Skeletal muscle cell populations. Separation and partial characterization of fibroblast-like cells from embryonic tissue using density centrifugation

Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll density centrifugation. Isolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle- and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flattened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.     

Isolation and clonal analysis of satellite cells from chicken pectoralis muscle

Satellite cells, liberated from the breast muscle of young adult chickens by sequential treatment with collagenase and trypsin, were fractionated by Percoll density centrifugation to remove myofibril fragments and cell debris which otherwise heavily contaminate the preparation. This procedure allowed direct measurements of cell yields (1.5-4 X 10(5) cells/g tissue), plating efficiencies (27-40%) and identification of single cells in culture. In mass cultures, satellite cells gave rise to myotubes on the fifth day, and the progeny of these cells were sequentially passaged several times without losing myogenic traits. In clonal studies, over 90% of the satellite cells gave rise to large clones of which more than 99% were myogenic as demonstrated by the appearance of myotubes. The results obtained with satellite cells differ from observations made using embryonic muscle cell preparation from chicks. In the embryonic system massive formation of myotubes was observed following the third day of culture; sequential subculturing led to overgrowth of fibroblast-like cells following the first passage; and cells gave rise to both small myogenic clones (up to 16 terminally differentiated cells per clone) and non-myogenic clones in addition to large myogenic clones. We conclude that the isolated satellite cells represent a homogeneous cell population and reside in a stem cell compartment.     

Discrimination of myogenic and nonmyogenic cells from embryonic skeletal muscle by 90 degrees light scattering

A myogenic cell suspension was isolated from the breast muscles of 10-day-old chicken embryos by trypsin digestion. The cell preparation was subjected to Percoll density centrifugation to reduce the number of fibroblast-like cells present. The Percoll-isolated, predominantly myogenic cell population was then fractionated by flow cytometry using 90 degrees light scattering as the parameter for sorting. Cells exhibiting lower scatter, with a peak of 45 units, produced cultures containing myotubes and gave rise only to myogenic clones. Cells exhibiting higher scatter (120-200 units) produced nonmyogenic cultures and gave rise to nonmyogenic clones. Cells with intermediate light scatter were also detected. The latter produced both myogenic and nonmyogenic clones. The differences in light scatter presumably reflect higher cytoplasmic complexity of the nonmyogenic cells compared with the myogenic cells. Moreover, the differences in light scattering properties of the different cell types offer a means for the isolation of pure populations of myogenic cells directly from the intact muscle.     

CD90-positive cells, an additional cell population, produce laminin alpha2 upon transplantation to dy(3k)/dy(3k) mice

Laminin alpha2 is a component of skeletal and cardiac muscle basal lamina. A defect of the laminin alpha2 chain leads to severe congenital muscular dystrophy (MDC1A) in humans and dy/dy mice. Myogenic cells including myoblasts, myotubes, and myofibers in skeletal muscle are a possible source of the laminin alpha2 chain, and myogenic cells are thus proposed as a cell source for congenital muscular dystrophy therapy. However, we observed production of laminin alpha2 in non-myogenic cells of normal mice, and we could enrich these laminin alpha2-producing cells in CD90(+) cell fractions. Intriguingly, the number of CD90(+) cells increased dramatically during skeletal muscle regeneration in mice. This fraction did not include myogenic cells but exhibited a fibroblast-like phenotype. Moreover, these cells were resident in skeletal muscle, not derived from bone marrow. Finally, the production of laminin alpha2 in CD90(+) cells was not dependent on fusion with myogenic cells. Thus, CD90(+) cells are a newly identified additional cell fraction that increased during skeletal muscle regeneration in vivo and could be another cell source for therapy for lama2-deficient muscular dystrophy.     

Biochemical and morphological differences between fibroblasts and myoblasts from embryonic chicken skeletal muscle

Summary Non-myogenic cells were isolated from the breast muscle of 10-day-old chicken embryos employing Percoll density centrifugation. In culture, these cells exhibited the spread out, stellate morphology of fibroblast-like cells. They also exhibited receptor-mediated binding of plateletderived growth factor (PDGF). Such binding was not detected in cultures of predominantly myogenic cells isolated by the Percoll density centrifugation from the same muscle. Percoll-isolated myogenic and fibrogenic cell populations were also analyzed by two-dimensional polyacrylamide gel electrophoresis immediately after removal from the muscle. This analysis revealed at least six polypeptides specific to the fibroblasts but not detected in the myogenic cell population. In addition, at least eight polypeptides found in the myogenic population were barely detectable, or lacking altogether from the fibroblast-like cells. Ultrastructural analysis of the freshly isolated cells demonstrated that the fibroblasts were larger than the myoblasts and that their cytoplasm contained many vesicles. We conclude that the fibrogenic and myogenic cells isolated by Percoll from embryonic muscle express cell type-specific characteristics. Moreover, based on the PDGF binding studies, the fibrogenic cells can be categorized as true fibroblasts.     

