Advertisement

Isolation of Muscle Stem Cells from Mouse Skeletal Muscle

  • Barbara Gayraud-MorelEmail author
  • Francesca Pala
  • Hiroshi Sakai
  • Shahragim Tajbakhsh
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1556)

Abstract

Isolation of muscle stem cells from skeletal muscle is a critical step for the study of skeletal myogenesis and regeneration. Although stem cell isolation has been performed for decades, the emergence of flow cytometry with defined cell surface markers, or transgenic mouse models, has allowed the efficient isolation of highly enriched stem cell populations. Here, we describe the isolation of mouse muscle stem cells using two different combinations of enzyme treatments allowing the release of mononucleated muscle stem cells from their niche. Mouse muscle stem cells can be further isolated as a highly enriched population by flow cytometry using fluorescent reporters or cell surface markers. We will present advantages and drawbacks of these different approaches.

Key words

Satellite cells Muscle stem cell isolation Enzymatic dissociation FACS 

Abbreviations

FACS

Fluorescent-activated cell sorting

TA

Tibialis anterior

GFP

Green fluorescent protein

FSC

Forward scatter

SSC

Side scatter

C/T

Collagenase D/Trypsin

C/D

Collagenase A/Dispase II

FBS

Fetal bovine serum

CD

Cluster of differentiation

Notes

Acknowledgments

We acknowledge the funding support from the Institut Pasteur, Centre National pour la Recherche Scientifique, Association Française contre les Myopathies, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir; ANR-10-LABX-73), Association pour la Recherche sur le Cancer, EU Advanced ERC grant, and Fondation pour la Recherche Médicale. H. Sakai is funded by the ERC and F. Pala by the LabEx Revive/Pasteur PPU program.

