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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Janssen I et al (2000) Skeletal muscle mass and distribution in 468 men and women aged 18–88yr. J Appl Physiol 89(1):81–88
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):39–49
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): 691–695
Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration. Physiol Rev 84(1):209–238
Studitsky AN (1964) Free auto- and homografts of muscle tissue in experiments on animals. Ann N Y Acad Sci 120:789–801
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):535–540
Scharner J, Zammit PS (2011) The muscle satellite cell at 50: the formative years. Skelet Muscle 1(1):28
Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495
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):289–301
Zammit PS et al (2004) Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 166(3):347–357
Tedesco FS et al (2010) Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells. J Clin Invest 120(1):11–19
Peault B et al (2007) Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol Ther 15(5):867–877
Dellavalle A et al (2011) Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells. Nat Commun 2:499
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):3639–3646
McCarthy JJ et al (2011) Effective fiber hypertrophy in satellite cell-depleted skeletal muscle. Development 138(17):3657–3666
Murphy MM et al (2011) Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 138(17):3625–3637
Sambasivan R et al (2011) Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 138(17):3647–3656
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
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):347–358
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):333–346
Bischoff R (1975) Regeneration of single skeletal muscle fibers in vitro. Anat Rec 182(2):215–235
Konigsberg UR, Lipton BH, Konigsberg IR (1975) The regenerative response of single mature muscle fibers isolated in vitro. Dev Biol 45(2):260–275
Kopriwa BM, Moss FP (1971) A radioautographic technique for whole mounts of muscle fibers. J Histochem Cytochem 19(1):51–55
Bekoff A, Betz WJ (1977) Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. J Physiol 271(1):25–40
Bekoff A, Betz W (1977) Properties of isolated adult rat muscle fibres maintained in tissue culture. J Physiol 271(2):537–547
Bischoff R (1986) Proliferation of muscle satellite cells on intact myofibers in culture. Dev Biol 115(1):129–139
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):588–603
Rosenblatt JD et al (1995) Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 31(10):773–779
Rosenblatt JD, Parry DJ, Partridge TA (1996) Phenotype of adult mouse muscle myoblasts reflects their fiber type of origin. Differentiation 60(1):39–45
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):1221–1234
Rosenblatt JD, Parry DJ (1992) Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J Appl Physiol 73(6):2538–2543
Collins CA, Zammit PS (2009) Isolation and grafting of single muscle fibres. Methods Mol Biol 482:319–330
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):651–662
Seale P et al (2000) Pax7 is required for the specification of myogenic satellite cells. Cell 102(6):777–786
Gnocchi VF et al (2009) Further characterisation of the molecular signature of quiescent and activated mouse muscle satellite cells. PLoS One 4(4):e5205
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Moyle, L.A., Zammit, P.S. (2014). Isolation, Culture and Immunostaining of Skeletal Muscle Fibres to Study Myogenic Progression in Satellite Cells. In: Kioussi, C. (eds) Stem Cells and Tissue Repair. Methods in Molecular Biology, vol 1210. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1435-7_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1435-7_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1434-0
Online ISBN: 978-1-4939-1435-7
eBook Packages: Springer Protocols