Abstract

During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7+ cells with satellite cell characteristics, including the ability to form dystrophin+ fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.

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References

  1. 1.

    & Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks. Transl. Res. 163, 409–417 (2013).

  2. 2.

    et al. Human ES- and iPS-derived myogenic progenitors restore DYSTROPHIN and improve contractility upon transplantation in dystrophic mice. Cell Stem Cell 10, 610–619 (2012).

  3. 3.

    et al. Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat. Med. 14, 134–143 (2008).

  4. 4.

    et al. Epigenetic reprogramming of human embryonic stem cells into skeletal muscle cells and generation of contractile myospheres. Cell Reports 3, 661–670 (2013).

  5. 5.

    et al. Generation of skeletal muscle cells from embryonic and induced pluripotent stem cells as an in vitro model and for therapy of muscular dystrophies. J. Cell. Mol. Med. 16, 1353–1364 (2012).

  6. 6.

    et al. Efficient and reproducible myogenic differentiation from human iPS cells: prospects for modeling Miyoshi Myopathy in vitro. PLoS ONE 8, e61540 (2013).

  7. 7.

    et al. Myogenic differentiation of muscular dystrophy-specific induced pluripotent stem cells for use in drug discovery. Stem Cells Transl. Med. 3, 149–160 (2014).

  8. 8.

    & ES-cells carrying two inactivated myf-5 alleles form skeletal muscle cells: activation of an alternative myf-5-independent differentiation pathway. Dev. Biol. 164, 24–36 (1994).

  9. 9.

    et al. Muscle cell differentiation of embryonic stem cells reflects myogenesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev. Biol. 164, 87–101 (1994).

  10. 10.

    & Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle cell lineages. EMBO J. 15, 310–318 (1996).

  11. 11.

    et al. Skeletal myogenesis by human embryonic stem cells. Cell Res. 16, 713–722 (2006).

  12. 12.

    et al. Generation of skeletal muscle stem/progenitor cells from murine induced pluripotent stem cells. FASEB J. 24, 2245–2253 (2010).

  13. 13.

    et al. BMP inhibition stimulates WNT-dependent generation of chondrogenic mesoderm from embryonic stem cells. Stem Cell Res. (Amst.) 3, 126–141 (2009).

  14. 14.

    et al. Directed in vitro myogenesis of human embryonic stem cells and their in vivo engraftment. PLoS ONE 8, e72023 (2013).

  15. 15.

    et al. Generation of transplantable, functional satellite-like cells from mouse embryonic stem cells. FASEB J. 23, 1907–1919 (2009).

  16. 16.

    et al. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species. Cell 155, 909–921 (2013).

  17. 17.

    et al. In vitro modeling of paraxial mesodermal progenitors derived from induced pluripotent stem cells. PLoS ONE 7, e47078 (2012).

  18. 18.

    et al. Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium. Stem Cell Res. (Amst.) 3, 157–169 (2009).

  19. 19.

    , , , & Paraxial mesodermal progenitors derived from mouse embryonic stem cells contribute to muscle regeneration via differentiation into muscle satellite cells. Stem Cells 26, 1865–1873 (2008).

  20. 20.

    , & Derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells. Stem Cell Reports 1, 620–631 (2013).

  21. 21.

    , , , & Derivation of myogenic progenitors directly from human pluripotent stem cells using a sphere-based culture. Stem Cells Transl. Med. 3, 564–574 (2014).

  22. 22.

    et al. In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity. PLoS Biol. 12, e1001937 (2014).

  23. 23.

    , , , & Efficient derivation of lateral plate and paraxial mesoderm subtypes from human embryonic stem cells through GSKi-mediated differentiation. Stem Cells Dev. 22, 1893–1906 (2013).

  24. 24.

    et al. Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells. Stem Cell Rev. 3, 516–529 (2013).

  25. 25.

    , & Spatiotemporal compartmentalization of key physiological processes during muscle precursor differentiation. Proc. Natl. Acad. Sci. USA 107, 4224–4229 (2010).

  26. 26.

    et al. Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6. EMBO Rep. 8, 784–789 (2007).

  27. 27.

    et al. R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis. Dev. Cell 7, 525–534 (2004).

  28. 28.

    , , & Mesodermal subdivision along the mediolateral axis in chicken controlled by different concentrations of BMP-4. Development 124, 1975–1984 (1997).

  29. 29.

    , , , & Embryonic and fetal limb myogenic cells are derived from developmentally distinct progenitors and have different requirements for beta-catenin. Genes Dev. 23, 997–1013 (2009).

