Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System

Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 798:21-52. DOI: 10.1007/978-1-61779-343-1_2
Source: PubMed


Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. Although emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells.

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    • "For the first strategy, muscle fibers were isolated from soleus and flexor digitorum brevis muscles by incubation in collagenase and subsequent trituration as described above and incubated until the satellite cells appeared in the dishes. For the second strategy satellite cells were isolated enzymatically according to the protocol of Yablonka-Reuveni [38] with minor changes (Figure 1). In brief, isolated muscles were placed directly into enzyme solution, without any additional mechanical disruption with scissors. "
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    ABSTRACT: Muscular dystrophies caused by defects in various genes are often associated with impairment of calcium homeostasis. Studies of calcium currents are hampered because of the lack of a robust cellular model. Primary murine myotubes, formed upon satellite cell fusion, were examined for their utilization as a model of adult skeletal muscle. We enzymatically isolated satellite cells and induced them to differentiation to myotubes. Myotubes displayed morphological and physiological properties resembling adult muscle fibers. Desmin and myosin heavy chain immunoreactivity in the differentiated myotubes were similar to the mature muscle cross-striated pattern. The myotubes responded to electrical and chemical stimulations with sarcoplasmic reticulum calcium release. Presence of L-type calcium channels in the myotubes sarcolemma was confirmed via whole-cell patch-clamp technique. To assess the use of myotubes for studying functional mutation effects lentiviral transduction was applied. Satellite cells easily underwent transduction and were able to retain a positive expression of lentivirally encoded GFP up to and after the formation of myotubes, without changes in their physiological and morphological properties. Thus, we conclude that murine myotubes may serve as a fruitful cell model for investigating calcium homeostasis in muscular dystrophy and the effects of gene modifications can be assessed due to lentiviral transduction.
    09/2015; 2015(2):594751. DOI:10.1155/2015/594751
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    • "Skeletal muscle myoblasts isolation, myotube differentiation, and oxygen consumption analysis Myoblasts were isolated from two pairs of young control and mutant mice (aged 6 months), and three pairs of age-matched old control and mutant mice (aged 22, 25 and 29 months), following the procedures described previously [27] but with modifications. The proliferation medium contained Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum, 10% horse serum, 1% chicken embryo extract, and 5 ng/ml bFGF, and the differentiation medium contained 10% horse serum without bFGF. "
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    ABSTRACT: Inner mitochondrial membrane peptidase 2-like (IMMP2L) protein is a mitochondrial inner membrane peptidase that cleaves the signal peptide sequences of cytochrome c1 (CYC1) and mitochondrial glycerol phosphate dehydrogenase (GPD2). Immp2l mutant mice show infertility and early signs of aging. It is unclear whether mitochondrial respiratory deficiency underlies this phenotype. Here we show that the intermediate forms of GPD2 and CYC1 have normal expression levels and enzymatic function in Immp2l mutants. Mitochondrial respiration is not diminished in isolated mitochondria and cells from mutant mice. Our data suggest that respiratory deficiency is not the cause of the observed Immp2l mutant phenotypes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
    08/2014; 2. DOI:10.1016/j.redox.2014.08.006
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    • "This is done, for example, by seeding a heterogeneous cell suspension on a common un-functionalized glass or plastic substrates. Under these conditions some cell types adhere to the bare substrate while others remain in suspension for a longer period of time (see for Danoviz and Yablonka-Reuveni, 2012). The selective cells-adhesivity reflects differences in the nature and rates of ECM secretion and CAM expression. "
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    ABSTRACT: Using a variety of proliferating cell types, it was shown that the surface of nanocrystalline diamond (NCD) provides a permissive substrate for cell adhesion and development without the need of complex chemical functionalization prior to cell seeding. In an extensive series of experiments we found that, unlike proliferating cells, post-mitotic primary neurons do not adhere to bare NCD surfaces when cultured in defined medium. These observations raise questions on the potential use of bare NCD as an interfacing layer for neuronal devices. Nevertheless, we also found that classical chemical functionalization methods render the "hostile" bare NCD surfaces with adhesive properties that match those of classically functionalized substrates used extensively in biomedical research and applications. Based on the results, we propose a mechanism that accounts for the conflicting results; which on one hand claim that un-functionalized NCD provides a permissive substrate for cell adhesion and growth, while other reports demonstrate the opposite.
    Frontiers in Neuroengineering 06/2014; 7. DOI:10.3389/fneng.2014.00017
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