| Our Faculty
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
2019
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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. -
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. -
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. -
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. 2018
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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. -
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. 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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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. -
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. -
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 -
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. -
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. -
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 -
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 -
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. -
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. -
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. -
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. -
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.