term reconstituting haematopoietic stem cells adopt only traditional haematopoietic fates. -
Christensen JL, Wright DE, Wagers AJ, Weissman IL. 2004. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS biology. 2(3):E75. Pubmed: 15024423 DOI:e75 Christensen JL, Wright DE, Wagers AJ, Weissman IL. 2004. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS biology. 2(3):E75. Pubmed: 15024423 DOI:e75 The major site of hematopoiesis transitions from the fetal liver to the spleen and bone marrow late in fetal development. To date, experiments have not been performed to evaluate functionally the migration and seeding of hematopoietic stem cells (HSCs) during this period in ontogeny. It has been proposed that developmentally timed waves of HSCs enter the bloodstream only during distinct windows to seed the newly forming hematopoietic organs. Using competitive reconstitution assays to measure HSC activity, we determined the localization of HSCs in the mid-to-late gestation fetus. We found that multilineage reconstituting HSCs are present at low numbers in the blood at all timepoints measured. Seeding of fetal bone marrow and spleen occurred over several days, possibly while stem cell niches formed. In addition, using dual-chamber migration assays, we determined that like bone marrow HSCs, fetal liver HSCs migrate in response to stromal cell-derived factor-1alpha (SDF-1alpha); however, unlike bone marrow HSCs, the migratory response of fetal liver HSCs to SDF-1alpha is greatly increased in the presence of Steel factor (SLF), suggesting an important role for SLF in HSC homing to and seeding of the fetal hematopoietic tissues. Together, these data demonstrate that seeding of fetal organs by fetal liver HSCs does not require large fluxes of HSCs entering the fetal bloodstream, and that HSCs constitutively circulate at low levels during the gestational period from 12 to 17 days postconception. Newly forming hematopoietic tissues are seeded gradually by HSCs, suggesting initial seeding is occurring as hematopoietic niches in the spleen and bone marrow form and become capable of supporting HSC self-renewal. We demonstrate that fetal and adult HSCs exhibit specific differences in chemotactic behavior. While both migrate in response to SDF-1alpha, fetal HSCs also respond significantly to the cytokine SLF. In addition, the combination of SDF-1alpha and SLF results in substantially enhanced migration of fetal HSCs, leading to migration of nearly all fetal HSCs in this assay. This finding indicates the importance of the combined effects of SLF and SDF-1alpha in the migration of fetal HSCs, and is, to our knowledge, the first demonstration of a synergistic effect of two chemoattractive agents on HSCs. -
Wagers AJ, Weissman IL. 2004. Plasticity of adult stem cells. Cell. 116(5):639-48. Pubmed: 15006347 Wagers AJ, Weissman IL. 2004. Plasticity of adult stem cells. Cell. 116(5):639-48. Pubmed: 15006347 Recent years have seen much excitement over the possibility that adult mammalian stem cells may be capable of differentiating across tissue lineage boundaries, and as such may represent novel, accessible, and very versatile effectors of therapeutic tissue regeneration. Yet studies proposing such "plasticity" of adult somatic stem cells remain controversial, and in general, existing evidence suggests that in vivo such unexpected transformations are exceedingly rare and in some cases can be accounted for by equally unexpected alternative explanations. -
Sherwood RI, Christensen JL, Weissman IL, Wagers AJ. 2004. Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells. Stem cells (Dayton, Ohio). 22(7):1292-304. Pubmed: 15579647 Sherwood RI, Christensen JL, Weissman IL, Wagers AJ. 2004. Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells. Stem cells (Dayton, Ohio). 22(7):1292-304. Pubmed: 15579647 To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c-kit+ Thy1.1lo Lin- Sca-1+ hematopoietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker-expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor-derived myofibers expressed both donor-specific and host-specific markers, suggesting that they arise by low-level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage. 2003
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Kondo M, Wagers AJ, Manz MG, Prohaska SS, Scherer DC, Beilhack GF, Shizuru JA, Weissman IL. 2003. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annual review of immunology. 21:759-806. Pubmed: 12615892 Kondo M, Wagers AJ, Manz MG, Prohaska SS, Scherer DC, Beilhack GF, Shizuru JA, Weissman IL. 2003. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annual review of immunology. 21:759-806. Pubmed: 12615892 Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases. 2002
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Wright DE, Bowman EP, Wagers AJ, Butcher EC, Weissman IL. 2002. Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. The Journal of experimental medicine. 195(9):1145-54. Pubmed: 11994419 Wright DE, Bowman EP, Wagers AJ, Butcher EC, Weissman IL. 2002. Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. The Journal of experimental medicine. 195(9):1145-54. Pubmed: 11994419 Although hematopoietic stem cell (HSC) migration into and out of sites of active hematopoiesis is poorly understood, it is a critical process that underlies modern clinical stem cell transplantation and may be important for normal hematopoietic homeostasis. Given the established roles of chemotactic cytokine (chemokine)-directed migration of other leukocyte subsets, the migration of murine HSC to a large panel of CC and CXC chemokines was investigated. HSC migrated only in response to stromal derived factor-1alpha, the ligand for the CXC chemokine receptor 4 (CXCR4). CXCR4 expression by HSC was confirmed by reverse transcription polymerase chain reaction analysis. Surprisingly, HSC also expressed mRNA for CCR3 and CCR9, although they failed to migrate to the ligands for these receptors. The sharply restricted chemotactic responsiveness of HSC is unique among leukocytes and may be necessary for the specific homing of circulating HSC to bone marrow, as well as for the maintenance of HSC in hematopoietic microenvironments. -
Wagers AJ, Allsopp RC, Weissman IL. 2002. Changes in integrin expression are associated with altered homing properties of Lin(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Experimental hematology. 30(2):176-85. Pubmed: 11823053 Wagers AJ, Allsopp RC, Weissman IL. 2002. Changes in integrin expression are associated with altered homing properties of Lin(-/lo)Thy1.1(lo)Sca-1(+)c-kit(+) hematopoietic stem cells following mobilization by cyclophosphamide/granulocyte colony-stimulating factor. Experimental hematology. 30(2):176-85. Pubmed: 11823053 Array -
Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL, Cyster JG, Engleman EG. 2002. Langerhans cells renew in the skin throughout life under steady-state conditions. Nature immunology. 3(12):1135-41. Pubmed: 12415265 Merad M, Manz MG, Karsunky H, Wagers A, Peters W, Charo I, Weissman IL, Cyster JG, Engleman EG. 2002. Langerhans cells renew in the skin throughout life under steady-state conditions. Nature immunology. 3(12):1135-41. Pubmed: 12415265 Langerhans cells (LCs) are bone marrow (BM)-derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors. -
Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. 2002. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science (New York, N.Y.). 297(5590):2256-9. Pubmed: 12215650 Wagers AJ, Sherwood RI, Christensen JL, Weissman IL. 2002. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science (New York, N.Y.). 297(5590):2256-9. Pubmed: 12215650 To rigorously test the in vivo cell fate specificity of bone marrow (BM) hematopoietic stem cells (HSCs), we generated chimeric animals by transplantation of a single green fluorescent protein (GFP)-marked HSC into lethally irradiated nontransgenic recipients. Single HSCs robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute appreciably to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Similarly, in GFP+:GFP- parabiotic mice, we found substantial chimerism of hematopoietic but not nonhematopoietic cells. These data indicate that "transdifferentiation" of circulating HSCs and/or their progeny is an extremely rare event, if it occurs at all. -
Wagers AJ, Christensen JL, Weissman IL. 2002. Cell fate determination from stem cells. Gene therapy. 9(10):606-12. Pubmed: 12032706 Wagers AJ, Christensen JL, Weissman IL. 2002. Cell fate determination from stem cells. Gene therapy. 9(10):606-12. Pubmed: 12032706 In the adult, tissue-specific stem cells are thought to be responsible for the replacement of differentiated cells within continuously regenerating tissues, such as the liver, skin, and blood system. In this review, we will consider the factors that influence stem cell fate, taking as a primary example the cell fate determination of hematopoietic stem cells. 2001
