Skip to main content
SpringerLink
Log in
Book cover

Basic Cell Culture Protocols pp 431–468Cite as

Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 946))

Abstract

Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing, and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

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

    Article  PubMed  CAS  Google Scholar 

  2. Yablonka-Reuveni Z, Day K, Vine A, Shefer G (2008) Defining the transcriptional signature of skeletal muscle stem cells. J Anim Sci 86:E207–E216

    Article  PubMed  CAS  Google Scholar 

  3. Hawke TJ, Garry DJ (2001) Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91:534–551

    PubMed  CAS  Google Scholar 

  4. Zammit PS, Partridge TA, Yablonka-Reuveni Z (2006) The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem 54:1177–1191

    Article  PubMed  CAS  Google Scholar 

  5. Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122:289–301

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  7. Day K, Shefer G, Shearer A, Yablonka-Reuveni Z (2010) The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny. Dev Biol 340:330–343

    Article  PubMed  CAS  Google Scholar 

  8. Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84:209–238

    Article  PubMed  CAS  Google Scholar 

  9. Shefer G, Yablonka-Reuveni Z (2008) Ins and outs of satellite cell myogenesis: the role of the ruling growth factors. In: Schiaffino S, Partridge T (eds) Skeletal muscle repair and regeneration. Springer, Dordrecht, The Netherlands, pp 107–144

    Chapter  Google Scholar 

  10. Morgan JE, Zammit PS (2010) Direct effects of the pathogenic mutation on satellite cell function in muscular dystrophy. Exp Cell Res 316:3100–3108

    Article  PubMed  CAS  Google Scholar 

  11. Yablonka-Reuveni Z, Day K (2011) Skeletal muscle stem cells in the spotlight: the satellite cell. In: Cohen I, Gaudette G (eds) Regenerating the Heart: Stem Cells and the Cardiovascular System. Springer, Humana Press. pp. 173–200

    Google Scholar 

  12. Muir AR, Kanji AH, Allbrook D (1965) The structure of the satellite cells in skeletal muscle. J Anat 99:435–444

    PubMed  CAS  Google Scholar 

  13. Yablonka-Reuveni Z (1995) Development and postnatal regulation of adult myoblasts. Microsc Res Tech 30:366–380

    Article  PubMed  CAS  Google Scholar 

  14. Boldrin L, Muntoni F, Morgan JE (2010) Are human and mouse satellite cells really the same? J Histochem Cytochem 58:941–955

    Google Scholar 

  15. 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 102:777–786

    Article  PubMed  CAS  Google Scholar 

  16. 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 304:246–259

    Article  PubMed  CAS  Google Scholar 

  17. Shefer G, Rauner G, Yablonka-Reuveni Z, Benayahu D (2010) Reduced satellite cell numbers and myogenic capacity in aging can be alleviated by endurance exercise. PLoS One 5:e13307

    Article  PubMed  Google Scholar 

  18. Allouh MZ, Yablonka-Reuveni Z, Rosser BW (2008) Pax7 reveals a greater frequency and concentration of satellite cells at the ends of growing skeletal muscle fibers. J Histochem Cytochem 56:77–87

    Article  PubMed  CAS  Google Scholar 

  19. 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 309:2064–2067

    Article  PubMed  CAS  Google Scholar 

  20. Beauchamp JR, Heslop L, Yu DS, Tajbakhsh S, Kelly RG, Wernig A, Buckingham ME, Partridge TA, Zammit PS (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151:1221–1234

    Article  PubMed  CAS  Google Scholar 

  21. Yablonka-Reuveni Z, Rivera AJ (1994) Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated adult rat fibers. Dev Biol 164:588–603

    Article  PubMed  CAS  Google Scholar 

  22. Shefer G, Van de Mark DP, Richardson JB, Yablonka-Reuveni Z (2006) Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle. Dev Biol 294:50–66

    Article  PubMed  CAS  Google Scholar 

  23. Zammit PS, Golding JP, Nagata Y, Hudon V, Partridge TA, Beauchamp JR (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166:347–357

    Article  PubMed  CAS  Google Scholar 

  24. Day K, Paterson B, Yablonka-Reuveni Z (2009) A distinct profile of myogenic regulatory factor detection within Pax7+ cells at S phase supports a unique role of Myf5 during posthatch chicken myogenesis. Dev Dyn 238:1001–1009

    Article  PubMed  CAS  Google Scholar 

  25. Yablonka-Reuveni Z, Quinn LS, Nameroff M (1987) Isolation and clonal analysis of satellite cells from chicken pectoralis muscle. Dev Biol 119:252–259

    Article  PubMed  CAS  Google Scholar 

  26. Kastner S, Elias MC, Rivera AJ, Yablonka-Reuveni Z (2000) Gene expression patterns of the fibroblast growth factors and their receptors during myogenesis of rat satellite cells. J Histochem Cytochem 48:1079–1096

    Article  PubMed  CAS  Google Scholar 

  27. Yablonka-Reuveni Z (2004) Isolation and culture of myogenic stem cells. In: Lanza R, Blau D, Melton D, Moore M, Thomas ED, Verfaillie C, Weissman I, West M (eds) Handbook of stem cells—vol 2: adult and fetal stem cells. Elsevier, San Diego

    Google Scholar 

  28. Ieronimakis N, Balasundaram G, Rainey S, Srirangam K, Yablonka-Reuveni Z, Reyes M (2010) Absence of CD34 on murine skeletal muscle satellite cells marks a reversible state of activation during acute injury. PLoS One 5:e10920

    Article  PubMed  Google Scholar 

  29. Danoviz ME, Yablonka-Reuveni Z (2012) Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system. Methods Mol Biol 798:21–52.

    Google Scholar 

  30. Shefer G, Yablonka-Reuveni Z (2005) Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 290:281–304

    PubMed  Google Scholar 

  31. Bekoff A, Betz W (1977) Properties of isolated adult rat muscle fibres maintained in tissue culture. J Physiol 271:537–547

    PubMed  CAS  Google Scholar 

  32. Bischoff R (1986) Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115:129–139

    Article  PubMed  CAS  Google Scholar 

  33. Bischoff R (1989) Analysis of muscle regeneration using single myofibers in culture. Med Sci Sports Exerc 21:S164–S172

    PubMed  CAS  Google Scholar 

  34. Yablonka-Reuveni Z, Rivera AJ (1997) Proliferative dynamics and the role of FGF2 during myogenesis of rat satellite cells on isolated fibers. Basic Appl Myol 7:189–202

    Google Scholar 

  35. Yablonka-Reuveni Z, Anderson JE (2006) Satellite cells from dystrophic (mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers. Dev Dyn 235:203–212

    Article  PubMed  CAS  Google Scholar 

  36. Yablonka-Reuveni Z, Rudnicki MA, Rivera AJ, Primig M, Anderson JE, Natanson P (1999) The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD. Dev Biol 210:440–455

    Article  PubMed  CAS  Google Scholar 

  37. Rosenblatt JD, Lunt AI, Parry DJ, Partridge TA (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31:773–779

    Article  PubMed  CAS  Google Scholar 

  38. Rosenblatt JD, Parry DJ, Partridge TA (1996) Phenotype of adult mouse muscle myoblasts reflects their fiber type of origin. Differentiation 60:39–45

    Article  PubMed  CAS  Google Scholar 

  39. Shefer G, Wleklinski-Lee M, Yablonka-Reuveni Z (2004) Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway. J Cell Sci 117:5393–5404

    Article  PubMed  CAS  Google Scholar 

  40. Kuang S, Kuroda K, Le Grand F, Rudnicki MA (2007) Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129:999–1010

    Article  PubMed  CAS  Google Scholar 

  41. Yablonka-Reuveni Z, Seger R, Rivera AJ (1999) Fibroblast growth factor promotes recruitment of skeletal muscle satellite cells in young and old rats. J Histochem Cytochem 47:23–42

    Article  PubMed  CAS  Google Scholar 

  42. Shefer G, Partridge TA, Heslop L, Gross JG, Oron U, Halevy O (2002) Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells. J Cell Sci 115:1461–1469

    PubMed  CAS  Google Scholar 

  43. Wozniak AC, Pilipowicz O, Yablonka-Reuveni Z, Greenway S, Craven S, Scott E, Anderson JE (2003) C-Met expression and mechanical activation of satellite cells on cultured muscle fibers. J Histochem Cytochem 51:1437–1445

    Article  PubMed  CAS  Google Scholar 

  44. Greene EC (1963) Anatomy of the rat. Hafner Publishing Company, New York, NY

    Google Scholar 

  45. Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR (1982) Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:6188–6193

    Article  PubMed  CAS  Google Scholar 

  46. Yablonka-Reuveni Z, Seifert RA (1993) Proliferation of chicken myoblasts is regulated by specific isoforms of platelet-derived growth factor: evidence for differences between myoblasts from mid and late stages of embryogenesis. Dev Biol 156:307–318

    Article  PubMed  CAS  Google Scholar 

  47. Yablonka-Reuveni Z (1995) Myogenesis in the chicken: the onset of differentiation of adult myoblasts is influenced by tissue factors. Basic Appl Myol 5:33–42

    Google Scholar 

  48. O’Neill MC, Stockdale FE (1972) A kinetic analysis of myogenesis in vitro. J Cell Biol 52:52–65

    Article  PubMed  Google Scholar 

  49. Stuelsatz P, Keire P, Almuly R, Yablonka-Reuveni Z (2012) A contemporary atlas of the mouse diaphragm: myogenicity, vascularity and the Pax3 connection. J Histochem Cytochem 60:638–657

    Google Scholar 

Download references

Acknowledgments

The authors are grateful to the granting agencies that funded this study. Our current research is supported by grants to Z.Y.R. from the National Institutes of Health (AG021566; AG035377; AR057794) and the Muscular Dystrophy Association (135908). The development the FDB myofiber isolation protocol described in this chapter could not be possible without the valuable contribution of our former lab member, Anthony Rivera, and previous funding from the Muscular Dystrophy Association, the Cooperative State Research, Education and Extension Service/US Department of Agriculture (National Research Initiative), the National Institutes of Health, and the Nathan Shock Center of Excellence in the Basic Biology of Aging, University of Washington.

Author information

Authors and Affiliations

  1. Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA, USA

    Paul Keire & Andrew Shearer

  2. Department of Biological Structure, Faculty of Medicine, University of Washington, Seattle, WA, USA

    Gabi Shefer

  3. Department of Biological Structure, University of Washington, Seattle, WA, USA

    Zipora Yablonka-Reuveni

Authors
  1. Paul Keire
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew Shearer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gabi Shefer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zipora Yablonka-Reuveni
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zipora Yablonka-Reuveni .

Editor information

Editors and Affiliations

  1. , Experimental Therapeutics, BC Cancer Agency, West 10th Ave. 675, Vancouver, V5Z 1L3, British Columbia, Canada

    Cheryl D. Helgason

  2. STEMCELL Technologies, Inc., W. Seventh Avenue 570, Vancouver, V5Z 1B3, British Columbia, Canada

    Cindy L. Miller

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Keire, P., Shearer, A., Shefer, G., Yablonka-Reuveni, Z. (2013). Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells. In: Helgason, C., Miller, C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology, vol 946. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-128-8_28

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-128-8_28

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-127-1

  • Online ISBN: 978-1-62703-128-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation