Myogenic cell formation in regenerating rat skeletal muscle injured by mincing I. A fine structural study

The degenerative and early regenerative events following mincing and autotransplantation of rat skeletal muscle were examined at the ultrastructural level. During the first eight hours after injury, myonuclei undergo pyknosis, mitochondria become enlarged and vesiculated, myofilaments appear less distinct than normal and the sarcolemmata either disappear or become extensively fragmented. Further degeneration of the myofibers progresses slowly until macrophages and polymorphonuclear neutrophils invade the degenerating sarcoplasm between one to four days after mincing. Scattered throughout the minced muscle implant during the first 24 hours after injury are small, viable-appearing, undifferentiated cells located between the external lamina and degenerating sarcoplasm. Such cells, which are structurally similar to satellite cells seen in uninjured muscle, are believed to be regenerating presumptive myoblasts due to their mesenchymal-like morphology and sublaminar position. External laminae of the injured muscle fibers do not undergo immediate degenerative changes, but rather persist during the first three to six days as laminar tubes within which spindle-shaped myoblasts and newly formed myotubes are frequently observed. Examples of regenerating myoblasts in the process of budding-off from damaged muscle fibers were not observed in this study. Therefore, the evidence suggests that satellite cells are the major source of regenerating myoblasts in skeletal muscle of the rat.     

Abortive myogenesis in denervated skeletal muscle: differentiative properties of satellite cells, their migration, and block of terminal differentiation

Little is known about the biological properties of myogenic satellite cells during postdenervation muscle atrophy. The present study investigated the differentiative capacity of satellite cells and their involvement in the compensatory regenerative process in long-term denervated rat muscle. Electron microscopy and immunocytochemical labeling of muscle tissue 1–18 months following denervation demonstrated that despite activation of satellite cells, myogenesis in denervated muscle is abortive and does not lead to the formation of normal muscle fibers. Small sizes, poor development of the contractile system in newly formed denervated myotubes, and the absence of satellite cells on the surface indicate that their differentiation typically does not progress to terminal stages. Many immature myotubes degenerate, and others survive but are embedded in a collagen lattice near their parent fibers. Interestingly, newly formed myotubes located on the surface of parent muscle fibers beneath the basal lamina typically did not contain developed myofibrils. This suggests that the contacts of daughter and parent muscle fibers block myofibrillogenesis. Assembly of sarcomeres in most cases occurs following complete spatial separation of daughter and parent muscle fibers. Another manifestation of the involvement of myogenic precursors in abortive myogenesis is the formation of clusters of underdeveloped branching myotubes surrounded by a common basal lamina. We found that myoblasts can also fuse directly with differentiated muscle fibers. The presence of satellite cells near the openings in the basal lamina and in the interstitial space indicates that myogenic precursors can migrate through the basal lamina and form myotubes at a distance from parent fibers. Our data may explain why long-term denervated skeletal muscle has a poor capacity for regeneration and functional restoration.     

Induction of HLA-DR expression on human myoblasts with interferon-gamma

Myoblast transplantation is an experimental therapy that may be useful in hereditary muscle diseases. One obstacle to this approach is immune rejection. We evaluated human myoblasts and myotubes for surface expression of HLA class I and II histocompatibility antigens. The myoblast preparations, cultured from muscle biopsy specimens, were more than 95% pure as assessed by immunostaining with the Leu19 monoclonal antibody that reacts with human myoblasts and regenerating muscle fibers. Myoblasts and myotubes constitutively expressed HLA-class I but not HLA-class II molecules. However, HLA-class II expression was induced on mononucleated myoblasts after culture for 5 days in the presence of recombinant human interferon-gamma. The results indicate that transplanted myoblasts can be rejected because of histoincompatibility at the HLA-class I and HLA class II locus. Furthermore, an aberrant expression of HLA-class II antigen on myoblasts in vivo may play a role in the pathogenesis of autoimmune muscle disorders.     

An ultrastructural study of the dufferentiation of skeletal muscle in the bovine fetus

Summary The differentiation of skeletal muscle was studied by electron microscopy in bovine fetuses from 47 days gestation to neonatal calves 3 days of age.Initally, the muscle was composed of clusters of myotubes with mononucleated myoblasts between them. In 2-month-old fetuses these myoblasts became apposed to the differentiating muscle cells and were enclosed within the rudimentary basal lamina of the myotubes. At this stage the clusters of myotubes consisted of central, larger diameter, more differentiated myotubes and also the mononucleated satellite cells. The differentiated myotubes separated from the clusters accompanied by satellite cells which continued proliferating and fused together to form new generations of satellite myotubes. In this manner new clusters of myotubes were formed. By 4–5 months some of the separating myotubes began to form individual myotubes and independent myofibers were prominent in fetuses of 5–8 months of age.The myofibers in the 8-month-old fetuses showed diversification into fiber types by differences in the thickness of the Z-line, the prominence of the sarcotubular system, the amount of glycogen and lipid droplets and also the number of mitochondria.     

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.     

Novel electrical stimulation sets the cultured myoblast contractile function to 'on'.

OBJECTIVE: In the present study, the effect of electrical stimulation was examined for the ability to induce morphological, physiological, and molecular biological effects on myoblasts during cell differentiation. METHODS: L6 rat myoblasts were electrically stimulated by newly developed methods on culture days 6, 8, 10 and 12. RESULTS: This electrical stimulation accelerated the appearance of myotubes, and subsequently produced spontaneously contracting muscle fibers. Measurement of membrane potential showed that the contracting cell had functional ion channels and gap junctional intercellular communication. In the electrically stimulated cells, an enhanced expression of MyoD family and M-cadherin was also observed. Expression of connexin 43 was increased and maintained at a high level in the electrically stimulated cells. CONCLUSION: This is the first demonstration of in vitro induction of myoblasts in spontaneously contractile muscle fibers by intermittent stimulation. This novel method for induction of myoblast differentiation represents an important advance in cell therapy.     

The interaction of myotropic and macrophagotropic strains ofTrypanosoma cruzi with myoblasts and fibers of skeletal muscle

The process of interaction of bloodstream trypomastigotes from the myotropic CL and Colombiana strains and the macrophagotropic Y strain ofTrypanosoma cruzi with mouse myoblasts and myotubes was analysed. After 24 h of parasite-host cell interaction, parasites from the CL and Colombiana strains appeared to be more infective to myoblasts than those from the Y strain. Parasites from the Colombiana strain were more infective for myotubes than those from the Y strain, while those from the CL strain showed very a low ability to infect the cells. For all strains the infectivity was low for short periods of interaction, increasing with time. Myoblasts infected with parasites from the Y strain fused with other infected and uninfected cells to form myotubes. However, the process of fusion was blocked when the myoblasts were infected with parasites from the CL and Colombiana strains. These data indicate a different behavior of muscle cells when in contact with myotropic or non-myotropic strains ofT. cruzi.     

Compressive Deformation and Damage of Muscle Cell Subpopulations in a Model System

To study the effects of compressive straining on muscle cell deformation and damage an in vitro model system was developed. Myoblasts were seeded in agarose constructs and cultured in growth medium for 4 days. Subsequently, the cells were allowed to fuse into multinucleated myotubes for 8 days in differentiation medium, resulting in a population of spherical myoblasts (50%), spherical myotubes (35%), and elongated myotubes (15%) with an overall viability of 90%. To evaluate cell deformation upon construct compression half-core shaped constructs were compressed up to 40% strain and the resulting cell shape was assessed from confocal scans through the central plane of spherical cells. The ratio of cell diameters measured parallel and perpendicular to the axis of compression was used as an index of deformation (DI). The average DI of myoblasts decreased with strain level (0.99±0.03, 0.70±0.04, and 0.56±0.10 at 0%, 20%, and 40% strain), whereas for myotubes DI decreased up to 20% strain and then remained fairly constant (0.99±0.06, 0.55±0.06, 0.50±0.11). The discrepancy in DI between spherical myoblasts and myotubes at 20% strain was explained by the relative sensitivity of the cell membrane to buckling, which is more pronounced in the myotubes. Sustained compression up to 24 h at 20% strain resulted in a significant increase in cell damage with time as compared to unstrained controls. Despite differences in membrane buckling no difference in damage between myoblasts and spherical myotubes was observed over time, whereas the elongated myotubes were more susceptible to damage. © 2001 Biomedical Engineering Society. PAC01: 8719Ff, 8719Rr, 8716Dg, 8717Aa, 8710+e, 8780Pa, 8764Rr     

Regeneration of single skeletal muscle fibers in vitro.

A culture system utilizing single skeletal muscle fibers from adult rats was developed to study the origin and behavior of mononucleated myoblasts during muscle regeneration. The single fibers are removed manually from the leg and thigh muscles at 300-400 gm rats and maintained in vitro embedded in a fibrin clot overlain with culture medium. Regenerative events were monitored by continuous observations of the cultured fibers. During the first few hours in vitro the fibers undergo degenerative changes including the formation of myofibrillar contraction clots and pyknosis of myonuclei. The endomysial tube (basement lamina) remains intact along the entire length of the fiber and forms transparent chambers bridging the contraction clots. Single fibers are free of endomysial cells and display no cellular outgrowth in cultures, except at the cut ends. In contrast, a rich outgrowth of endomysial connective tissue cells is found with bundles consisting of two or more fibers. Isolated mononucleated cells, presumed to be muscle satellite cells, are present within the endomysial tube of single fibers at the onset of the culture period. There is no evidence that myonuclei contribute to the formation of mononucleated cells. The satellite cells enlarge and begin to proliferate during the second day in vitro to form clones of presumptive myoblasts within the endomysial tube. The early clones have a cell doubling time of about 22 hours and exhibit mitotic synchrony. After 5-7 days in vitro the satellite cell progeny begin to fuse to form multinucleated myotubes within the endomysial tube of the original fiber. The myotubes display spontaneous contractile activity and may extend throughout the length of the endomysial tube.     

