Article

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

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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.

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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

  1. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA, USA

    • Michael R. Hicks
    • , Majib Jan
    • , Haibin Xi
    • , Courtney S. Young
    • , Melissa J. Spencer
    •  & April D. Pyle
  2. Center for Duchenne Muscular Dystrophy, University of California, Los Angeles, CA, USA

    • Michael R. Hicks
    • , Ascia Eskin
    • , Majib Jan
    • , Haibin Xi
    • , Courtney S. Young
    • , Stanley F. Nelson
    • , Melissa J. Spencer
    •  & April D. Pyle
  3. Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA, USA

    • Michael R. Hicks
    • , Julia Hiserodt
    • , Katrina Paras
    • , Wakana Fujiwara
    • , Majib Jan
    • , Haibin Xi
    •  & April D. Pyle
  4. Department of Human Genetics, University of California, Los Angeles, CA, USA

    • Ascia Eskin
    •  & Stanley F. Nelson
  5. Molecular Biology Interdepartmental Program, University of California, Los Angeles, CA, USA

    • Courtney S. Young
    • , Stanley F. Nelson
    • , Melissa J. Spencer
    •  & April D. Pyle
  6. Department of Orthopaedic Surgery, Keck School of Medicine, Stem Cell Research and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA

    • Denis Evseenko
    •  & Ben Van Handel
  7. Department of Neurology, University of California, Los Angeles, CA, USA

    • Melissa J. Spencer

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to April D. Pyle.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–7 and Supplementary References.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

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

  4. Supplementary Table 2

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

  5. Supplementary Table 3

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

  6. Supplementary Table 4

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

  7. Supplementary Table 5

    Gene primer lists.

  8. Supplementary Table 6

    Antibody lists for IF and FACS.

  9. Supplementary Table 7

    Statistical source data.