  30. 30.

    & Cellular and molecular regulation of muscle regeneration. Physiol. Rev. 84, 209–238 (2004).

  31. 31.

    , , & Dynamics of muscle fibre growth during postnatal mouse development. BMC Dev. Biol. 10, 21 (2010).

  32. 32.

    et al. Nfix regulates fetal-specific transcription in developing skeletal muscle. Cell 140, 554–566 (2010).

  33. 33.

    , , & Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435, 948–953 (2005).

  34. 34.

    & Skeletal muscle stem cell birth and properties. Semin. Cell Dev. Biol. 18, 870–882 (2007).

  35. 35.

    & The cell biology of disease: cellular and molecular mechanisms underlying muscular dystrophy. J. Cell Biol. 201, 499–510 (2013).

  36. 36.

    & Duchenne muscular dystrophy--what causes the increased membrane permeability in skeletal muscle? Int. J. Biochem. Cell Biol. 43, 290–294 (2011).

  37. 37.

    , & Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle. J. Biochem. 118, 959–964 (1995).

  38. 38.

    et al. Microarchitecture is severely compromised but motor protein function is preserved in dystrophic mdx skeletal muscle. Biophys. J. 98, 606–616 (2010).

  39. 39.

    et al. A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions. Cell Rep. 2, 1448–1460 (2012).

  40. 40.

    , & Cellular heterogeneity during vertebrate skeletal muscle development. Dev. Biol. 308, 281–293 (2007).

  41. 41.

    et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).

  42. 42.

    et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).

  43. 43.

    & Human pluripotent stem cells: decoding the naive state. Sci. Transl. Med. 3, 76ps10 (2011).

  44. 44.

    , , & Muscular dystrophy begins early in embryonic development deriving from stem cell loss and disrupted skeletal muscle formation. Dis. Model. Mech. 2, 374–388 (2009).

  45. 45.

    & The role of branched fibres in the pathogenesis of Duchenne muscular dystrophy. Exp. Physiol. 96, 564–571 (2011).

  46. 46.

    , , , & Structural and functional evaluation of branched myofibers lacking intermediate filaments. Am. J. Physiol. Cell Physiol. 303, C224–C232 (2012).

  47. 47.

    A new function for odorant receptors: MOR23 is necessary for normal tissue repair in skeletal muscle. Cell Adh. Migr. 4, 502–506 (2010).

  48. 48.

    et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 309, 2064–2067 (2005).

  49. 49.

    et al. Myoblast transfer therapy: is there any light at the end of the tunnel? Acta Myol. 24, 128–133 (2005).

  50. 50.

    et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289–301 (2005).

  51. 51.

    et al. Tbx6-mediated Notch signaling controls somite-specific Mesp2 expression. Proc. Natl. Acad. Sci. USA 103, 3651–3656 (2006).

  52. 52.

    et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

  53. 53.

    , , , & Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428 (1993).

  54. 54.

    et al. The transcriptional activator PAX3-FKHR rescues the defects of Pax3 mutant mice but induces a myogenic gain-of-function phenotype with ligand-independent activation of Met signaling in vivo. Genes Dev. 17, 2950–2965 (2003).

  55. 55.

    , , , & Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a single backcross to 129/Sv strain: establishment of ES cell lines bearing the Omd conditional lethal mutation. Mamm. Genome 9, 998–1001 (1998).

  56. 56.

    , , & Separable regulatory elements governing myogenin transcription in mouse embryogenesis. Science 261, 215–218 (1993).

  57. 57.

    et al. Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates. Dev. Cell 16, 810–821 (2009).

  58. 58.

    et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008).

  59. 59.

    et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011).

  60. 60.

    et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

  61. 61.

    et al. A complex oscillating network of signaling genes underlies the mouse segmentation clock. Science 314, 1595–1598 (2006).

  62. 62.

    et al. GenePattern 2.0. Nat. Genet. 38, 500–501 (2006).

  63. 63.

    & Manteia, a predictive data mining system for vertebrate genes and its applications to human genetic diseases. Nucleic Acids Res. 42 (Database issue), D882–D891 (2014).

  64. 64.

    et al. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790 (1995).

  65. 65.

    , , & Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827–1839 (1997).

  66. 66.

    , , & Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. Nat. Genet. 21, 444–448 (1999).

  67. 67.

    & The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121, 439–451 (1995).

  68. 68.

    et al. Connective tissue fibroblasts and Tcf4 regulate myogenesis. Development 138, 371–384 (2011).

