Advertisement

Isolation, Culture and Immunostaining of Skeletal Muscle Fibres to Study Myogenic Progression in Satellite Cells

  • Louise A. Moyle
  • Peter S. Zammit
Part of the Methods in Molecular Biology book series (MIMB, volume 1210)

Abstract

Satellite cells are the resident stem cells of skeletal muscle, located on the surface of a myofibre, beneath the surrounding basal lamina. Satellite cells are responsible for the homeostasis, hypertrophy and repair of skeletal muscle fibres, being activated to enter proliferation and generate myoblasts that either fuse to existing myofibres, or fuse together for de novo myofibre formation. Isolating muscle fibres allows the associated satellite cells to be obtained while remaining in their anatomical niche beneath the basal lamina, free of interstitial and vascular tissue. Myofibres can then be immunostained to examine gene expression in quiescent satellite cells, or cultured to activate satellite cells before immunostaining to investigate gene expression dynamics during myogenic progression and self-renewal. Here, we describe methods for the isolation, culture and immunostaining of muscle fibres for examining satellite cell biology.

Key words

Satellite cell Stem cell Skeletal muscle Muscle fibre Myofibre Culture Immunostaining Self-renewal 

Notes

Acknowledgements

We would like to thank Farah Patell for the confocal image of a satellite cell (Fig. 2e). Louise Moyle is supported by a Muscular Dystrophy Campaign PhD studentship (RA4/817). The laboratory of Pete Zammit is currently also supported by the Medical Research Council (G1100193), and Association Française Contre les Myopathies (SB/CP/2012-0218/15814 and SB/CF/2012-0910), together with OPTISTEM (223098) and BIODESIGN (262948-2) from the European Commission 7th Framework Programme.

References

  1. 1.
    Janssen I et al (2000) Skeletal muscle mass and distribution in 468 men and women aged 18–88yr. J Appl Physiol 89(1):81–88PubMedGoogle Scholar
  2. 2.
    Zammit PS et al (2002) Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Exp Cell Res 281(1):39–49PubMedCrossRefGoogle Scholar
  3. 3.
    Luz MA, Marques MJ, Santo Neto H (2002) Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells. Braz J Med Biol Res 35(6): 691–695Google Scholar
  4. 4.
    Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84(1):209–238PubMedCrossRefGoogle Scholar
  5. 5.
    Studitsky AN (1964) Free auto- and homografts of muscle tissue in experiments on animals. Ann N Y Acad Sci 120:789–801PubMedCrossRefGoogle Scholar
  6. 6.
    Snow MH (1978) An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res 186(3):535–540PubMedCrossRefGoogle Scholar
  7. 7.
    Scharner J, Zammit PS (2011) The muscle satellite cell at 50: the formative years. Skelet Muscle 1(1):28Google Scholar
  8. 8.
    Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Collins CA et al (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122(2):289–301PubMedCrossRefGoogle Scholar
  10. 10.
    Zammit PS et al (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166(3):347–357PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Tedesco FS et al (2010) Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 120(1):11–19PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Peault B et al (2007) Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol Ther 15(5):867–877PubMedCrossRefGoogle Scholar
  13. 13.
    Dellavalle A et al (2011) Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells. Nat Commun 2:499PubMedCrossRefGoogle Scholar
  14. 14.
    Lepper C, Partridge TA, Fan CM (2011) An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Development 138(17):3639–3646PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    McCarthy JJ et al (2011) Effective fiber hypertrophy in satellite cell-depleted skeletal muscle. Development 138(17):3657–3666PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Murphy MM et al (2011) Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 138(17):3625–3637PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Sambasivan R et al (2011) Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 138(17):3647–3656PubMedCrossRefGoogle Scholar
  18. 18.
    Relaix F, Zammit PS (2012) Satellite cells are essential for skeletal muscle regeneration: the cell on the edge returns centre stage. Development 139(16):2845–2856PubMedCrossRefGoogle Scholar
  19. 19.
    Cardasis CA, Cooper GW (1975) An analysis of nuclear numbers in individual muscle fibers during differentiation and growth: a satellite cell-muscle fiber growth unit. J Exp Zool 191(3):347–358PubMedCrossRefGoogle Scholar
  20. 20.
    Cardasis CA, Cooper GW (1975) A method for the chemical isolation of individual muscle fibers and its application to a study of the effect of denervation on the number of nuclei per muscle fiber. J Exp Zool 191(3):333–346PubMedCrossRefGoogle Scholar
  21. 21.
    Bischoff R (1975) Regeneration of single skeletal muscle fibers in vitro. Anat Rec 182(2):215–235PubMedCrossRefGoogle Scholar
  22. 22.
    Konigsberg UR, Lipton BH, Konigsberg IR (1975) The regenerative response of single mature muscle fibers isolated in vitro. Dev Biol 45(2):260–275PubMedCrossRefGoogle Scholar
  23. 23.
    Kopriwa BM, Moss FP (1971) A radioautographic technique for whole mounts of muscle fibers. J Histochem Cytochem 19(1):51–55PubMedCrossRefGoogle Scholar
  24. 24.
    Bekoff A, Betz WJ (1977) Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. J Physiol 271(1):25–40PubMedCentralPubMedGoogle Scholar
  25. 25.
    Bekoff A, Betz W (1977) Properties of isolated adult rat muscle fibres maintained in tissue culture. J Physiol 271(2):537–547PubMedCentralPubMedGoogle Scholar
  26. 26.
    Bischoff R (1986) Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115(1):129–139PubMedCrossRefGoogle Scholar
  27. 27.
    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(2):588–603PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Rosenblatt JD et al (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31(10):773–779PubMedCrossRefGoogle Scholar
  29. 29.
    Rosenblatt JD, Parry DJ, Partridge TA (1996) Phenotype of adult mouse muscle myoblasts reflects their fiber type of origin. Differentiation 60(1):39–45PubMedCrossRefGoogle Scholar
  30. 30.
    Beauchamp JR et al (2000) Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 151(6):1221–1234PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Rosenblatt JD, Parry DJ (1992) Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J Appl Physiol 73(6):2538–2543PubMedGoogle Scholar
  32. 32.
    Collins CA, Zammit PS (2009) Isolation and grafting of single muscle fibres. Methods Mol Biol 482:319–330Google Scholar
  33. 33.
    Calhabeu F et al (2013) Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells. Oncogene 32(5):651–662Google Scholar
  34. 34.
    Seale P et al (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102(6):777–786PubMedCrossRefGoogle Scholar
  35. 35.
    Gnocchi VF et al (2009) Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells. PLoS One 4(4):e5205Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Randall Division of Cell and Molecular Biophysics, New Hunt’s HouseKing’s College LondonLondonUK

Personalised recommendations