Functionally heterogeneous human satellite cells identified by single cell RNA sequencing

Abstract

Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.

Data availability

Single cell rna sequencing data were uploaded to Dryad and can be accessed under the doi: 10.7272/Q65X273X

The following data sets were generated

Article and author information

Author details

  1. Emilie Barruet

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Steven M Garcia

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Katharine Striedinger

    Departments of Surgery and Orofacial Sciences, Division of Plastic and Reconstructive Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jake Wu

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Solomon Lee

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Lauren Byrnes

    Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Alvin Wong

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Sun Xuefeng

    Orthopedic Surgery, Eli and Edythe Broad Center of Regeneration Medicine, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Stanley Tamaki

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Andrew S Brack

    Department of Orthopaedic Surgery, University of California, San Francisco, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Jason H Pomerantz

    Departments of Surgery and Orofacial Sciences, Division of Plastic Surgery, Program in Craniofacial Biology, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, United States
    For correspondence
    jason.pomerantz@ucsf.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5107-1883

Funding

California Institute for Regenerative Medicine (New Faculty Physician Scientist Award RN3-06504)

  • Jason H Pomerantz

National Institutes of Health (R01AR072638-03)

  • Jason H Pomerantz

University of California, San Francisco (UCSF PROF-PATH program via NIH R25MD006832)

  • Steven M Garcia

University of California, San Francisco (Research Allocation Program for trainees)

  • Solomon Lee

Eli and Edythe Broad Foundation (Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research Fellowship)

  • Alvin Wong

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All procedures were approved and performed in accordance with the UCSF Institutional Animal Care and Use Committee (Protocols #181101).

Human subjects: This study was conducted under the approval of the Institutional Review Board at The University of California San Francisco (UCSF). Written informed consent was obtained from all subjects.

Reviewing Editor

  1. Shahragim Tajbakhsh, Institut Pasteur, France

Publication history

  1. Received: September 4, 2019
  2. Accepted: March 27, 2020
  3. Accepted Manuscript published: April 1, 2020 (version 1)
  4. Version of Record published: April 17, 2020 (version 2)

Copyright

© 2020, Barruet et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,087
    Page views
  • 664
    Downloads
  • 27
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Emilie Barruet
  2. Steven M Garcia
  3. Katharine Striedinger
  4. Jake Wu
  5. Solomon Lee
  6. Lauren Byrnes
  7. Alvin Wong
  8. Sun Xuefeng
  9. Stanley Tamaki
  10. Andrew S Brack
  11. Jason H Pomerantz
(2020)
Functionally heterogeneous human satellite cells identified by single cell RNA sequencing
eLife 9:e51576.
https://doi.org/10.7554/eLife.51576

Further reading

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Giacomo Leon Glotzer et al.
    Research Article

    The expression of Fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescen in situ hybridization and a semi-automated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similarly to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl.

    1. Stem Cells and Regenerative Medicine
    Rafael Jesus Fernandez III et al.
    Research Article

    Dyskeratosis congenita (DC) is a rare genetic disorder characterized by deficiencies in telomere maintenance leading to very short telomeres and the premature onset of certain age-related diseases, including pulmonary fibrosis (PF). PF is thought to derive from epithelial failure, particularly that of type II alveolar epithelial (AT2) cells, which are highly dependent on Wnt signaling during development and adult regeneration. We use human iPSC-derived AT2 (iAT2) cells to model how short telomeres affect AT2 cells. Cultured DC mutant iAT2 cells accumulate shortened, uncapped telomeres and manifest defects in the growth of alveolospheres, hallmarks of senescence, and apparent defects in Wnt signaling. The GSK3 inhibitor, CHIR99021, which mimics the output of canonical Wnt signaling, enhances telomerase activity and rescues the defects. These findings support further investigation of Wnt agonists as potential therapies for DC related pathologies.