Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs

Abstract

Human pluripotent stem cells (hPSCs) can be directed to differentiate into skeletal muscle progenitor cells (SMPCs). However, the myogenicity of hPSC-SMPCs relative to human fetal or adult satellite cells remains unclear. We observed that hPSC-SMPCs derived by directed differentiation are less functional in vitro and in vivo compared to human satellite cells. Using RNA sequencing, we found that the cell surface receptors ERBB3 and NGFR demarcate myogenic populations, including PAX7 progenitors in human fetal development and hPSC-SMPCs. We demonstrated that hPSC skeletal muscle is immature, but inhibition of transforming growth factor-β signalling during differentiation improved fusion efficiency, ultrastructural organization and the expression of adult myosins. This enrichment and maturation strategy restored dystrophin in hundreds of dystrophin-deficient myofibres after engraftment of CRISPR–Cas9-corrected Duchenne muscular dystrophy human induced pluripotent stem cell-SMPCs. The work provides an in-depth characterization of human myogenesis, and identifies candidates that improve the in vivo myogenic potential of hPSC-SMPCs to levels that are equal to directly isolated human fetal muscle cells.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: hPSC-SMPCs have reduced in vitro and in vivo myogenic potential relative to fetal or adult SCs.
Fig. 2: HNK1NCAM+ increases myogenic cell numbers but does not increase myogenicity in vivo.
Fig. 3: RNA-seq identifies unique gene signatures in fetal and hPSC-derived muscle.
Fig. 4: Increased myogenic ability resides in the ERBB3+NGFR+ fraction of human fetal muscle during primary and secondary myogenesis.
Fig. 5: Increased myogenic ability resides in the ERBB3+NGFR+ fraction of SMPCs from multiple hPSC lines and directed differentiation protocols.
Fig. 6: Inhibition of TGF-β signalling induces hPSC skeletal muscle maturation.
Fig. 7: In vivo engraftment of CRISPR–Cas9-corrected DMD hiPSC-SMPCs restores dystrophin to levels approaching uncultured fetal muscle.

References

  1. 1.

    Chong, J. J. H. et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts. Nature 510, 273–277 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Schwartz, S. D. et al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet 385, 509–516 (2014).

    Article  PubMed  Google Scholar 

  3. 3.

    Steinbeck, J. A. et al. Optogenetics enables functional analysis of human embryonic stem cell-derived grafts in a Parkinson’s disease model. Nat. Biotechnol. 33, 204–209 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Hrvatin, S. et al. Differentiated human stem cells resemble fetal, not adult, β cells. Proc. Natl Acad. Sci. USA 111, 3038–3043 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Witty, A. D. et al. Generation of the epicardial lineage from human pluripotent stem cells. Nat. Biotechnol. 32, 1026–1035 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Yang, X., Pabon, L. & Murry, C. E. Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes. Circ. Res. 114, 511–523 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Jonsson, M. K. et al. Application of human stem cell-derived cardiomyocytes in safety pharmacology requires caution beyond hERG. J. Mol. Cell. Cardiol. 52, 998–1008 (2012).

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Borchin, B., Chen, J. & Barberi, T. Derivation and FACS-mediated purification of PAX3+/PAX7+ skeletal muscle precursors from human pluripotent stem cells. Stem Cell Rep. 21, 620–631 (2013).

    Article  Google Scholar 

  9. 9.

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Chal, J. et al. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat. Biotechnol. 33, 962–969 (2015).

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Shelton, M. et al. Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells. Stem Cell Rep. 3, 516–529 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Chal, J. et al. Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nat. Protoc. 11, 1833–1850 (2016).

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Swartz, E. W. et al. A novel protocol for directed differentiation of C9orf72-associated human induced pluripotent stem cells into contractile skeletal myotubes. Stem Cells Transl. Med. 5, 1461–1472 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Abujarour, R. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Kimura, E. et al. Cell-lineage regulated myogenesis for dystrophin replacement: a novel therapeutic approach for treatment of muscular dystrophy. Hum. Mol. Genet. 17, 2507–2517 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Darabi, R. 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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

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

    CAS  Article  PubMed  Google Scholar 

  18. 18.

