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Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells

Nature Protocols volume 10, pages 941–958 (2015)Cite this article

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A Corrigendum to this article was published on 27 August 2015

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Abstract

Skeletal muscle is the most abundant human tissue; therefore, an unlimited availability of myogenic cells has applications in regenerative medicine and drug development. Here we detail a protocol to derive myogenic cells from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, and we also provide evidence for its extension to human iPS cells cultured without feeder cells. The procedure, which does not require the generation of embryoid bodies or prospective cell isolation, entails four stages with different culture densities, media and surface coating. Pluripotent stem cells are disaggregated to single cells and then differentiated into expandable cells resembling human mesoangioblasts. Subsequently, transient Myod1 induction efficiently drives myogenic differentiation into multinucleated myotubes. Cells derived from patients with muscular dystrophy and differentiated using this protocol have been genetically corrected, and they were proven to have therapeutic potential in dystrophic mice. Thus, this platform has been demonstrated to be amenable to gene and cell therapy, and it could be extended to muscle tissue engineering and disease modeling.

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Figure 1: Workflow of the protocol to generate myogenic cells from human pluripotent stem cells via differentiation into mesoangioblast-like cells and transient Myod1 activation.
Figure 2: Representative images showing the expected cell morphology along the protocol.
Figure 3: Representative number of cells obtained in 1 month of culture.
Figure 4: Analysis of HEDEM/HIDEM surface markers by flow cytometry.
Figure 5: Myogenic differentiation of HEDEMs and HIDEMs with MyoD-ER(T).

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  • 05 August 2015

     In the version of this article initially published, the concentration of ROCK inhibitor Y27362 in Step 2 was incorrectly given as 10 mM. The correct concentration to be used is 10 µM. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank G. Cossu (University of Manchester, UK) and all co-authors of the original article describing this technology for their initial support and contribution. We are also grateful to the following biobanks and colleagues for some of the cells used in this study and/or in its relative original article: (i) P. Andrews, H. Moore (University of Sheffield, UK) and the UK Stem Cell Bank for kindly supplying Shef 6 human ES cells; (ii) M. Mora and The 'Cells, tissues and DNA from patients with neuromuscular diseases Biobank', member of the Telethon Network of Genetic Biobanks (project no. GTB12001), funded by Telethon Italy and of the EuroBioBank network; (iii) B. Schoser, P. Schneiderat and The Muscle Tissue Culture Collection, part of the German network on muscular dystrophies (service structure S1, 01GM0601) and the German network for mitochondrial disorders (project D2, 01GM0862) funded by the German ministry of education and research (BMBF, Bonn, Germany). The Muscle Tissue Culture Collection is a partner of EuroBioBank (http://www.eurobiobank.org) and TREAT-NMD (http://www.treat-nmd.eu); (iv) Y. Torrente, M. Moggio and the Telethon Network of Genetic Biobanks at Ospedale Maggiore Policlinico, Milan, Italy; (v) M. Oshimura and Y. Kazuki (the Tottori University, Yonago, Japan) for the DMD iPS cells (generated from Coriell Institute fibroblasts GM05169, US National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository). We also thank J. Chamberlain (University of Washington, Seattle, WA) for the original MyoD-ER(T) construct. This project has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 602423 (PluriMes) and from Takeda New Frontier Science program. Work in the Tedesco laboratory is also supported by the UK Medical Research Council (MRC) and the Biotechnology and Biological Sciences Research Council (BBSRC), Duchenne Parent Project Onlus, Muscular Dystrophy UK, Duchenne Children's Trust and the Duchenne Research Fund. M.K.C., T.V.D., S.D. and M.L. were supported by the Fund for Scientific Research (FWO), Association Française contre les Myopathies and the Willy Gepts Fund (VUB).

AUTHOR CONTRIBUTIONS

S.M.M. and M.F.M.G. performed the experiments with the help of M.R., analyzed the data and wrote the manuscript; S.D. performed experiments on feeder-free iPS cells and HIDEMs, and discussed and analyzed results with T.V.D. and M.K.C.; S.B. performed the proliferation assay and analyzed and discussed results; M.L. performed live imaging experiments and analyzed and discussed results; S.D. and M.L. performed most of the work described here in a collaborative project as visiting PhD students at University College London; F.S.T. developed the protocol, discussed and analyzed the results with all coauthors, and wrote the paper.

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Author notes

  1. Sara Benedetti

    Present address: Present address: Institute of Child Health, University College London, London, UK.,

  2. Sara M Maffioletti and Mattia F M Gerli: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell and Developmental Biology, University College London, London,, UK

    Sara M Maffioletti, Mattia F M Gerli, Martina Ragazzi, Sara Benedetti & Francesco Saverio Tedesco

  2. Department of Gene Therapy and Regenerative Medicine, Free University of Brussels (VUB), Brussels, Belgium

    Sumitava Dastidar, Mariana Loperfido, Thierry VandenDriessche & Marinee K Chuah

  3. Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven (KU Leuven), Leuven, Belgium

    Mariana Loperfido, Thierry VandenDriessche & Marinee K Chuah

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Correspondence to Francesco Saverio Tedesco.

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Competing interests

The work described in this article is partially covered by patent application no. PCT/GB2013/050112 (publication no. WO2013108039 A1). F.S.T. is the principal investigator of a grant funded by the Takeda New Frontier Science program and provided speaking and consulting services to Takeda Pharmaceuticals International, Inc. via UCL Consultants. The remaining authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Additional cell characterization: pluripotency analysis and alkaline phosphatase staining.

(a) The histogram on the left-hand side shows absence of alkaline phosphatase (AP) signal in one HIDEM line, whereas the picture on the right-hand side shows AP activity detected with enzymatic reaction (purple/black precipitate; scale bar 50 μm). (b) Representative immunofluorescence staining for the pluripotency factors NANOG, OCT4 and SOX2. Feeder-free HIDEMs (FF HIDEMs) are negative for all the markers and are shown in the left part of the panel. On the right hand side, stained feeder-free iPS (FF hiPS) cells are shown as positive control (scale bar 50 μm).

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Supplementary Text and Figures

Supplementary Figure 1, Supplementary Tables 1 and 2, Supplementary Methods (PDF 264 kb)

The video shows a myotube derived from differentiated DMD HIDEMs twitching in culture at day 8 in myogenic differentiation medium.

Sarcomeric striations are visible in their characteristic structure of alternate dark and light bands (640X magnification). (MP4 17149 kb)

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Maffioletti, S., Gerli, M., Ragazzi, M. et al. Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells. Nat Protoc 10, 941–958 (2015). https://doi.org/10.1038/nprot.2015.057

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