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Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL. 2001. Physiological migration of hematopoietic stem and progenitor cells. Science (New York, N.Y.). 294(5548):1933-6. Pubmed: 11729320 Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL. 2001. Physiological migration of hematopoietic stem and progenitor cells. Science (New York, N.Y.). 294(5548):1933-6. Pubmed: 11729320 Hematopoietic stem cells (HSCs) reside predominantly in bone marrow, but low numbers of HSCs are also found in peripheral blood. We examined the fate of blood-borne HSCs using genetically marked parabiotic mice, which are surgically conjoined and share a common circulation. Parabionts rapidly established stable, functional cross engraftment of partner-derived HSCs and maintained partner-derived hematopoiesis after surgical separation. Determination of the residence time of injected blood-borne progenitor cells suggests that circulating HSCs/progenitors are cleared quickly from the blood. These data demonstrate that HSCs rapidly and constitutively migrate through the blood and play a physiological role in, at least, the functional reengraftment of unconditioned bone marrow. -
Wright DE, Cheshier SH, Wagers AJ, Randall TD, Christensen JL, Weissman IL. 2001. Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood. 97(8):2278-85. Pubmed: 11290588 Wright DE, Cheshier SH, Wagers AJ, Randall TD, Christensen JL, Weissman IL. 2001. Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle. Blood. 97(8):2278-85. Pubmed: 11290588 Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB HSC) are widely used for transplantation in the treatment of malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G(0) or G(1) phase of the cell cycle. This has raised the question of whether a subset of noncycling BM HSC is selectively released, or whether cycling BM HSC are mobilized after M phase, but before the next S phase of the cell cycle. To distinguish between these possibilities, mice were treated with one dose of CY followed by daily doses of G-CSF, and dividing cells were marked by administration of bromodeoxyuridine (BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB (LT)-HSC stained positively for BrdU, and therefore derived from cells that divided during the treatment interval. Next, LT-HSC from mice previously treated with a single dose of CY, which kills cycling cells, and 3 daily doses of G-CSF, were nearly all killed by a second dose of CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle. Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA content of MPB HSC suggests that these cells are mostly in G(1) phase. After CY/G-CSF treatment, virtually all BM LT-HSC enter the cell cycle; some of these HSC then migrate into the blood, specifically after M phase, and are rapidly recruited to particular hematopoietic organs. 2000
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Wagers AJ, Kansas GS. 2000. Potent induction of alpha(1,3)-fucosyltransferase VII in activated CD4+ T cells by TGF-beta 1 through a p38 mitogen-activated protein kinase-dependent pathway. Journal of immunology (Baltimore, Md. : 1950). 165(9):5011-6. Pubmed: 11046029 Wagers AJ, Kansas GS. 2000. Potent induction of alpha(1,3)-fucosyltransferase VII in activated CD4+ T cells by TGF-beta 1 through a p38 mitogen-activated protein kinase-dependent pathway. Journal of immunology (Baltimore, Md. : 1950). 165(9):5011-6. Pubmed: 11046029 Homing of effector T cells to sites of inflammation, particularly in the skin, is dependent on T cell expression of ligands for the endothelial selectins. Underlying expression of these ligands is the expression of alpha(1,3)-fucosyltransferase VII (FucT-VII), a FucT essential for biosynthesis of selectin ligands. FucT-VII is sharply induced in activated T cells by IL-12, but cytokines other than IL-12 that induce FucT-VII and functional selectin ligands have not been identified, and are likely to be important in homing of T cells to other selectin-dependent sites. Screening of a number of cytokines known to be active on T cells identified only TGF-beta1 as able to up-regulate FucT-VII mRNA levels and selectin ligands on activated CD4 T cells. The sharp increase in FucT-VII induced by TGF-beta1 in activated T cells was completely blocked by pharmacologic inhibition of p38 mitogen-activated protein kinase, but was unaffected by mitogen-activated protein/extracellular signal-related kinase kinase inhibitors. The selective ability of TGF-beta1 to induce selectin ligands on activated T cells is likely important for T cell homing to the gut, which is a strongly selectin-dependent site, and correlates with the ability of TGF-beta1 to coordinately induce other gut-associated homing pathways. 1999
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Lim YC, Henault L, Wagers AJ, Kansas GS, Luscinskas FW, Lichtman AH. 1999. Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4. Journal of immunology (Baltimore, Md. : 1950). 162(6):3193-201. Pubmed: 10092770 Lim YC, Henault L, Wagers AJ, Kansas GS, Luscinskas FW, Lichtman AH. 1999. Expression of functional selectin ligands on Th cells is differentially regulated by IL-12 and IL-4. Journal of immunology (Baltimore, Md. : 1950). 162(6):3193-201. Pubmed: 10092770 Immune responses may be qualitatively distinct depending on whether Th1 or Th2 cells predominate at the site of Ag exposure. T cell subset-specific expression of ligands for vascular selectins may underlie the distinct patterns of recruitment of Th1 or Th2 cells to peripheral inflammatory sites. Here we examine the regulation of selectin ligand expression during murine T helper cell differentiation. Large numbers of Th1 cells interacted with E- and P-selectin under defined flow conditions, while few Th2 and no naive T cells interacted. Th1 cells also expressed more fucosyltransferase VII mRNA than naive or Th2 cells. IL-12 induced expression of P-selectin ligands on Ag-activated naive T cells, even in the presence of IL-4, and on established Th2 cells restimulated in the presence of IL-12 and IFN-gamma. In contrast, Ag stimulation alone induced only E-selectin ligand. Interestingly, restimulation of established Th2 cells in the presence of IL-12 and IFN-gamma induced expression of P-selectin ligands but not E-selectin ligands; IFN-gamma alone did not enhance expression of either selectin ligand. In summary, functional P- and E-selectin ligands are expressed on most Th1 cells, few Th2 cells, but not naive T cells. Furthermore, selectin ligand expression is regulated by the cytokine milieu during T cell differentiation. IL-12 induces P-selectin ligand, while IL-4 plays a dominant role in down-regulating E-selectin ligand. 1998
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Wagers AJ, Stoolman LM, Craig R, Knibbs RN, Kansas GS. 1998. An sLex-deficient variant of HL60 cells exhibits high levels of adhesion to vascular selectins: further evidence that HECA-452 and CSLEX1 monoclonal antibody epitopes are not essential for high avidity binding to vascular selectins. Journal of immunology (Baltimore, Md. : 1950). 160(10):5122-9. Pubmed: 9590264 Wagers AJ, Stoolman LM, Craig R, Knibbs RN, Kansas GS. 1998. An sLex-deficient variant of HL60 cells exhibits high levels of adhesion to vascular selectins: further evidence that HECA-452 and CSLEX1 monoclonal antibody epitopes are not essential for high avidity binding to vascular selectins. Journal of immunology (Baltimore, Md. : 1950). 160(10):5122-9. Pubmed: 9590264 Selectins are carbohydrate-binding cell adhesion molecules that play a key role in the initiation of inflammatory responses. Several studies have suggested that the sialylated, fucosylated tetrasaccharide sialyl Lewis X (sLex) is an important component of leukocyte ligands for E- and P-selectin. We have identified a stable variant of the HL60 cell line, HL60var, which displays a nearly complete absence of staining with several mAb directed against sLex and/or sLex-related structures. HL60var also exhibits a concomitant increase in reactivity with mAb directed against the unsialylated Lewis X (Lex/CD15) structure. Despite this sLex deficiency, HL60var binds well to both E- and P-selectin. No significant differences in expression of alpha1,3-fucosyltransferases, C2GnT (Core2 transferase), or P-selectin glycoprotein ligand-1 between HL60var and typical sLex(high) HL60 cells were detected. Although the precise molecular basis for the sLex(-/low) phenotype of HL60var remains uncertain, flow cytometric analysis with the sialic acid-specific Limax flavus lectin revealed a sharp reduction in HL60var surface sialylation. Thus, the loss in mAb reactivity may result from a loss of sialic acid residues from the mAb carbohydrate epitope. However, binding of HL60var to E- and P-selectin remains sensitive to neuraminidase treatment. Taken together, these data indicate that high levels of surface sLex and/or related epitopes are not essential for interactions with vascular selectins, implying that as yet unidentified sialylated, fucosylated structures serve as physiologically relevant ligands for E- and P-selectin. -
Wagers AJ, Waters CM, Stoolman LM, Kansas GS. 1998. Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha1, 3-fucosyltransferase VII gene expression. The Journal of experimental medicine. 188(12):2225-31. Pubmed: 9858509 Wagers AJ, Waters CM, Stoolman LM, Kansas GS. 1998. Interleukin 12 and interleukin 4 control T cell adhesion to endothelial selectins through opposite effects on alpha1, 3-fucosyltransferase VII gene expression. The Journal of experimental medicine. 188(12):2225-31. Pubmed: 9858509 The alpha1,3-fucosyltransferase, FucT-VII, is crucial for the formation of ligands for all three selectins, and its expression regulates the synthesis of these ligands. Short-term polarized T helper (Th)1, but not Th2 or naive CD4(+) T cells, can home to sites of inflammation, but the molecular basis for this difference has remained unclear. Here we show that naive CD4(+) T cells do not express FucT-VII and fail to bind vascular selectins. We also show that when CD4(+) T cells are activated in the presence of the Th1 polarizing cytokine interleukin (IL)-12, levels of FucT-VII mRNA and binding to E- and P-selectin are significantly augmented. In contrast, activation of CD4(+) T cells in the presence of IL-4, a Th2 polarizing cytokine, inhibited FucT-VII expression and binding to vascular selectins. T cell activation upregulated expression of the Core2 transferase, C2GnT, equivalently regardless of the presence or absence of polarizing cytokines. These data indicate that the selective ability of Th1 cells, as opposed to Th2 cells or naive CD4(+) T cells, to recognize vascular selectins and home to sites of inflammation is controlled principally by the expression of a single gene, FucT-VII. 1997
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Snapp KR, Wagers AJ, Craig R, Stoolman LM, Kansas GS. 1997. P-selectin glycoprotein ligand-1 is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines. Blood. 89(3):896-901. Pubmed: 9028320 Snapp KR, Wagers AJ, Craig R, Stoolman LM, Kansas GS. 1997. P-selectin glycoprotein ligand-1 is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines. Blood. 89(3):896-901. Pubmed: 9028320 P-selectin (CD62P) is a member of the selectin family of adhesion molecules involved in the regulation of leukocyte traffic. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like molecule that is thought to be a primary ligand for P-selectin. The interaction of P-selectin with PSGL-1 results in leukocyte rolling and recruitment of leukocytes to sites of inflammation and tissue injury. However, expression of PSGL-1 protein alone is insufficient for binding to P-selectin. Several posttranslational modifications of PSGL-1, including sialylation, sulfation, and fucosylation by alpha 1,3-fucosyltransferase(s) (FucT), are required for functional interaction with P-selectin. Recently, several groups have reported that PSGL-1 might also serve as a ligand for E-selectin. Differential posttranslational modifications of PSGL-1 may determine whether it can interact with either P- or E-selectin or both. To determine whether PSGL-1 is essential for adhesion to P- or E-selectin, we have constructed and analyzed a panel of stably transfected K562 cells. K562 cells express FucT-IV but not FucT-VII or PSGL-1, and do not bind to either E- or P-selectin. K562 cells transfected with PSGL-1 cDNA also did not bind to either P- or E-selectin. Binding to P-selectin occurred only when K562 cells were cotransfected with both FucT-VII and PSGL-1. In contrast, expression of FucT-VII alone was sufficient for E-selectin binding. These data demonstrate that expression of PSGL-1 is not required for adhesion of a stably transfected hematopoietic cell line to E-selectin, and suggest that FucT-IV alone cannot properly modify PSGL-1, expressed in transfected K562 cells, to bind P-selectin. -