  69. 69.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

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Acknowledgements

We thank C. Henderson, K. Hnia, M. Knockaert and members of the Pourquié laboratory for comments. We are grateful to J. Pace, T. Knauer-Meyer, G. Vilhais-Neto, and M. McLaird for help. We thank C. Ebel, C. Thibault-Carpentier, A. Magloth-Roth and the Imaging Facility. We thank the microinjection and phenotyping teams of the Mouse Clinical Institute. We thank M. Durnin from the Stowers Institute Cell Culture and Animal Facilities. This work was supported by an advanced grant from the European Research Council to O.P., by the Stowers Institute for Medical Research, by the Howard Hughes Medical Institute, by the FP7 EU grant Plurimes (agreement no. 602423) and by a strategic grant from the French Muscular Dystrophy Association (AFM) to O.P. The Venus Plasmid was a kind gift of A. Miyawaki. The anti-Tbx6 antibody was a kind gift of Y. Saga51.

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Affiliations

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

    • Jérome Chal
    • , Masayuki Oginuma
    • , Ziad Al Tanoury
    • , Bénédicte Gobert
    • , Olga Sumara
    • , Yasmine Zidouni
    • , Caroline Mursch
    • , Philippe Moncuquet
    • , Olivier Tassy
    • , Stéphane Vincent
    • , Ayako Miyanari
    • , Agata Bera
    • , Jean-Marie Garnier
    • , Leif Kennedy
    • , Thomas Cherrier
    •  & Olivier Pourquié
  2. Stowers Institute for Medical Research, Kansas City, Missouri, USA.

    • Jérome Chal
    •  & Olivier Pourquié
  3. Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • Jérome Chal
    • , Getzabel Guevara
    • , Marie Hestin
    •  & Olivier Pourquié
  4. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Jérome Chal
    • , Getzabel Guevara
    • , Marie Hestin
    •  & Olivier Pourquié
  5. Harvard Stem Cell Institute, Boston, Massachusetts, USA.

    • Jérome Chal
    • , Getzabel Guevara
    • , Marie Hestin
    •  & Olivier Pourquié
  6. Anagenesis Biotechnologies, Parc d'innovation, Illkirch Graffenstaden, France.

    • Aurore Hick
    •  & Fanny Bousson
  7. UPMC Paris 06, UMRS 787, INSERM, Avenir team, Pitié-Salpêtrière, Paris, France.

    • Shinichiro Hayashi
    • , Bernadette Drayton
    •  & Frédéric Relaix
  8. Institut de Myologie, Paris, France.

    • Shinichiro Hayashi
    • , Bernadette Drayton
    •  & Frédéric Relaix
  9. Institut Pasteur, CNRS URA 2578, Paris, France.

    • Barbara Gayraud-Morel
    •  & Shahragim Tajbakhsh
  10. Division of Genetics and Genomics Boston Children's Hospital, Boston, Massachusetts, USA.

    • Emanuela Gussoni
  11. Howard Hughes Medical Institute, Kansas City, Missouri, USA.

    • Olivier Pourquié

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Contributions

J.C. designed and performed experiments, analyzed data and coordinated the project. M.O. performed the PSM microdissection series. Z.A.T. transposed the differentiation protocol to hiPS cells and characterized the human cultures with help from C.M. and B.G. B.G., A.M. and G.G. carried out most of the mouse ES cell differentiation experiments, and M.H. carried out hiPS cell and hES cell experiments, under J.C.'s supervision. O.S. validated the Myog-repV line. A.H. helped coordinate experiments. F.B. contributed to hiPS cell culture and differentiation. Y.Z. helped develop the serum-free protocol. P.M. and O.T. helped with microarray data analysis. T.C. helped analyze mdx cultures. A.B. contributed experimentally to the early project. L.K. provided technical support. J.-M.G. generated reporter constructs. S.H., B.D. and F.R. provided the Pax3-GFP ES cells. B.G.-M. and S.T. provided expertise and the Pax7-GFP ES cells. S.V. helped establish mdx ES cells. E.G. provided Rag1-mdx mice and performed transplantation. O.P. conceived and supervised the overall project. O.P. and J.C. performed the final data analysis and wrote the manuscript.

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 co-founders and shareholders of Anagenesis Biotechnologies, a startup company specializing in production of muscle cells in vitro for cell therapy and drug screening.

Corresponding author

Correspondence to Olivier Pourquié.

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https://doi.org/10.1038/nbt.3297