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.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting

Nature Protocols volume 10, pages 1612–1624 (2015)Cite this article

Subjects

Abstract

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM+CD31−CD45−Sca1−). The isolation process takes ∼5–6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Representative FACS profiles of quiescent and activated MuSCs.
Figure 2: Dissection of hind limb muscles.
Figure 3: Confirmation of the myogenicity of isolated MuSCs.

References

  1. Morgan, J.E. & Partridge, T.A. Muscle satellite cells. Int. J. Biochem. Cell Biol. 35, 1151–1156 (2003).

    Article  CAS  Google Scholar 

  2. Rando, T.A. Stem cells, ageing and the quest for immortality. Nature 441, 1080–1086 (2006).

    Article  CAS  Google Scholar 

  3. Mauro, A. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, 493–495 (1961).

    Article  CAS  Google Scholar 

  4. Seale, P. et al. Pax7 is required for the specification of myogenic satellite cells. Cell 102, 777–786 (2000).

    Article  CAS  Google Scholar 

  5. Cheung, T.H. & Rando, T.A. Molecular regulation of stem cell quiescence. Nat. Rev. Mol. Cell Biol. 14, 329–340 (2013).

    Article  CAS  Google Scholar 

  6. Liu, L. & Rando, T.A. Manifestations and mechanisms of stem cell aging. J. Cell Biol. 193, 257–256 (2011).

    Article  CAS  Google Scholar 

  7. Rinaldi, F. & Perlingeiro, R.C. Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks. Transl. Res. 163, 409–417 (2014).

    Article  CAS  Google Scholar 

  8. Tedesco, F.S. & Cossu, G. Stem cell therapies for muscle disorders. Curr. Opin. Neurol. 25, 597–603 (2012).

    Article  Google Scholar 

  9. Sherwood, R.I. et al. Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell 119, 543–554 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Fukada, S. et al. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25, 2448–2459 (2007).

    Article  CAS  Google Scholar 

  12. Joe, A.W. et al. Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis. Nat. Cell Biol. 12, 153–163 (2010).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Boutet, S.C. et al. Alternative polyadenylation mediates microRNA regulation of muscle stem cell function. Cell Stem Cell 10, 327–336 (2012).

    Article  CAS  Google Scholar 

  15. Liu, L. et al. Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging. Cell Rep. 4, 189–204 (2013).

    Article  CAS  Google Scholar 

  16. Conboy, M.J., Cerletti, M., Wagers, A.J. & Conboy, I.M. Immuno-analysis and FACS sorting of adult muscle fiber-associated stem/precursor cells. Methods Mol. Biol. 621, 165–173 (2010).

    Article  CAS  Google Scholar 

  17. Conboy, M.J. & Conboy, I.M. Preparation of adult muscle fiber-associated stem/precursor cells. Methods Mol. Biol. 621, 149–163 (2010).

    Article  CAS  Google Scholar 

  18. Rocheteau, P., Gayraud-Morel, B., Siegl-Cachedenier, I., Blasco, M.A. & Tajbakhsh, S. A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division. Cell 148, 112–125 (2012).

    Article  CAS  Google Scholar 

  19. Bosnakovski, D. et al. Prospective isolation of skeletal muscle stem cells with a Pax7 reporter. Stem Cells 26, 3194–3204 (2008).

    Article  CAS  Google Scholar 

  20. Bjornson, C.R. et al. Notch signaling is necessary to maintain quiescence in adult muscle stem cells. Stem Cells 30, 232–242 (2012).

    Article  CAS  Google Scholar 

  21. Cheung, T.H. et al. Maintenance of muscle stem-cell quiescence by microRNA-489. Nature 482, 524–528 (2012).

    Article  CAS  Google Scholar 

  22. Rodgers, J.T. et al. mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510, 393–396 (2014).

    Article  CAS  Google Scholar 

  23. Tang, A.H. & Rando, T.A. Induction of autophagy supports the bioenergetic demands of quiescent muscle stem cell activation. EMBO J. 33, 2782–2797 (2014).

