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

Department of Biological Structure, School of Medicine, University of Washington, Seattle, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2005; 290:281-304. DOI: 10.1007/978-1-62703-128-8_28
Source: PubMed


Myofibers are the functional contractile units of skeletal muscle. Mononuclear satellite cells located between the basal lamina and the plasmalemma of the myofiber are the primary source of myogenic precursor cells in postnatal muscle. This chapter describes protocols used in our laboratory for isolation, culturing, and immunostaining of single myofibers from mouse skeletal muscle. The isolated myofibers are intact and retain their associated satellite cells underneath the basal lamina. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. Myofibers are cultured in dishes coated with Vitrogen collagen, and 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). Different from the FDB myofibers, the longer EDL myofibers tend to tangle and break when cultured together; therefore, EDL myofibers are cultured individually. These myofibers are cultured in dishes coated with Matrigel. The satellite cells initially remain associated with the myofiber and later migrate away to its vicinity, resulting in extensive cell proliferation and differentiation. These protocols allow studies on the interplay between the myofiber and its associated satellite cells.

    • "Given that the muscle tissue is directly minced, the result is a myoblast culture containing of a variety of cell types, such as fibroblasts and adipocytes. Alternatively, purer myoblast cultures can be achieved through the isolation of live myofibers (Keire, Shearer, Shefer, & Yablonka-Reuveni, 2013), to which satellite cells are still attached. In culture, the satellite cells can be allowed to migrate out from the fiber, or alternatively, the myofibers can be grown in floating conditions, allowing satellite cells to activate and proliferate on the surface of the myofibers in a more natural environment. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Current research utilizes the specific expression pattern of intermediate filaments (IF) for identifying cellular state and origin, as well as for the purpose of disease diagnosis. Nestin is commonly utilized as a specific marker and driver for CNS progenitor cell types, but in addition, nestin can be found in several mesenchymal progenitor cells, and it is constitutively expressed in a few restricted locations, such as muscle neuromuscular junctions and kidney podocytes. Alike most other members of the IF protein family, nestin filaments are dynamic, constantly being remodeled through posttranslational modifications, which alter the solubility, protein levels, and signaling capacity of the nestin filaments. Through its interactions with kinases and other signaling executors, resulting in a complex and bidirectional regulation of cell signaling events, nestin has the potential to determine whether cells divide, differentiate, migrate, or stay in place. In this review, the broad and similar roles of IFs as dynamic signaling scaffolds, is exemplified by observations of nestin functions and its interaction with the cyclin- dependent kinase 5, the atypical kinase in the family of cyclin-dependent kinases.
    No preview · Article · Nov 2015
  • Source
    • "First, in ex vivo culturing experiments, we did not observe significant changes in domain size or dissipation of the mito-Dendra2 signal over the course of 72 hr (Figures S4A and S4B). Thus, mitochondrial domains are stable under standard culturing conditions that maintain myofibers in a healthy and functional state (Casas et al., 2010;Eisner et al., 2014;Keire et al., 2013). Second, we observed a significant increase in the frequency of mitochondrial domains in aged animals, suggesting that domains are stably accumulating over time (Figures S4C and S4D). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Skeletal muscle fibers differentiate into specific fiber types with distinct metabolic properties determined by their reliance on oxidative phosphorylation (OXPHOS). Using in vivo approaches, we find that OXPHOS-dependent fibers, compared to glycolytic fibers, contain elongated mitochondrial networks with higher fusion rates that are dependent on the mitofusins Mfn1 and Mfn2. Switching of a glycolytic fiber to an oxidative IIA type is associated with elongation of mitochondria, suggesting that mitochondrial fusion is linked to metabolic state. Furthermore, we reveal that mitochondrial proteins are compartmentalized to discrete domains centered around their nuclei of origin. The domain dimensions are dependent on fiber type and are regulated by the mitochondrial dynamics proteins Mfn1, Mfn2, and Mff. Our results indicate that mitochondrial dynamics is tailored to fiber type physiology and provides a rationale for the segmental defects characteristic of aged and diseased muscle fibers.
    Full-text · Article · Oct 2015 · Cell metabolism
  • Source
    • "In the first strategy, satellite cells were allowed to branch out of muscle fibers and attach to the dish bottom. In the second strategy, satellite cells were isolated as a " pure " culture by enzymatic dissociation of muscle fibers [28] [36] [37]. 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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Sep 2015
Show more