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Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro

Nature Protocols volume 11, pages 1833–1850 (2016)Cite this article

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Abstract

Progress toward finding a cure for muscle diseases has been slow because of the absence of relevant cellular models and the lack of a reliable source of muscle progenitors for biomedical investigation. Here we report an optimized serum-free differentiation protocol to efficiently produce striated, millimeter-long muscle fibers together with satellite-like cells from human pluripotent stem cells (hPSCs) in vitro. By mimicking key signaling events leading to muscle formation in the embryo, in particular the dual modulation of Wnt and bone morphogenetic protein (BMP) pathway signaling, this directed differentiation protocol avoids the requirement for genetic modifications or cell sorting. Robust myogenesis can be achieved in vitro within 1 month by personnel experienced in hPSC culture. The differentiating culture can be subcultured to produce large amounts of myogenic progenitors amenable to numerous downstream applications. Beyond the study of myogenesis, this differentiation method offers an attractive platform for the development of relevant in vitro models of muscle dystrophies and drug screening strategies, as well as providing a source of cells for tissue engineering and cell therapy approaches.

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Figure 1: Maintenance and stepwise protocol for myogenic differentiation in vitro and phase-contrast images of differentiating cultures (Steps 1–25).
Figure 2: Immunophenotyping of hPSC-derived myogenic cultures (Steps 26–36).
Figure 3: Subculturing of hPSC-derived myogenic culture (Steps 37–54).
Figure 4: Immunophenotyping of myogenic subcultures (Steps 26–36).
Figure 5: Example of downstream applications for hPSC-derived myogenic cultures.

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Acknowledgements

We thank all coauthors of the original article describing this technology for their initial support and contribution. We thank G. Guevara for lab assistance. We thank C. Fugier and F. Bousson for comments and feedback. We are grateful to L. Daheron and members of the Pourquié laboratory for comments. We thank A. Freismuth and M. Humbert from the IGBMC cell culture service for hiPSC culture assistance. This work was supported by an advanced grant from the European Research Council to O.P., by the FP7 EU grant Plurimes (agreement no. 602423 to O.P.), by a strategic grant from the French Muscular Dystrophy Association (AFM) to O.P. and by the INGESTEM project (ANR).

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  1. Jérome Chal and Ziad Al Tanoury: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut de Génétique et de Biologie Moléculaireet Cellulaire (IGBMC), CNRS (UMR 7104), Inserm U964, Université de Strasbourg, Illkirch-Graffenstaden, France

    Jérome Chal, Ziad Al Tanoury, Bénédicte Gobert, Thomas Cherrier & Olivier Pourquié

  2. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA

    Jérome Chal, Marie Hestin, Suvi Aivio & Olivier Pourquié

  3. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Jérome Chal, Marie Hestin, Suvi Aivio & Olivier Pourquié

  4. Harvard Stem Cell Institute, Boston, Massachusetts, USA

    Jérome Chal, Marie Hestin, Suvi Aivio & Olivier Pourquié

  5. Anagenesis Biotechnologies, Parc d'innovation,, Illkirch-Graffenstaden, France

    Aurore Hick

  6. Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA

    Alexander P Nesmith & Kevin K Parker

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Contributions

J.C. and Z.A.T. designed and performed the experiments and protocol optimizations, analyzed data and coordinated the project. M.H. and S.A. carried out hPSC differentiation and protocol optimization under the supervision of J.C. B.G. carried out some hPSC optimization experiments under the supervision of Z.A.T. A.H. contributed to hPSC cell culture analysis and data interpretation. T.C. contributed to hPSC culture differentiation and analysis. A.P.N. manufactured micropatterned substrates and performed the fiber structural analysis. K.K.P. provided expertise. O.P. supervised the overall project. J.C., Z.A.T. and O.P. performed the final data analysis and wrote the manuscript.

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Correspondence to Olivier Pourquié.

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Competing interests

The work described in this article is partially covered by patent application no. PCT/EP2012/066793 (publication no. WO2013030243 A1). O.P. and J.C. are cofounders and shareholders of Anagenesis Biotechnologies, a startup company specializing in the production of muscle cells in vitro for cell therapy and drug screening.

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Chal, J., Al Tanoury, Z., Hestin, M. et al. Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nat Protoc 11, 1833–1850 (2016). https://doi.org/10.1038/nprot.2016.110

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