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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. While 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.
This chapter aims to provide simple protocols for the isolation, culture and analysis of satellite cells from adult skeletal muscle. We first detail background information about satellite cells (Subheadings 1.1. through 1.3.) and the range of cell isolation approaches developed over the years by us and others to analyze satellite cells (Subheading 1.4.). We then introduce an overview of our basic satellite cell isolation and culture protocol (Subheading 2.) followed by practical details (starting with Subheading 3.). We provide what we consider the simplest protocol that can be performed in any basic tissue culture laboratory, and in Subheading 1.4. we briefly discuss alternative approaches to purifying satellite cells. The basic approach provided in this chapter is an excellent means for analysis of satellite cells in culture when extreme purity is not needed. With careful attention to minimize connective tissue contribution, our standard protocol can yield cultures that are 80– 95% pure based on staining for protein markers Pax7 and MyoD on culture day 4 (for additional details about these markers see Subheading 1.3.). Collectively, our simple protocol for satellite cell isolation and culture has allowed detailed analyses of tissue-dissociated satellite cells. Standardized protocols for the isolation and culture of satellite cells are essential tools to enhance our understanding of cell autonomous and extrinsic factors that regulate their performance.
The functional units responsible for skeletal muscle contraction are cylindrical, multinucleated muscle fibers (myofibers). These contractile structures are established during embryogenesis, when mononuclear cells known as myoblasts fuse into immature muscle fibers or myotubes. Myonuclei (the myofiber nuclei) are postmitotic and under normal conditions cannot re-enter a proliferative state to contribute additional nuclei. During postnatal life, myofiber growth, homeostasis and repair rely on a population of mononuclear myogenic cells known as satellite cells (1–3). Satellite cells were initially described fifty years ago by their anatomical location on the surface of muscle fibers, between the myofiber plasmalemma and the basal lamina (4, 5) (for a schematic and electron microscope image see Fig. 1). However, the ultimate experimental proof that satellite cells are indeed myogenic progenitors has only been obtained by showing that cells derived from isolated myofibers produce myogenic progeny, able to proliferate, differentiate and self-renew in vitro and in vivo (6–13).
Satellite cells were initially described using electron microscopy (4, 5, 14, 15). More recent methods facilitate monitoring these cells by light microscopy based on expression of a range of specific markers that can be detected by immunostaining (16, 17). In particular, specific expression of the paired box transcription factor Pax7 and availability of an excellent antibody for immunodetection of this protein provides a uniform means to identify satellite cells in their native position in a range of species including mouse (10, 12, 13, 18, 19), rat (20), chicken (21, 22), and human (23, 24).
Additionally, genetically manipulated reporter mice permit direct detection of satellite cells based on specific expression of a fluorophore or β-galactosidase (β-gal) (13, 17, 19, 25, 26). We demonstrated that transgenic expression of GFP under the control of nestin regulatory elements (NES-GFP) allows detection of satellite cells in freshly isolated myofibers. NES-GFP mice also facilitate isolation of satellite cells using fluorescent-activated cell sorting (FACS) and subsequent studies of purified populations (13, 19). The Myf5nLacZ/+ mouse has also provided a means to identify satellite cells in intact muscle and isolated myofibers (2, 11, 19, 26, 27). In this mouse, one of the Myf5 alleles was modified to direct lacZ expression, resulting in β-gal expression in satellite cells as originally reported by Beauchamp and colleagues (26). We frequently use crosses of NES-GFP with Myf5nLacZ/+ mice, allowing the detection of satellite cells by means of direct fluorescence and X-gal staining (19).
Satellite cells are considered the major, if not only, source of myogenic progeny in adult muscle (2, 3). Other cell types isolated from skeletal muscle, such as mesoangioblasts, pericytes, and myoendothelial cells also seem to have some myogenic potency (28–30), but whether these cell types participate in normal muscle maintenance and repair remains unclear. The isolation of the latter cell types require special enrichment approaches and these cells do not appear to contribute to our myogenic preparations. The majority of cells in our standard preparations of freshly isolated myogenic progenitors display the satellite cell phenotype; i.e., preparations from Myf5nLacZ/+/NES-GFP mice are enriched with Pax7+/β-gal+/GFP+ cells (shown by cytospin and mRNA expression analyses of freshly isolated cells). Hence, we refer to our freshly isolated cells prepared by the basic approach detailed herein as preparations of satellite cells or myogenic progenitors. Once satellite cells are cultured and proliferate, the resulting cells are referred to as myogenic progeny.
In the juvenile growth phase, when muscles enlarge, satellite cells are proliferative and add nuclei to growing myofibers (21, 31–34). In most adult muscles, satellite cells are typically quiescent until their activation is invoked by muscle injury (1, 35–37). Subtle injuries may lead to minimal proliferation of activated satellite cells whereas major trauma can recruit greater numbers of satellite cells and promote prolonged proliferation prior to differentiation. As small myofiber injuries can occur routinely during daily activity, a mechanism for repair is essential for muscle maintenance throughout life.
Activation of myogenic precursors is controlled by proximal signals from the muscle niche, microvasculature and from inflammatory cells (38–41). Systemic factors may also regulate satellite cell activation (42–44). Following their activation, satellite cells may contribute to repair of damaged myofibers and also generate new myofibers following cell division and fusion of myoblast progeny. Satellite cell behavior is under stringent regulatory control in order to balance various actively maintained states, including quiescence, entry into proliferation and continuity of the cell cycle, and terminal differentiation (45, 46). Furthermore, apart from their ability to fortify myofibers and contribute to muscle regeneration, satellite cells have the capacity to replenish a reserve pool and self-renew, qualifying them as tissue-specific stem cells (11, 47). It is not known, however, to what extent individual satellite cells differ with regard to their amplification and renewal potential (19, 47).
During early growth, muscle satellite cells may represent about 30% of the nuclei, whereas in the healthy adult satellite cells represent approximately 2–7% of nuclei within skeletal muscle (1, 21). The number of satellite cells per myofiber or per cross-sectional area may vary immensely between muscles. For example, the fast twitch extensor digitorum longus (EDL) contains fewer satellite cells compared to the slow twitch soleus (1, 12, 48). Additionally, myofiber ends may have a higher concentration of satellite cells than the rest of the myofiber (22). There are also reports of an age-associated decline in satellite cell number, where the presence and extent of decline may vary by muscle (12, 19, 20, 49)). Satellite cell performance may also decline in the aging environment, a possible contributory factor to age-associated muscle deterioration, also known as sarcopenia (20, 50). However, additional studies suggest that initial performance of skeletal muscle progenitors is delayed, but not necessarily impaired in old age and that factors beyond satellite cell activity alone may play a role in reducing muscle repair in old age (44, 51). Indeed, satellite cell activity can be rejuvenated upon exposure of old muscle to a juvenile environment by cross-transplantation or by parabiosis of young and old mice (42, 52). Muscle wasting associated with muscular dystrophy is also thought to lead to exhaustion of satellite cells due to the continuous demand for reparative myogenic cells (53–55). Overall, satellite cells are vital to skeletal muscle homeostasis and regeneration throughout life, and understanding the regulation of myogenic stem cells will likely provide valuable insights into muscle wasting in aging and disease.
At the molecular level, myogenesis of satellite cells is highly orchestrated to ensure that specific genes are regulated in a temporally organized manner according to genetic blueprints, cell cycle requirements, and environmental factors. The resulting pattern of gene expression yields terminally differentiated myoblasts, capable of adding myonuclei to existing myofibers in addition to fusing together to form new myofibers during muscle growth and repair (3, 45, 56, 57). To monitor various stages of satellite cell myogenesis in culture, we focus primarily on the expression patterns of Pax7 and the myogenic regulatory factors MyoD, myogenin and Myf5. As demonstrated in our published studies, the temporal expression patterns of these genes do not vary for mouse, rat, or chicken satellite cell progeny. For additional background information about the functional roles of Pax7 and the myogenic regulatory factors in myogenesis the reader should refer to additional publications (e.g., (58–60); for a comprehensive review see (3)).
Satellite cell progeny can be distinguished from their quiescent progenitors based on distinctive gene expression patterns (2, 3, 57). In particular, expressions of MyoD and myogenin have been used extensively in conjunction with Pax7 (8, 10, 12, 46) (Fig. 2). Proliferating progeny (myoblasts) continue to express Pax7, but distinctly from their quiescent progenitors, also express MyoD. A decline in Pax7 along with the induction of the muscle-specific transcription factor myogenin marks myoblasts that have entered the differentiation phase and initiated cell cycle withdrawal. Coinciding with or soon after the upregulation of myogenin, differentiating myoblasts initiate expression of various genes encoding structural proteins, such as sarcomeric myosin, and fuse into myotubes (12, 21, 39, 61). During myoblast differentiation, a subpopulation of mononucleated cells downregulate MyoD expression and exit the cell cycle, but maintain Pax7 expression. These cells define a reserve population that presumably reflects satellite cell self-renewal (10–12, 19, 46, 47, 57).
Both quiescent and proliferating satellite cells also express the myogenic regulatory factor Myf5 as determined by mRNA analysis (13, 19, 62). Myf5 promoter activity can also be observed through β-gal detection in satellite cells and their proliferating progeny in myogenic cultures from the aforementioned Myf5nlacZ/+ mice (19, 26). However, detection of the Myf5 protein has not been reported in quiescent satellite cells, though proliferating progeny do express Myf5 protein (46, 63). Thus, it is possible that while the Myf5 promoter is active in quiescent satellite cells, Myf5 protein is not produced until cells begin to proliferate. Ultimately, Myf5 expression declines when myoblasts enter differentiation, while MyoD expression persists well into the differentiation stage when satellite cells are maintained in our standard culture conditions (3, 12, 46).
Much of our understanding of satellite cell biology has arisen from cell culture studies. The information provided in this section focuses on primary cultures of bona fide satellite cells. Studies with myogenic cell lines (including rat L6 and L8, and mouse C2, C2C12 and MM14) have also permitted extensive biochemical and molecular analyses of aspects of myogenesis, though these models do not always fully adhere to the biology of satellite cells (64–68). A comprehensive description of myogenic cells lines from the American Tissue Culture Collection (ATCC) and other sources can be found in our recent review (3).
Two main cell culture approaches have been employed by us and other investigators in the study of bona fide satellite cells:
Alternatively, satellite cells can be enriched from whole muscle cell suspensions by various approaches that reduce the presence of fibroblastic cells, typically present to some degree in the preparation, and remove myofibril debris present in the initial cell suspension. Such approaches have included: (a) initial plating on uncoated tissue cultures dishes that results in separation of cells based on adhesion characteristics, where cells that remain in suspension after a short period are collected for culturing (i.e., differential plating) (73–75); (b) fractionation on Percoll density gradients (62, 76–78); c) cell sorting by forward and side scatter (79, 80).
In studies where further enrichment of satellite cells is warranted, cells can be isolated by FACS using antibodies that react with satellite cell surface antigens (47). First, cells are released from the muscle tissue using collagenase or collagenase-dispase, enzyme preparations that preserve cell surface antigens compared to Pronase or trypsin digestion methods. Studies from various laboratories (performed mainly with mouse tissue) have established that satellite cells can be isolated based on negative selection for CD45, CD31 and Sca1, and positive selection for CD34 and α7 integrin (25, 47, 81). Additional cell surface antigens, including CXCR4, β1 integrin, and syndecan-4 have also been used for isolation from adult muscle (82–84).
A range of fluorescence-based reporter systems in genetically manipulated mouse strains have also permitted reliable isolation of purified populations of satellite cells. For example, we have isolated satellite cells from different muscle groups of transgenic NES-GFP mice (13, 19), and Pax3- / Pax7-driven GFP reporter expression has also been used for isolation by FACS (25, 85), with the limitation that the Pax3 reporter is only expressed in satellite cells from selective muscles (25). Mice with a GFP reporter gene inserted into the Myf5 locus also permit isolation of myogenic cells by FACS (86–88); however, GFP expression is below detection level in many of the satellite cells, which reduces the usefulness of these Myf5GFP mice for satellite cell isolation by FACS.
Additionally, Cre-Lox mouse models are useful for isolating satellite cells and identifying their progeny. Fluorescent reporters can be permanently turned on in cells derived from myogenic progenitors upon expression of Cre-recombinase driven by promoters of myogenic genes such as Pax3, Myf5 and MyoD (89–92). When using such Cre-Lox mouse models to sort satellite cells, one should be careful to ensure that the reporter is not expressed in additional cell types during embryogenesis. For example, Myf5-Cre expression has been reported in nonmyogenic regions (93–95). It is also important to note that some head muscles (e.g., extraocular muscles) develop via Pax3-independent pathways and satellite cells in these muscles do not express the Pax3-Cre-driven reporter ((17, 96) and our unpublished studies).
In this chapter we describe the basic methodologies regularly used in our laboratory for the isolation, culture and characterization of myogenic progenitors from adult mouse skeletal muscle. As detailed in the previous section, we also use contemporary approaches for satellite cell isolation that are based on fluorescence reporter expression and/or based on expression of cell surface antigens. However, such approaches require the availability of special resources and reagents. Here, we describe a basic and straightforward method that we frequently use to isolate and characterize satellite cell performance in culture. This procedure can be performed in any tissue culture facility, using wildtype and mutant mouse muscles of various ages (12), and is suitable for satellite cell isolation from rat (62) and chicken (46) muscles. Fig. 3 shows representative micrographs of myogenic cultures emanating from satellite cells isolated using our basic procedure from adult mouse hindlimb muscles.
Our standard protocol for immuncytochemical analysis of satellite cell cultures provides quantitative insight into the “myogenicity” of the cell preparation (i.e., the presence and frequency of myogenic cells) and progression of satellite cell progeny from proliferation to differentiation and production of reserve cells. Table 1 summarizes the source and characteristics of a set of monoclonal antibodies used in our laboratory for the analysis of myogenesis in primary cultures of mouse satellite cells, which are also applicable to rat satellite cells (8, 12, 13, 19, 20, 62). For analysis of chicken satellite cells, we rely on the same Pax7 and MF20 antibodies as in Table 1, but for the detection of myogenic regulatory factors we use rabbit polyclonal antibodies developed against the chicken proteins (21, 46, 97).
In the following subheadings, we discuss some important considerations that should be taken in mind when establishing satellite cell primary cultures.
The protocols in this chapter focus on the isolation and culture of myogenic progenitors from adult (3–6 month-old) C57BL/6 mice. Aged mice and other mouse strains have also been used in our studies following the same procedures (12, 19). However, muscles from younger mice may contribute more cells due to age-associated decline in satellite cells in some muscles (12, 19). Also, the contribution of nonmyogenic cells in the preparation may increase with age or in different mouse strains, and consequently, some conditions may need modification (i.e. duration of enzymatic digestion, extent of tissue trituration, cell straining conditions to remove debris, centrifugation speed of harvested suspension, etc.) to minimize the proportion of undesired cell types.
Herein we detail our standard procedure for the isolation of satellite cells from hindlimb muscles of adult mice. For this preparation, we typically pool the fast twitch muscles tibialis anterior (TA) and gastrocnemius from both hindlimbs, using one mouse per preparation. For additional details about TA and gastrocnemius anatomy and isolation procedures see Notes 1 and 2. This approach can also be used for isolating myogenic progenitors from limb, body and head muscles. However, the contribution of connective tissue and vasculature may vary between muscles, and the tissue isolation procedure should be modified accordingly to minimize cells derived from such structures. The purity of the resultant preparation of isolated satellite cells (and cultures emanating from this preparation) is directly dependent on the amount of effort spent meticulously cleaning the muscle of these additional structures.
Our procedure is based on cell dissociation from whole muscle using Pronase digestion (see item 7 in Subheading 3.4. and items 4 and 13 in Subheading 4.1.). Pronase (available from Calbiochem) consists of a mixture of proteases isolated from the extracellular fluid of Streptomyces griseus. Due to its particular protease content, which includes several types of endo- and exopeptidases, Pronase has a broad activity (103, 104).
Pronase digestion may not be optimal for prospective satellite cell enrichment by antigen-based cell sorting because of extended digestion of surface antigens. However, myogenic cell preparations isolated by Pronase digestions show a lower level of nonmyogenic cells compared to that observed when collagenase or collagenase/dispase enzyme solutions are used. It is possible that certain nonmyogenic populations do not survive well after Pronase digestion and this may lead to the increased purity of these cultures.
Cell yields can vary depending on the age of the animal. Muscles from neonatal and young mice (1-month old or less) yield considerably more myogenic progenitors than muscles from adult mice. As mentioned before, variations are also observed when working with different muscles. For the mouse strain (C57BL/6) and hindlimb muscles (TA and gastrocnemius) used for the protocol described herein, each preparation typically yields 2–5 × 105 cells.
We commonly use 24-well or 35-mm culture dishes. We generally use 35-mm dishes for training or when performing single comparisons. In such cases, we initiate the cultures at 5–10 × 104 cells per plate. For multiple replicates across multiple time points, we use 24-well plates where starting cell density can be proportionally matched with that of the 35-mm plates based on surface area. Alternatively, seeding densities can be further reduced and depending on experimental goals, may range from 5 × 104 to 1 × 103 for primary cultures. While not further detailed below in the protocol section, it is noteworthy that we also use in some of our studies 48-well trays where we seed 2–10 cells per well; in such studies we aim to achieve clonal growth for monitoring progeny of individual satellite cells.
The standard growth medium used for our mouse satellite cell cultures consists of high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum, 10% horse serum and 1% chicken embryo extract (CEE). This serum-rich growth medium supports both proliferation and differentiation of myogenic cells (12). See Subheading 3.4. and Notes 3–6 for details about recommended cell culture reagents, our protocol for preselection of optimal sera lots and preparation of CEE, and final medium preparation.
Some variations can be found from laboratory to laboratory with regard to the basic culture media (e.g., Ham's F10 instead of DMEM, or a mixture of the two), serum type and concentration, and source of growth factors (e.g., purified growth factors, especially fibroblast growth factor, instead of CEE). Differences in culture conditions may explain some divergences in satellite cell behavior among different laboratories. For example, some published protocols rely on first using serum-rich growth medium that supports proliferation followed by a switch to serum-poor medium to support differentiation. There are also reported variations in medium composition when preparing cultures from other species. For example, for primary cultures of chicken satellite cells we typically use medium containing 10% horse serum and 5% chicken embryo extract (21, 46, 76, 97).
To study the effects of specific growth factors on myogenic cell performance, we typically maintain the cells for 3 days in our standard rich growth medium to allow for optimal cell adherence, then switch the cells into serum-low (e.g., DMEM containing 2% horse serum) or serum-deprived media. Prior to switching to serum-low medium, the cultures are rinsed extensively with DMEM to remove traces of the rich medium that otherwise adhere to the cell layer and reduce the observed effect of the additives being examined.
Adhesion of myogenic progenitors to cell culture dishes can be significantly improved by coating the plastic substrate with a variety of extracellular matrix constituents or derivatives. In addition to cell adhesion, matrix components can influence the extent of myogenic cell proliferation, differentiation and renewal (12, 13, 105, 106). In our laboratory, the main matrices used for coating tissue culture plates for satellite cell cultures are Matrigel and gelatin.
Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins. Its major component is laminin, followed by collagen IV, entactin, and heparan sulfate proteoglycan (107). Matrigel is available from BD Biosciences and can be obtained in its standard format or in its growth factor reduced format. In our studies we have typically used the growth factor reduced format. Matrigel must be carefully handled on ice when aliquoting and coating tissue culture dishes with dilutions. For additional details on Matrigel source and handling, see item 8 in Subheading 3.4. and Notes 7 and 8 for additional details.
Gelatin is produced by partial hydrolysis of type I collagen extracted from connective tissues. It can be purchased in a tissue culture grade powder form and easily reconstituted in water to the desired concentration. For specific details about gelatin source and our preparation of gelatin solution, see item 8 in Subheading 3.4. and Notes 9 and 10.
Gelatin is readily available, inexpensive and easy to use, which makes it an ideal product for training new team members and for use in standard cultures. However, long-term high-density myogenic cultures may spontaneously detach from plates coated with gelatin. In addition, satellite cell progeny typically demonstrate a more limited proliferative period, earlier differentiation, smaller myotubes and more meager development of reserve cells when grown on this substrate compared to Matrigel-coated dishes. Matrigel also allows a more even cell distribution upon initial cell plating compared to that observed when cells are seeded on gelatin-coated dishes. Additionally, when plated on Matrigel-coated dishes, myogenic progenitors can reach high cell densities and form complex myotube networks, typically without detaching from the substrate. The latter features have prompted us to use Matrigel especially when seeding cells at low density or when aiming to obtain single cell clones. Disadvantages of Matrigel include higher cost and the requirement for more careful handling. Other commercially available matrices that we have tested in pilot experiments that may provide reasonable alternatives include: (a) GelTrex (a Matrigel-like product from Invitrogen) and (b) Attachment Factor (Invitrogen), a ready made gelatin-based product.
For immunostaining analyses using the antibodies listed in Table 1, we typically fix the cultures with a paraformaldehyde-sucrose solution that is prepared in our laboratory. For further details about fixation approach and fixative composition, see Subheading 4.2. and Notes 11 and 12. It should be noted that fixatives should be optimized for preservation of both the cells and the antigens being analyzed. We perform all immunostaining steps in a manner that maintains sterility; handling antibodies strictly in the tissue culture hood minimizes possible bacterial contamination and helps maintain antibody stocks for years.
The following facilities are required for the cultures described in this chapter:
Unless otherwise stated, the following solutions are stored at 4°C and pre-warmed at room temperature before use.
We thank Lindsey Muir for reviewing this manuscript and providing valuable comments. We are also grateful to the granting agencies that funded this study. Our current research is supported by grants to Z.Y.R. from the National Institutes of Health (AG021566; AG035377; AR057794) and the Muscular Dystrophy Association (135908). M.E.D is supported by the Genetic Approaches to Aging Training Program (T32 AG000057). The development of the protocols described here could not be possible without early support to Z.Y.R from the American Heart Association, the USDA Cooperative State Research, Education and Extension Service, and the National Institutes of Health (AG013798).