Differential response among cells in the chick embryo myogenic lineage to photosensitization by Merocyanine 540

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.     

Developmental regulation of cell-surface glycoproteins in clonal cultures of human skeletal muscle satellite cells

Pure populations of myogenic cells were obtained by cloning satellite cells from human skeletal muscle biopsies. Cell-surface glycoproteins at various stages of myogenesis were analysed by one- and two-dimensional gel electrophoresis. A total of 14 distinct proteins were detectable at the cell surface, on the basis of their susceptibility to desialation by exogenous neuraminidase or their iodination by exogenous lactoperoxidase. Reproducible changes in lectin binding or iodination of eight of these proteins occurred during myogenesis. Only two of the developmentally regulated proteins were components of the detergent-insoluble extracellular matrix fraction. Developmental regulation of these two proteins was unaffected by growth of cultures in 5-bromo-2'-deoxyuridine to inhibit myogenesis. In contrast, developmental regulation of the other cell-surface proteins was inhibited by growth in 5-bromo-2'-deoxyuridine, suggesting that changes in these proteins are tightly coupled to satellite cell differentiation. These studies represent the first systematic analysis of the surface proteins of pure, clonally derived, primary cultures of normal myogenic cells.     

Muscle-bound primordial stem cells give rise to myofiber-associated myogenic and non-myogenic progenitors

Myofiber cultures give rise to myogenic as well as to non-myogenic cells. Whether these myofiber-associated non-myogenic cells develop from resident stem cells that possess mesenchymal plasticity or from other stem cells such as mesenchymal stem cells (MSCs) remain unsolved. To address this question, we applied a method for reconstructing cell lineage trees from somatic mutations to MSCs and myogenic and non-myogenic cells from individual myofibers that were cultured at clonal density.Our analyses show that (i) in addition to myogenic progenitors, myofibers also harbor non-myogenic progenitors of a distinct, yet close, lineage; (ii) myofiber-associated non-myogenic and myogenic cells share the same muscle-bound primordial stem cells of a lineage distinct from bone marrow MSCs; (iii) these muscle-bound primordial stem-cells first part to individual muscles and then differentiate into myogenic and non-myogenic stem cells.     

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD^iCre/+;R26R^EYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.     

Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.     

Paracrine control of myoblast proliferation and differentiation by fibroblasts

Double-muscling (DM) is a hereditary (apparently single-gene) skeletal muscle hyperplasia which occurs in beef cattle. In order to investigate the cellular basis of this phenotype, cell cultures from developing muscle tissue of normal and DM fetal calves were studied. In cultures composed of both myogenic cells and nonmyogenic, fibroblast-like cells, DM myoblasts exhibited a prolonged proliferative phase. This resulted in delayed, but increased production of fused myotubes in the DM cultures. "Conditioned" media experiments indicated that the fibroblast-like cells in the cultures produced soluble myoblast growth factor activity. Both normal and DM fibroblast-like cells produced the growth factor activity, but the mutant fibroblast-like cells produced a greater level of such activity. The conditioned media failed to increase proliferation of bovine muscle fibroblasts and did not stimulate quiescent Swiss 3T3 cells to divide, indicating that the myoblast trophic activity is distinct from bFGF or PDGF. Also, the myotrophic activity present in the conditioned media acted in an additive fashion with saturating doses of bFGF and of IGF-1, suggesting that the activity is not due to either of these known myogenic growth factors. Both normal and DM fibroblast-like cells produced myoblast trophic activity when the cells were proliferating, but did not produce myotrophic activity when the fibroblasts were mitotically quiescent. These findings indicate that the proliferative state of the connective tissue cells in muscle may have a controlling influence on myoblast proliferation and differentiation during development.     

Synthesis of type IV collagen and laminin in cultures of skeletal muscle cells and their assembly on the surface of myotubes

The synthesis of two components of the basal lamina, laminin and type IV collagen, and their extracellular deposition on the surface of myotubes was studied in cultures of embryonic mouse and quail skeletal muscle cells and in the rat myoblast cell line L6. Production of type IV collagen and laminin by myoblasts and muscle fibroblasts was demonstrated by incorporation of radioactive amino acids into proteins and by immunoprecipitation with specific antibodies and electrophoretic analysis of labeled proteins. Immunofluorescence staining experiments revealed strong intracellular reactions with antibodies to laminin and type IV collagen in mononucleated myogenic and fibrogenic cells. Cells of fibroblast-like morphology showed a more intense staining than bipolar, spindle-shaped cells which perhaps represented postmitotic myoblasts. Myotubes did not show detectable intracellular staining. The formation of a basal lamina on myotubes was indicated by the deposition of laminin and type IV collagen on the surface of myotubes as viewed by immunofluorescence examination of unfixed cells. Staining for extracellular laminin was stronger in mass cultures than in myogenic clones, suggesting that secretion and deposition of components of the basal lamina on the myotube surface are complex processes which may involve cooperation between myogenic and fibrogenic cells.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

Analysis of myogenesis by somatic cell hybridization : I. Myogenic competence of homotypic hybrids derived from rat L6 myoblasts

Several authors have described the extinction of myogenic competence in hybrids produced by fusion of myogenic and non-myogenic cells. Interpretations of such experiments rest upon the assumption that extinction does not occur with any appreciable frequency as a non-specific consequence of the cell hybridization process itself. In order to test this assumption we have analyzed the myogenic competence of over 140 independent homotypic hybrid clones produced by PEG-mediated fusion of rat L6 myoblasts. Based upon an evaluation of myotube formation in hybrid colonies, we demonstrate that 99% of primary hybrid clones are myogenic. The fact that 97% of secondary hybrid colonies also differentiate indicates that myogenic competence is a stable characteristic of the hybrids. Four hybrid clones were isolated and expanded for analyses of chromosome numbers, myotube formation, creatine kinase activities, and microfluorimetric DNA determinations of myotube nuclei. Our results demonstrate that polyploid homotypic hybrid cells produced by fusion of non-neoplastic, developmentally determined rat myoblasts retain and express their program of differentiation. This work provides a foundation for future studies which will investigate the expression of myogenic properties in hybrids between myogenic and non-myogenic cells.     

Invited review: mesenchymal progenitor cells in intramuscular connective tissue development

The abundance and cross-linking of intramuscular connective tissue contributes to the background toughness of meat, and is thus undesirable. Connective tissue is mainly synthesized by intramuscular fibroblasts. Myocytes, adipocytes and fibroblasts are derived from a common pool of progenitor cells during the early embryonic development. It appears that multipotent mesenchymal stem cells first diverge into either myogenic or non-myogenic lineages; non-myogenic mesenchymal progenitors then develop into the stromal-vascular fraction of skeletal muscle wherein adipocytes, fibroblasts and derived mesenchymal progenitors reside. Because non-myogenic mesenchymal progenitors mainly undergo adipogenic or fibrogenic differentiation during muscle development, strengthening progenitor proliferation enhances the potential for both intramuscular adipogenesis and fibrogenesis, leading to the elevation of both marbling and connective tissue content in the resulting meat product. Furthermore, given the bipotent developmental potential of progenitor cells, enhancing their conversion to adipogenesis reduces fibrogenesis, which likely results in the overall improvement of marbling (more intramuscular adipocytes) and tenderness (less connective tissue) of meat. Fibrogenesis is mainly regulated by the transforming growth factor (TGF) β signaling pathway and its regulatory cascade. In addition, extracellular matrix, a part of the intramuscular connective tissue, provides a niche environment for regulating myogenic differentiation of satellite cells and muscle growth. Despite rapid progress, many questions remain in the role of extracellular matrix on muscle development, and factors determining the early differentiation of myogenic, adipogenic and fibrogenic cells, which warrant further studies.     

A contemporary atlas of the mouse diaphragm: myogenicity, vascularity, and the pax3 connection

The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this "contemporary atlas" can be further explored in the context of diaphragm pathology and genetic disorders.     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

Myogenic specification of side population cells in skeletal muscle

Skeletal muscle contains myogenic progenitors called satellite cells and muscle-derived stem cells that have been suggested to be pluripotent. We further investigated the differentiation potential of muscle-derived stem cells and satellite cells to elucidate relationships between these two populations of cells. FACS(R) analysis of muscle side population (SP) cells, a fraction of muscle-derived stem cells, revealed expression of hematopoietic stem cell marker Sca-1 but did not reveal expression of any satellite cell markers. Muscle SP cells were greatly enriched for cells competent to form hematopoietic colonies. Moreover, muscle SP cells with hematopoietic potential were CD45 positive. However, muscle SP cells did not differentiate into myocytes in vitro. By contrast, satellite cells gave rise to myocytes but did not express Sca-1 or CD45 and never formed hematopoietic colonies. Importantly, muscle SP cells exhibited the potential to give rise to both myocytes and satellite cells after intramuscular transplantation. In addition, muscle SP cells underwent myogenic specification after co-culture with myoblasts. Co-culture with myoblasts or forced expression of MyoD also induced muscle differentiation of muscle SP cells prepared from mice lacking Pax7 gene, an essential gene for satellite cell development. Therefore, these data document that satellite cells and muscle-derived stem cells represent distinct populations and demonstrate that muscle-derived stem cells have the potential to give rise to myogenic cells via a myocyte-mediated inductive interaction.