Wagers AJ, Stoolman LM, Kannagi R, Craig R, Kansas GS. 1997. Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes. Journal of immunology (Baltimore, Md. : 1950). 159(4):1917-29. Pubmed: 9257857 Wagers AJ, Stoolman LM, Kannagi R, Craig R, Kansas GS. 1997. Expression of leukocyte fucosyltransferases regulates binding to E-selectin: relationship to previously implicated carbohydrate epitopes. Journal of immunology (Baltimore, Md. : 1950). 159(4):1917-29. Pubmed: 9257857 E-selectin is a carbohydrate-binding endothelial cell adhesion molecule that reportedly interacts with several related sialylated and fucosylated carbohydrates. The activity of leukocyte alpha1,3-fucosyltransferases (FucT-IV or FucT-VII) is an essential step in the synthesis of E-selectin ligands. Using a panel of stably transfected hemopoietic cell lines, we have investigated the role of alpha1,3-fucosyltransferases in generating E-selectin ligands, and the relationship between adhesion to E-selectin and expression of mAb-defined carbohydrates. Expression of FucT-VII was always sufficient for binding to E- and P-selectin, while the ability of FucT-IV to construct E-selectin ligands varied among different cell types. Furthermore, FucT-IV was unable to support any binding to P-selectin in a lymphoid cell line, even when expressed at levels equivalent to those in myeloid cells. FucT-IV expression generated high levels of surface Le(x)/CD15 and CDw65, whereas expression of FucT-VII correlated with a subset of mAb-defined sialyl Lewis X (sLex)-like structures. FucT-IV-associated epitopes were present on both binding and nonbinding cells, whereas all cells that expressed FucT-VII-associated epitopes bound E-selectin. However, treatment of HL60 cells with neuraminidase destroyed FucT-VII-associated epitopes at a faster rate than E-selectin binding sites. Surface expression of a subset of mAb-defined sLex-like carbohydrates is therefore a good marker for high levels of FucT-VII activity, but these carbohydrates are not themselves required for recognition of E-selectin. 1996
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Wagers AJ, Lowe JB, Kansas GS. 1996. An important role for the alpha 1,3 fucosyltransferase, FucT-VII, in leukocyte adhesion to E-selectin. Blood. 88(6):2125-32. Pubmed: 8822932 Wagers AJ, Lowe JB, Kansas GS. 1996. An important role for the alpha 1,3 fucosyltransferase, FucT-VII, in leukocyte adhesion to E-selectin. Blood. 88(6):2125-32. Pubmed: 8822932 E-selectin is an adhesion molecule expressed on the surface of activated endothelial cells, which has been shown to be important in the initial steps of leukocyte extravasation into inflamed tissues. E-selectin binds neutrophils, monocytes, eosinophils, basophils, natural killer (NK) cells, and subsets of lymphocytes, although the precise ligand(s) on these cells have not been identified. Several studies have proposed certain carbohydrate structures, including sLex and related structures, as E-selectin ligands. In contrast to these studies, we report here the identification of several human B cell lines that exhibit binding to E-selectin without expression of any of the previously identified E-selectin binding carbohydrate epitopes. The unique carbohydrate phenotype of these B cell lines suggests that they may express a novel, sialylated carbohydrate structure(s) that binds to E-selectin. To investigate the enzymatic basis of this novel form of E-selectin binding, we examined the expression of the two principal leukocyte alpha 1,3 fucosyltransferases, FucT-IV and FucT-VII, in a panel of human hematopoietic cell lines. FucT-VII was expressed in all E-selectin binding cell lines except one, whereas FucT-IV was expressed by nearly all cell lines, regardless of their ability to bind E-selectin. In addition, transfection of cells with cDNA encoding FucT-VII conferred E-selectin binding ability. Taken together, these data suggest that, regardless of surface carbohydrate phenotype, E-selectin binding ability is determined largely by expression of FucT-VII. 1954
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BRECKENRIDGE RL, WAGERS AJ, BALTZELL WH. 1954. Lethal granuloma of the midline facial tissues; granuloma gangraenescens. The Annals of otology, rhinology, and laryngology. 63(2):278-95. Pubmed: 13189300 BRECKENRIDGE RL, WAGERS AJ, BALTZELL WH. 1954. Lethal granuloma of the midline facial tissues; granuloma gangraenescens. The Annals of otology, rhinology, and laryngology. 63(2):278-95. Pubmed: 13189300