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

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Xi, H. et al. In vivo human somitogenesis guides somite development from hPSCs. Cell Rep. 18, 1573–1585 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Loh, K. M. et al. Mapping the pairwise choices leading from pluripotency to human bone, heart, and other mesoderm cell types. Cell 166, 451–467 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Yin, H., Price, F. & Rudnicki, M. A. Satellite cells and the muscle stem cell niche. Physiol. Rev. 93, 23–67 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Biressi, S. et al. Myf5 expression during fetal myogenesis defines the developmental progenitors of adult satellite cells. Dev. Biol. 379, 195–207 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Tierney, M. T. et al. Autonomous extracellular matrix remodeling controls a progressive adaptation in muscle stem cell regenerative capacity during development. Cell Rep. 14, 1940–1952 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S. & Blau, H. M. Self-renewal and expansion of single transplanted muscle stem cells. Nature 456, 502–506 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Young, C. S. et al. A single CRISPR–Cas9 deletion strategy that targets the majority of DMD patients restores dystrophin function in hiPSC-derived muscle cells. Cell Stem Cell 18, 533–540 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Tierney, M. T. & Sacco, A. Satellite cell heterogeneity in skeletal muscle homeostasis. Trends Cell Biol. 26, 434–444 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Castiglioni, A. et al. Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle. Stem Cell Rep. 2, 92–106 (2014).

    CAS  Article  PubMed Central  Google Scholar 

  28. 28.

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

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Cerletti, M. et al. Highly efficient, functional engraftment of skeletal muscle stem cells in dystrophic muscles. Cell 134, 37–47 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Godfrey, C. et al. How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse. Hum. Mol. Genet. 24, 4225–4237 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Sharp, P. S., Bye-a-Jee, H. & Wells, D. J. Physiological characterization of muscle strength with variable levels of dystrophin restoration in mdx mice following local antisense therapy. Mol. Ther. 19, 165–171 (2011).

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Chan, Y. & Walmsley, R. P. Learning and understanding the Kruskal–Wallis one-way analysis-of-variance-by-ranks test for differences among three or more independent groups. Phys. Ther. 77, 1755–1762 (1997).

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Choi, I. Y. et al. Concordant but varied phenotypes among Duchenne muscular dystrophy patient-specific myoblasts derived using a human iPSC-based model. Cell Rep. 15, 2301–2312 (2016).

    CAS  Article  PubMed  Google Scholar 

  34. 34.

    Xu, X. et al. Human satellite cell transplantation and regeneration from diverse skeletal muscles. Stem Cell Rep. 5, 419–434 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Cahan, P. & Daley, G. Q. Origins and implications of pluripotent stem cell variability and heterogeneity. Nat. Rev. Mol. Cell Biol. 14, 357–368 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Osafune, K. et al. Marked differences in differentiation propensity among human embryonic stem cell lines. Nat. Biotechnol. 26, 313–315 (2008).

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Cusella-De Angelis, M. G. et al. Differential response of embryonic and fetal myoblasts to TGF beta: a possible regulatory mechanism of skeletal muscle histogenesis. Development 120, 925–933 (1994).

    CAS  PubMed  Google Scholar 

  38. 38.

    Schiaffino, S., Rossi, A. C., Smerdu, V., Leinwand, L. A. & Reggiani, C. Developmental myosins: expression patterns and functional significance. Skelet. Muscle 5, 22 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Pagliuca, F. W. et al. Generation of functional human pancreatic β cells in vitro. Cell 159, 428–439 (2104).

    Article  Google Scholar 

  40. 40.

    Maroof, A. M. et al. Directed differentiation and functional maturation of cortical interneurons from human embryonic stem cells. Cell Stem Cell 12, 559–572 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Evseenko, D. et al. Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells. Proc. Natl Acad. Sci. USA 107, 13742–13747 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Alexander, M. S. et al. CD82 is a marker for prospective isolation of human muscle satellite cells and is linked to muscular dystrophies. Cell Stem Cell 19, 800–807 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Figeac, N., Serralbo, O., Marcelle, C. & Zammit, P. S. ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells. Dev. Biol. 386, 135–151 (2014).

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Golding, J. P., Calderbank, E., Partridge, T. A. & Beauchamp, J. R. Skeletal muscle stem cells express anti-apoptotic ErbB receptors during activation from quiescence. Exp. Cell Res. 313, 341–356 (2007).

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Van Ho, A. T. et al. Neural crest cell lineage restricts skeletal muscle progenitor cell differentiation through neuregulin1–ErbB3 signaling. Dev. Cell 21, 273–287 (2011).

    CAS  Article  PubMed  Google Scholar 

  46. 46.

    Deponti, D. et al. The low-affinity receptor for neurotrophins p75NTR plays a key role for satellite cell function in muscle repair acting via RhoA. Mol. Biol. Cell 20, 3620–3627 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Esteves de Lima, J., Bonnin, M. A., Birchmeier, C. & Duprez, D. Muscle contraction is required to maintain the pool of muscle progenitors via YAP and NOTCH during fetal myogenesis. eLife 5, e15593 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Manceau, M. et al. Myostatin promotes the terminal differentiation of embryonic muscle progenitors. Genes Dev. 22, 668–681 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. 49.

    Arnett, A. L. et al. Adeno-associated viral (AAV) vectors do not efficiently target muscle satellite cells. Mol. Ther. Methods Clin. Dev. 1, 14038 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Quarta, M. et al. An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy. Nat. Biotechnol. 34, 752–759 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Rommel, C. et al. Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways. Nat. Cell Biol. 3, 1009–1013 (2001).

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Young, C. S. et al. Creation of a novel humanized dystrophic mouse model of Duchenne muscular dystrophy and application of a CRISPR/Cas9 gene editing therapy. J. Neuromuscul. Dis. 4, 139–145 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Nguyen, H. T. & Morris, G. E. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy. Am. J. Hum. Genet. 52, 1057–1066 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56.

    da Huang, W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).

    Article  Google Scholar 

  57. 57.

    da Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    CAS  Article  Google Scholar 

  58. 58.

    Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Lapan, A. D., Rozkalne, A. & Gussoni, E. Human fetal skeletal muscle contains a myogenic side population that expresses the melanoma cell-adhesion molecule. Hum. Mol. Genet. 21, 3668–3680 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Inman, G. J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Tojo, M. et al. The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-β. Cancer Sci. 96, 791–800 (2005).

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank S. Younesi, J. Marshall, M. Emami, E. Korsakova, K. Saleh, V. Rezek, J. Wen and C. Kumagai-Cresse for helping with stem cell culture and mouse experiments. Furthermore, we thank E. Mokhonova for performing the western blots. We also thank J. Morgan’s laboratory for training on cell engraftments. The following cores were used: CDMD Muscle Phenotyping and Imaging Core, High Throughput and Cell Repository Core, and Bioinformatics and Genomics Core; the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA (BSCRC) Flow Cytometry Core; the UCLA Technology Center for Genomics and Bioinformatics, JCCC Electron Microscopy Core, the CFAR Flow Cytometry Core (NIH P30CA016042, 5P30AI028697); and the UCLA Humanized Mouse Core (CFAR, NIAID AI028697). M.R.H. is the recipient of a BSCRC and Schaffer Fellowship, a CDMD-Cure Duchenne Fellowship and a CDMD-NIH Paul Wellstone Center Training Fellowship (U54 AR052646). D.E. was funded by an NIH grant K01AR061415, Department of Defense (DoD) grant W81XWH-13-1-0465 and California Institute for Regenerative Medicine (CIRM) grant RB5-07230. Funding was provided by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR064327 to A.D.P., and NIAMS 5P30AR05723 to M.J.S., the CDMD at UCLA, the NIH/NCATS, the UCLA CTSI (UL1TR000124), the BSCRC Research Award (A.D.P.), the Rose Hills Foundation Research Award to A.D.P., and a CIRM Inception and CIRM Quest (DISC1-08823 and DISC2-08824 to A.D.P.).

Author information

Affiliations

Authors

Contributions

M.R.H., B.V.H. and A.D.P. conceived the study. M.R.H., B.V.H. and M.J.S. designed the experiments. A.E., B.V.H., S.F.N. and M.R.H. analysed the data. M.R.H., B.V.H., J.H., K.P., W.F., M.J., H.X. and C.S.Y. conducted the experiments. M.R.H. and A.D.P. wrote the manuscript. M.R.H., A.D.P., B.V.H., J.H., K.P., C.S.Y., M.J.S. and S.F.N. edited the manuscript. Funding acquisition, M.R.H., A.D.P. and D.E.; Resources, D.E.; A.D.P. supervised the study.

Corresponding author

Correspondence to April D. Pyle.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–7 and Supplementary References.

Life Sciences Reporting Summary

Supplementary Table 1

Engraftment quantification of all human fetal and hPSC skeletal muscle in all mdx-NSG mice.

Supplementary Table 2

Gene Lists of key biological processes upregulated by CD31-CD45-NCAM+ cultured fetal muscle cells and HNK1-NCAM+ hPSC-SMPCs.

Supplementary Table 3

Gene Lists of key biological processes upregulated by directly-isolated fetal muscle cells and hPSC-SMPCs.

Supplementary Table 4

Gene Lists of key biological processes upregulated by directly-isolated fetal muscle cells and CD31-CD45-NCAM+ cultured fetal muscle cells.

Supplementary Table 5

Gene primer lists.

Supplementary Table 6

Antibody lists for IF and FACS.

Supplementary Table 7

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hicks, M.R., Hiserodt, J., Paras, K. et al. ERBB3 and NGFR mark a distinct skeletal muscle progenitor cell in human development and hPSCs. Nat Cell Biol 20, 46–57 (2018). https://doi.org/10.1038/s41556-017-0010-2

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing