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

  • Paul Keire
  • Andrew Shearer
  • Gabi Shefer
  • Zipora Yablonka-Reuveni
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Skeletal muscle Satellite cells Stem cells Collagen Matrigel Myofiber isolation Flexor digitorum brevis Extensor digitorum longus Diaphragm Masseter Extraocular Mouse Immunostaining Pax7 

Notes

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.

References

  1. 1.
    Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495PubMedCrossRefGoogle Scholar
  2. 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–E216PubMedCrossRefGoogle Scholar
  3. 3.
    Hawke TJ, Garry DJ (2001) Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91:534–551PubMedGoogle Scholar
  4. 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 54:1177–1191PubMedCrossRefGoogle Scholar
  5. 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 122:289–301PubMedCrossRefGoogle Scholar
  6. 6.
    Sacco A, Doyonnas R, Kraft P, Vitorovic S, Blau HM (2008) Self-renewal and expansion of single transplanted muscle stem cells. Nature 456:502–506PubMedCrossRefGoogle Scholar
  7. 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 340:330–343PubMedCrossRefGoogle Scholar
  8. 8.
    Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84:209–238PubMedCrossRefGoogle Scholar
  9. 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 107–144CrossRefGoogle Scholar
  10. 10.
    Morgan JE, Zammit PS (2010) Direct effects of the pathogenic mutation on satellite cell function in muscular dystrophy. Exp Cell Res 316:3100–3108PubMedCrossRefGoogle Scholar
  11. 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. 173–200Google Scholar
  12. 12.
    Muir AR, Kanji AH, Allbrook D (1965) The structure of the satellite cells in skeletal muscle. J Anat 99:435–444PubMedGoogle Scholar
  13. 13.
    Yablonka-Reuveni Z (1995) Development and postnatal regulation of adult myoblasts. Microsc Res Tech 30:366–380PubMedCrossRefGoogle Scholar
  14. 14.
    Boldrin L, Muntoni F, Morgan JE (2010) Are human and mouse satellite cells really the same? J Histochem Cytochem 58:941–955Google Scholar
  15. 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 102:777–786PubMedCrossRefGoogle Scholar
  16. 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 304:246–259PubMedCrossRefGoogle Scholar
  17. 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:e13307PubMedCrossRefGoogle Scholar
  18. 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 56:77–87PubMedCrossRefGoogle Scholar
  19. 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 309:2064–2067PubMedCrossRefGoogle Scholar
  20. 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 151:1221–1234PubMedCrossRefGoogle Scholar
  21. 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 164:588–603PubMedCrossRefGoogle Scholar
  22. 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 294:50–66PubMedCrossRefGoogle Scholar
  23. 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 166:347–357PubMedCrossRefGoogle Scholar
  24. 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 238:1001–1009PubMedCrossRefGoogle Scholar
  25. 25.
    Yablonka-Reuveni Z, Quinn LS, Nameroff M (1987) Isolation and clonal analysis of satellite cells from chicken pectoralis muscle. Dev Biol 119:252–259PubMedCrossRefGoogle Scholar
  26. 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 48:1079–1096PubMedCrossRefGoogle Scholar
  27. 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 DiegoGoogle Scholar
  28. 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:e10920PubMedCrossRefGoogle Scholar
  29. 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. 30.
    Shefer G, Yablonka-Reuveni Z (2005) Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 290:281–304PubMedGoogle Scholar
  31. 31.
    Bekoff A, Betz W (1977) Properties of isolated adult rat muscle fibres maintained in tissue culture. J Physiol 271:537–547PubMedGoogle Scholar
  32. 32.
    Bischoff R (1986) Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115:129–139PubMedCrossRefGoogle Scholar
  33. 33.
    Bischoff R (1989) Analysis of muscle regeneration using single myofibers in culture. Med Sci Sports Exerc 21:S164–S172PubMedGoogle Scholar
  34. 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 7:189–202Google Scholar
  35. 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 235:203–212PubMedCrossRefGoogle Scholar
  36. 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 210:440–455PubMedCrossRefGoogle Scholar
  37. 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 31:773–779PubMedCrossRefGoogle Scholar
  38. 38.
    Rosenblatt JD, Parry DJ, Partridge TA (1996) Phenotype of adult mouse muscle myoblasts reflects their fiber type of origin. Differentiation 60:39–45PubMedCrossRefGoogle Scholar
  39. 39.
    Shefer G, Wleklinski-Lee M, Yablonka-Reuveni Z (2004) Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway. J Cell Sci 117:5393–5404PubMedCrossRefGoogle Scholar
  40. 40.
    Kuang S, Kuroda K, Le Grand F, Rudnicki MA (2007) Asymmetric self-renewal and commitment of satellite stem cells in muscle. Cell 129:999–1010PubMedCrossRefGoogle Scholar
  41. 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 47:23–42PubMedCrossRefGoogle Scholar
  42. 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 115:1461–1469PubMedGoogle Scholar
  43. 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 51:1437–1445PubMedCrossRefGoogle Scholar
  44. 44.
    Greene EC (1963) Anatomy of the rat. Hafner Publishing Company, New York, NYGoogle Scholar
  45. 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 21:6188–6193PubMedCrossRefGoogle Scholar
  46. 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 156:307–318PubMedCrossRefGoogle Scholar
  47. 47.
    Yablonka-Reuveni Z (1995) Myogenesis in the chicken: the onset of differentiation of adult myoblasts is influenced by tissue factors. Basic Appl Myol 5:33–42Google Scholar
  48. 48.
    O’Neill MC, Stockdale FE (1972) A kinetic analysis of myogenesis in vitro. J Cell Biol 52:52–65PubMedCrossRefGoogle Scholar
  49. 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 60:638–657Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Paul Keire
    • 1
  • Andrew Shearer
    • 1
  • Gabi Shefer
    • 2
  • Zipora Yablonka-Reuveni
    • 3
  1. 1.Department of Biological Structure, School of MedicineUniversity of WashingtonSeattleUSA
  2. 2.Department of Biological Structure, Faculty of MedicineUniversity of WashingtonSeattleUSA
  3. 3.Department of Biological StructureUniversity of WashingtonSeattleUSA

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