    Article  CAS  Google Scholar 

  24. Nishijo, K. et al. Biomarker system for studying muscle, stem cells, and cancer in vivo. FASEB J. 23, 2681–2690 (2009).

    Article  CAS  Google Scholar 

  25. Yi, L. & Rossi, F. Purification of progenitors from skeletal muscle. J. Vis. Exp. 10.3791/2476 (2011).

  26. Conboy, I.M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760–764 (2005).

    Article  CAS  Google Scholar 

  27. Gibson, M.C. & Schultz, E. Age-related differences in absolute numbers of skeletal muscle satellite cells. Muscle Nerve 6, 574–580 (1983).

    Article  CAS  Google Scholar 

  28. Sajko, S. et al. Frequency of M-cadherin–stained satellite cells declines in human muscles during aging. J. Histochem. Cytochem. 52, 179–185 (2004).

    Article  CAS  Google Scholar 

  29. Pasut, A., Oleynik, P. & Rudnicki, M.A. Isolation of muscle stem cells by fluorescence-activated cell sorting cytometry. Methods Mol. Biol. 798, 53–64 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Huang for technical assistance of dissection and L. Rott for technical assistance with FACS. This work was supported by grants from The Glenn Foundation for Medical Research, the US National Institutes of Health (P01 AG036695, R37 AG023806 and R01 AR062185) and the Department of Veterans Affairs to T.A.R.

Author information

Author notes

  1. Tom H Cheung

    Present address: Present address: Division of Life Science, Hong Kong University of Science and Technology, Kowloon, Hong Kong.,

Authors and Affiliations

  1. The Glenn Center for the Biology of Aging and the Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, California, USA

    Ling Liu, Tom H Cheung, Gregory W Charville & Thomas A Rando

  2. Neurology Service and Rehabilitation, Research and Development Center of Excellence, Veterans Affairs (VA) Palo Alto Health Care System, Palo Alto, California, USA

    Thomas A Rando

Authors
  1. Ling Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tom H Cheung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gregory W Charville
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas A Rando
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the design and interpretation of the experiments; L.L., T.H.C. and G.W.C. conducted the experiments; and L.L. and T.A.R. wrote the manuscript.

Corresponding author

Correspondence to Thomas A Rando.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of the VCAM1+Sca1-CD31-CD45- population from muscle as MuSCs using a genetic lineage tracer.

Pax7CreER/+; ROSA26eYFP/+ mice were injected with tamoxifen at 8-10 weeks of age. Mononucleated cells were isolated from limb muscles of these mice at different ages (indicated in parentheses) in the absence of injury or at different times following the injury, as indicated. Cells were then stained with APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Sca1 and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. Cells positive for CD31 and CD45 (CD31+/CD45+), cells positive for Sca1 but negative for CD31/CD45 (CD31-/CD45-, Sca1+), and cells positive for VCAM1 but negative for CD31/CD45/Sca1 (CD31-/CD45-/Sca1-, VCAM1+) were analyzed for YFP-expressing cells. The FITC channel (for the detection of YFP) is shown on the Y axis and FSC-S is shown on the X axis of all plots. qMuSCs: quiescent MuSCs; aMuSCs: activated MuSCs and their progeny; mo: months; DPI: days post-injury; MPI: months post-injury.

Supplementary Figure 2 Typical FACS profiles for single channel staining controls.

The suspension of resident mononucleated cells from uninjured limb muscles of a 2-month-old C57BL/6 mouse was stained with (A) APC anti-mouse CD31 and APC anti-mouse CD45, (B) Pacific Blue anti-mouse Ly-6A/E (Sca1), and (C) Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. The population hierarchy is shown under the plots.

Supplementary Figure 3 Typical FACS profiles for Fluorescence Minus One (FMO) staining controls.

The suspension of resident mononucleated cells from uninjured limb muscles of a 2-month-old C57BL/6 mouse was stained with (A) Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, (B) APC anti-mouse CD31, APC anti-mouse CD45 and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, and (C) APC anti-CD31, APC anti-mouse CD45 and Pacific Blue anti-mouse Ly-6A/E (Sca1). The population hierarchy is shown under the plots.

Supplementary Figure 4 Typical FACS profiles for staining using isotype control antibodies.

The suspension of resident mononucleated cells from uninjured limb muscles of a 2-month-old C57BL/6 mouse was stained with (A) APC IgG2a, APC IgG2b, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, (B) APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue IgG2a, and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin, and (C) APC anti-CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1), and Biotin IgG2a followed by PE/Cy7 Streptavidin. The population hierarchy is shown under the plots.

Supplementary Figure 5 Representative FACS profiles of quiescent and activated MuSCs without PI staining.

The digested tissue preparations, as described in this protocol, from uninjured or injured limb muscles were stained with APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. Cells in the P4 gate are MuSCs. (A) Profiles of the unstained quiescent MuSCs. (B) Profiles of quiescent MuSCs after antibody staining. The population hierarchy is shown under the plots. (C) VCAM1+/CD31-/CD45-/Sca1- SCs were isolated from a 2-month-old C57BL/6 mouse according to the protocol described. Cells were then incubated with 0.3 μg/ml PI and analyzed by FACS. (D) Profiles of the unstained activated MuSCs and their progeny. (E) Profiles of activated MuSCs and their progeny after antibody staining. The population hierarchy is shown under the plots. (F) VCAM1+/CD31-/CD45-/Sca1- SCs were isolated from a 2-month-old C57BL/6 mouse injured by BaCl2 injection according to the protocol described. Cells were then incubated with 0.3 μg/ml PI and analyzed by FACS for the percentage of PI-positive cells.

Supplementary Figure 6 Identification and isolation of different cell-types in skeletal muscle.

(A) Distinct separation of mononucleated cells from myofiber debris following isolation from uninjured skeletal muscle. 2,000 events are shown in the FSC-A and SSC plot. The region of the plot containing cellular debris is highlighted in red. (B) Representative FACS profiles to isolate highly pure quiescent MuSCs (P6) and muscle-derived mesenchymal stem cells (P5) simultaneously. The suspension of resident mononucleated cells from uninjured limb muscles was stained with APC anti-mouse CD31, APC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. 20,000 events were recorded and are shown in the plots. The population hierarchy is shown under the plots. Note the size difference of the P1 gate in comparison to that in Figure 1A and 1B. Mesenchymal stem cells are much larger than MuSCs and therefore a larger gate is needed to acquire these cells. Therefore we recommend using the FSC-W parameter to further select for small cells within the CD31-/CD45-/Sca1-/VCAM1+ population (P4). (C) Typical FACS profiles to identify and isolate highly pure quiescent MuSCs (P8) and muscle-derived mesenchymal stem cells (P5), as well as populations enriched for hematopoietic (P6) and endothelial (P7) cells simultaneously. The suspension of resident mononucleated cells from uninjured limb muscles was stained with APC anti-mouse CD31, FITC anti-mouse CD45, Pacific Blue anti-mouse Ly-6A/E (Sca1) and Biotin anti-mouse CD106 (VCAM1) followed by PE/Cy7 Streptavidin. 20,000 events were recorded and are shown in the plots. The population hierarchy is shown under the plots. The FSC-W parameter further selected for small cells within the CD31-/CD45-/Sca1-/VCAM1+ population (P4) in order to maximize the purity of MuSCs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-6 (PDF 1270 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Liu, L., Cheung, T., Charville, G. et al. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc 10, 1612–1624 (2015). https://doi.org/10.1038/nprot.2015.110

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.110

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced 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