Adult satellite cells are quiescent, but are poised for activation in response to exercise, injury, or disease allowing adult muscle growth or repair. Once activated, satellite cells proliferate extensively to produce enough myogenic progenitors in order to regenerate the muscles. In order to self-renew, a subset of activated satellite cells can resist the myogenic differentiation and return to quiescence to replenish the satellite cell pool. These cellular processes that normally occur during skeletal muscle regeneration can be recapitulated ex vivo using isolated and cultured myofibers. Here, we describe a protocol to isolate single myofibers from the extensor digitorum longus muscle. Moreover, we detail experimental conditions for analyzing satellite cells in quiescence and progression through the myogenic lineage.
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C.E.B. is supported by a Postdoctoral Fellowship from the Ontario Institute for Regenerative Medicine. Y.X.W. is supported by fellowships from the Queen Elizabeth II Graduate Scholarships in Science and Technology and the CIHR. M.A.R. holds a Canada Research Chair in Molecular Genetics. These studies were carried out with support of grants to M.A.R. from the US National Institutes for Health [R01AR044031], the Canadian Institutes for Health Research [MOP-12080, MOP-81288, FDN-148387], E-Rare-2: Canadian Institutes of Health Research/Muscular Dystrophy Canada [ERA-132935], the Muscular Dystrophy Association, and the Stem Cell Network.
1.Regenerative Medicine Program, Department of Cellular and Molecular Medicine, Faculty of Medicine, Sprott Center for Stem Cell Research, Ottawa Hospital Research InstituteUniversity of OttawaOttawaCanada