References

  1. 1.
    Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Relaix F, Zammit PS (2012) Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development 139(16):2845–2856. doi: 10.1242/dev.069088 CrossRefPubMedGoogle Scholar
  3. 3.
    Yablonka-Reuveni Z (2011) The skeletal muscle satellite cell: still young and fascinating at 50. J Histochem Cytochem 59(12):1041–1059. doi: 10.1369/0022155411426780 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, Tanimura N, Yamamoto H, Miyagoe-Suzuki Y, Takeda S (2007) Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells 25(10):2448–2459. doi:2007-0019 [pii]5.1634/stemcells.2007-0019Google Scholar
  5. 5.
    Kuang S, Rudnicki MA (2008) The emerging biology of satellite cells and their therapeutic potential. Trends Mol Med 14(2):82–91Google Scholar
  6. 6.
    Motohashi N, Asakura A (2014) Muscle satellite cell heterogeneity and self-renewal. Front Cell Dev Biol 2:1. doi: 10.3389/fcell.2014.00001 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Rocheteau P, Vinet M, Chretien F (2015) Dormancy and quiescence of skeletal muscle stem cells. Results Probl Cell Differ 56:215–235. doi: 10.1007/978-3-662-44608-9_10 CrossRefPubMedGoogle Scholar
  8. 8.
    Sambasivan R, Tajbakhsh S (2015) Adult skeletal muscle stem cells. Results Probl Cell Differ 56:191–213. doi: 10.1007/978-3-662-44608-9_9 CrossRefPubMedGoogle Scholar
  9. 9.
    Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102(6):777–786CrossRefPubMedGoogle Scholar
  10. 10.
    Sambasivan R, Comai G, Le Roux I, Gomes D, Konge J, Dumas G, Cimper C, Tajbakhsh S (2013) Embryonic of adult muscle stem cells are primed by the determination gene Mrf4. Dev Biol 381(1):241–255. doi: 10.1016/j.ydbio.2013.04.018 CrossRefPubMedGoogle Scholar
  11. 11.
    Sambasivan R, Gayraud-Morel B, Dumas G, Cimper C, Paisant S, Kelly RG, Tajbakhsh S (2009) Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates. Dev Cell 16(6):810–821CrossRefPubMedGoogle Scholar
  12. 12.
    Bosnakovski D, Xu Z, Li W, Thet S, Cleaver O, Perlingeiro RC, Kyba M (2008) Prospective isolation of skeletal muscle stem cells with a Pax7 reporter. Stem Cells 26(12):3194–3204CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gunther S, Kim J, Kostin S, Lepper C, Fan CM, Braun T (2013) Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells. Cell Stem Cell 13(5):590–601. doi: 10.1016/j.stem.2013.07.016 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lepper C, Fan CM (2010) Inducible lineage tracing of Pax7-descendant cells reveals embryonic origin of adult satellite cells. Genesis 48(7):424–436. doi: 10.1002/dvg.20630 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mourikis P, Sambasivan R, Castel D, Rocheteau P, Bizzarro V, Tajbakhsh S (2012) A critical requirement for notch signaling in maintenance of the quiescent skeletal muscle stem cell state. Stem Cells 30(2):243–252. doi: 10.1002/stem.775 CrossRefPubMedGoogle Scholar
  16. 16.
    Murphy MM, Lawson JA, Mathew SJ, Hutcheson DA, Kardon G (2011) Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 138(17):3625–3637. doi: 10.1242/dev.064162 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Relaix F, Rocancourt D, Mansouri A, Buckingham M (2005) A Pax3/Pax7-dependent population of skeletal muscle progenitor cells. Nature 435(7044):948–953CrossRefPubMedGoogle Scholar
  18. 18.
    Gayraud-Morel B, Chretien F, Jory A, Sambasivan R, Negroni E, Flamant P, Soubigou G, Coppee JY, Di Santo J, Cumano A, Mouly V, Tajbakhsh S (2012) Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells. J Cell Sci 125(Pt 7):1738–1749. doi: 10.1242/jcs.097006 CrossRefPubMedGoogle Scholar
  19. 19.
    Comai G, Sambasivan R, Gopalakrishnan S, Tajbakhsh S (2014) Variations in the efficiency of lineage marking and ablation confound distinctions between myogenic cell populations. Dev Cell 31(5):654–667. doi: 10.1016/j.devcel.2014.11.005 CrossRefPubMedGoogle Scholar
  20. 20.
    Haldar M, Hancock JD, Coffin CM, Lessnick SL, Capecchi MR (2007) A conditional mouse model of synovial sarcoma: insights into a myogenic origin. Cancer Cell 11(4):375–388. doi: 10.1016/j.ccr.2007.01.016 CrossRefPubMedGoogle Scholar
  21. 21.
    Kuang S, Kuroda K, Le Grand F, Rudnicki MA (2007) Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129(5):999–1010CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Tallquist MD, Weismann KE, Hellstrom M, Soriano P (2000) Early myotome specification regulates PDGFA expression and axial skeleton development. Development 127(23):5059–5070PubMedGoogle Scholar
  23. 23.
    Day K, Shefer G, Richardson JB, Enikolopov G, Yablonka-Reuveni Z (2007) Nestin-GFP reporter expression defines the quiescent state of skeletal muscle satellite cells. Dev Biol 304(1):246–259. doi: 10.1016/j.ydbio.2006.12.026 CrossRefPubMedGoogle Scholar
  24. 24.
    Keire P, Shearer A, Shefer G, Yablonka-Reuveni Z (2013) Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 946:431–468. doi: 10.1007/978-1-62703-128-8_28 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Shinin V, Gayraud-Morel B, Tajbakhsh S (2009) Template DNA-strand co-segregation and asymmetric cell division in skeletal muscle stem cells. Methods Mol Biol 482:295–317. doi: 10.1007/978-1-59745-060-7_19 CrossRefPubMedGoogle Scholar
  26. 26.
    Rocheteau P, Gayraud-Morel B, Siegl-Cachedenier I, Blasco MA, Tajbakhsh S (2012) A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division. Cell 148(1–2):112–125. doi: 10.1016/j.cell.2011.11.049 CrossRefPubMedGoogle Scholar
  27. 27.
    Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM (2008) Self-renewal and expansion of single transplanted muscle stem cells. Nature 456(7221):502–506CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, Partridge T, Buckingham M (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science 309(5743):2064–2067CrossRefPubMedGoogle Scholar
  29. 29.
    Chakkalakal JV, Jones KM, Basson MA, Brack AS (2012) The aged niche disrupts muscle stem cell quiescence. Nature 490(7420):355–360. doi: 10.1038/nature11438 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Cheung TH, Quach NL, Charville GW, Liu L, Park L, Edalati A, Yoo B, Hoang P, Rando TA (2012) Maintenance of muscle stem-cell quiescence by microRNA-489. Nature 482(7386):524–528. doi: 10.1038/nature10834 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Fukada S, Higuchi S, Segawa M, Koda K, Yamamoto Y, Tsujikawa K, Kohama Y, Uezumi A, Imamura M, Miyagoe-Suzuki Y, Takeda S, Yamamoto H (2004) Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. Exp Cell Res 296(2):245–255. doi: 10.1016/j.yexcr.2004.02.018 CrossRefPubMedGoogle Scholar
  32. 32.
    Sherwood RI, Christensen JL, Conboy IM, Conboy MJ, Rando TA, Weissman IL, Wagers AJ (2004) Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell 119(4):543–554CrossRefPubMedGoogle Scholar
  33. 33.
    Tanaka KK, Hall JK, Troy AA, Cornelison DD, Majka SM, Olwin BB (2009) Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration. Cell Stem Cell 4(3):217–225CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Rodgers JT, King KY, Brett JO, Cromie MJ, Charville GW, Maguire KK, Brunson C, Mastey N, Liu L, Tsai CR, Goodell MA, Rando TA (2014) mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510(7505):393–396. doi: 10.1038/nature13255 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Motohashi N, Asakura Y, Asakura A (2014) Isolation, culture, and transplantation of muscle satellite cells. J Vis Exp (86). doi: 10.3791/50846
  36. 36.
    Pisani DF, Dechesne CA, Sacconi S, Delplace S, Belmonte N, Cochet O, Clement N, Wdziekonski B, Villageois AP, Butori C, Bagnis C, Di Santo JP, Kurzenne JY, Desnuelle C, Dani C (2010) Isolation of a highly myogenic CD34-negative subset of human skeletal muscle cells free of adipogenic potential. Stem Cells 28(4):753–764. doi: 10.1002/stem.317 CrossRefPubMedGoogle Scholar
  37. 37.
    Marg A, Escobar H, Gloy S, Kufeld M, Zacher J, Spuler A, Birchmeier C, Izsvak Z, Spuler S (2014) Human satellite cells have regenerative capacity and are genetically manipulable. J Clin Invest 124(10):4257–4265. doi: 10.1172/JCI63992 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Bareja A, Holt JA, Luo G, Chang C, Lin J, Hinken AC, Freudenberg JM, Kraus WE, Evans WJ, Billin AN (2014) Human and mouse skeletal muscle stem cells: convergent and divergent mechanisms of myogenesis. PLoS One 9(2):e90398. doi: 10.1371/journal.pone.0090398 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Castiglioni A, Hettmer S, Lynes MD, Rao TN, Tchessalova D, Sinha I, Lee BT, Tseng YH, Wagers AJ (2014) Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle. Stem Cell Reports 2(1):92–106. doi: 10.1016/j.stemcr.2013.12.006 CrossRefPubMedCentralGoogle Scholar
  40. 40.
    Tamaki T, Uchiyama Y, Hirata M, Hashimoto H, Nakajima N, Saito K, Terachi T, Mochida J (2015) Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution. Front Physiol 6:165. doi: 10.3389/fphys.2015.00165 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Mitchell KJ, Pannerec A, Cadot B, Parlakian A, Besson V, Gomes ER, Marazzi G, Sassoon DA (2010) Identification and characterization of a non-satellite cell muscle resident progenitor during postnatal development. Nat Cell Biol 12(3):257–266PubMedGoogle Scholar
  42. 42.
    Sambasivan R, Yao R, Kissenpfennig A, Van Wittenberghe L, Paldi A, Gayraud-Morel B, Guenou H, Malissen B, Tajbakhsh S, Galy A (2011) Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 138(17):3647–3656CrossRefPubMedGoogle Scholar
  43. 43.
    Schiaffino S, Reggiani C (2011) Fiber types in mammalian skeletal muscles. Physiol Rev 91(4):1447–1531. doi: 10.1152/physrev.00031.2010 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Open Access This chapter is distributed under the terms of the Creative Commons Attribution Noncommercial License, which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Authors and Affiliations

  • Barbara Gayraud-Morel
    • 1
    Email author
  • Francesca Pala
    • 1
  • Hiroshi Sakai
    • 1
  • Shahragim Tajbakhsh
    • 1
  1. 1.Department of Developmental and Stem Cell Biology, Stem Cells and Development, CNRS URA 2578Institut PasteurParisFrance

Personalised recommendations