Embryonic stem (ES) cells differentiate into multiple lineages during in vitro embryoid body (EB) formation. This makes the ES/EB system a powerful tool to study early embryonic developmental pathways and to generate specific cell populations for regenerative medicine. Although this system has been available for more than two decades, studies on skeletal muscle development, until our recent work, had progressed no farther than documenting muscle marker gene expression. The system had not been used to investigate the molecular mechanisms of skeletal muscle lineage determination acting in nascent mesoderm. Similarly, in spite of the interest in and hype surrounding the potential use of ES cells to treat diseases of muscle degeneration, the generation of an ES-derived myogenic population with proliferative and regenerative potential had not been accomplished. This is in part due to the rarity of skeletal muscle precursors within the EB, as well as the lack of reliable identification and isolation criteria. We have developed an ES cell line in which expression of Pax3, the master regulator of the embryonic myogenic program, can be induced by doxycycline (dox). Our results show that induction of Pax3 during EB development enhances paraxial mesoderm, and a cell population endowed with myogenic potential within this fraction. Accordingly, a homogenous population of proliferating myogenic progenitors can be isolated from heterogeneous EB cultures by sorting for PDGF1R, a paraxial mesoderm marker, and absence of Flk-1, a lateral plate mesoderm marker. Upon dox withdrawal, these cells differentiate into muscle in vitro. When transplanted into cardiotoxin-injured immunodeficient or dystrophin-deficient immunosuppressed mice (intramuscular or systemic), Pax3-induced cells demonstrate an exceptional potential for skeletal muscle regeneration, differentiating robustly into functional adult myofibers. This is the first demonstration that therapeutic skeletal muscle progenitors can be derived from ES cells. Here we propose studies aimed at assessing in further detail the long-term therapeutic potential of ES/Pax3-derived myogenic progenitors, including the transplantation into more severe mouse models of muscular dystrophy as well as understanding the mechanism underlying their generation within paraxial mesoderm during ES/EB differentiation.

Public Health Relevance

Embryonic stem cells hold great promise for the treatment of degenerative diseases, however to date studies on their potential use in the treatment of muscular dystrophies have been hampered by the difficulty of differentiating ES cells into skeletal muscle progenitors. This application builds on a novel method we have developed to generate muscle progenitors from ES cells. We have shown that such progenitors can be transplanted into normal injured and dystrophic mice, where they contribute to muscle fiber regeneration, and improve muscle function after injury. In these studies, we will (Aim 1) test whether these ES-derived cells, in addition to contributing to regenerating fibers, are capable of contributing to the muscle stem cell pool, (Aim 2) test whether they improve muscle function in more severe animal models of muscular dystrophy, and (Aim 3) use this system to dissect the molecular events involved in the generation of the earliest muscle progenitor cells of the embryo.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MOSS-H (14))
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Nuckolls, Glen H
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University of Minnesota Twin Cities
Internal Medicine/Medicine
Schools of Medicine
United States
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Xu, Bin; Magli, Alessandro; Anugrah, Yoska et al. (2018) Nanotopography-responsive myotube alignment and orientation as a sensitive phenotypic biomarker for Duchenne Muscular Dystrophy. Biomaterials 183:54-66
Mondragon-Gonzalez, Ricardo; Perlingeiro, Rita C R (2018) Recapitulating muscle disease phenotypes with myotonic dystrophy 1 induced pluripotent stem cells: a tool for disease modeling and drug discovery. Dis Model Mech 11:
Magli, Alessandro; Perlingeiro, Rita R C (2017) Myogenic progenitor specification from pluripotent stem cells. Semin Cell Dev Biol 72:87-98
Lilja, Karin C; Zhang, Nan; Magli, Alessandro et al. (2017) Pax7 remodels the chromatin landscape in skeletal muscle stem cells. PLoS One 12:e0176190
Kim, Jaemin; Oliveira, Vanessa K P; Yamamoto, Ami et al. (2017) Generation of skeletal myogenic progenitors from human pluripotent stem cells using non-viral delivery of minicircle DNA. Stem Cell Res 23:87-94
Magli, Alessandro; Incitti, Tania; Kiley, James et al. (2017) PAX7 Targets, CD54, Integrin ?9?1, and SDC2, Allow Isolation of Human ESC/iPSC-Derived Myogenic Progenitors. Cell Rep 19:2867-2877
Magli, Alessandro; Incitti, Tania; Perlingeiro, Rita C R (2016) Myogenic Progenitors from Mouse Pluripotent Stem Cells for Muscle Regeneration. Methods Mol Biol 1460:191-208
Baik, June; Magli, Alessandro; Tahara, Naoyuki et al. (2016) Endoglin integrates BMP and Wnt signalling to induce haematopoiesis through JDP2. Nat Commun 7:13101
Carrió, Elvira; Magli, Alessandro; Muñoz, Mar et al. (2016) Muscle cell identity requires Pax7-mediated lineage-specific DNA demethylation. BMC Biol 14:30
Rinaldi, Fabrizio; Zhang, Yu; Mondragon-Gonzalez, Ricardo et al. (2016) Treatment with rGDF11 does not improve the dystrophic muscle pathology of mdx mice. Skelet Muscle 6:21

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