Regeneration of single skeletal muscle fibers in vitro

A culture system utilizing single skeletal muscle fibers from adult rats was developed to study the origin and behavior of mononucleated myoblasts during muscle regeneration. The single fibers are removed manually from the leg and thigh muscles of 300–400 gm rats and maintained in vitro embedded in a fibrin clot overlain with culture medium. Regenerative events were monitored by continuous observation of the cultured fibers. During the first few hours in vitro the fibers undergo degenerative changes including the formation of myofibrillar contraction clots and pyknosis of myonuclei. The endomysial tube (basement lamina) remains intact along the entire length of the fiber and forms transparent chambers bridging the contraction clots. Single fibers are free of endomysial cells and display no cellular outgrowth in culture, except at the cut ends. In contrast, a rich outgrowth of endomysial connective tissue cells is found with bundles consisting of two or more fibers. Isolated mononucleated cells, presumed to be muscle satellite cells, are present within the endomysial tube of single fibers at the onset of the culture period. There is no evidence that myonuclei contribute to the formation of mononucleated cells. The satellite cells enlarge and begin to proliferate during the second day in vitro to form clones of presumptive myoblasts within the endomysial tube. The early clones have a cell doubling time of about 22 hours and exhibit mitotic synchrony. After 5–7 days in vitro the satellite cell progeny begin to fuse to from multinucleated myotubes within the endomysial tube of the original fiber. The myotubes display spontaneous contractile activity and may extend throughout the length of the endomysial tube.     

CD34在体外培养大鼠骨骼肌卫星细胞发育的免疫组织化学研究

目的 观察CD34在发育中的体外培养大鼠骨骼肌卫星细胞中的分布，探讨CD34在发育中的大鼠骨骼肌卫星细胞中的作用。方法 原代培养骨骼肌卫星细胞，第1次传代后，取贴壁2、4、8、16、24、48和72h的细胞行CD34多克隆抗体的免疫组织化学染色。结果 CD34免疫阳性反应存在于发育中的骨骼肌卫星细胞中，但在形成肌管后，细胞核免疫反应转为阴性，同时成纤维细胞呈弱阳性反应。结论 CD34在发育中的骨骼肌卫星细胞中有一定量的分布，它可能在骨骼肌的生理发育中起某种调节作用。     

干细胞因子在体外培养大鼠骨骼肌卫星细胞表达和发育的免疫组织化学研究

目的 观察干细胞因子(SCF)在发育中的体外培养大鼠骨骼肌卫星细胞中的分布，探讨SCF在大鼠骨骼肌卫星细胞中的作用。方法 原代培养骨骼肌卫星细胞，经两次传代后，取培养2、4、8、16、24、48、72h的细胞行SCF单克隆抗体的免疫组织化学染色。结果SCF免疫阳性反应物存在于发育中的骨骼肌卫星细胞中，形成肌管后，细胞核呈强阳性反应。结论 SCF在发育的骨骼肌卫星细胞中有表达，它可能在骨骼肌的生理发育中起某种调节作用。     

Regenerating skeletal muscle cells express insulin-like growth factor I

The expression of the trophic peptide insulin-like growth factor I (IGF-I; somatomedin C) was investigated in the regenerating soleus muscle of mice after injury by the snake venom taipoxin. No specific IGF-I immunoreactivity was observed in muscle cells during control conditions. Within 2 days after taipoxin injection, IGF-I immunoreactivity could be demonstrated in activated satellite cells. Myoblasts and myotubes expressed high IGF-I immunoreactivity. The IGF-I immunoreactivity was strictly cytoplasmatic and obviously associated with polyribosomes. No vesicular or membraneous IGF-I immunoreactivity could be demonstrated. It is concluded that IGF-I is synthesized in myogenic cells during skeletal muscle regeneration. It is suggested that IGF-I exerts its effects on skeletal muscle mainly by autocrine mechanisms.     

Adult bone marrow-derived stem cells in muscle connective tissue and satellite cell niches

Skeletal muscle includes satellite cells, which reside beneath the muscle fiber basal lamina and mainly represent committed myogenic precursor cells, and multipotent stem cells of unknown origin that are present in muscle connective tissue, express the stem cell markers Sca-1 and CD34, and can differentiate into different cell types. We tracked bone marrow (BM)-derived stem cells in both muscle connective tissue and satellite cell niches of irradiated mice transplanted with green fluorescent protein (GFP)-expressing BM cells. An increasing number of GFP+ mononucleated cells, located both inside and outside of the muscle fiber basal lamina, were observed 1, 3, and 6 months after transplantation. Sublaminal cells expressed unambiguous satellite cell markers (M-cadherin, Pax7, NCAM) and fused into scattered GFP+ muscle fibers. In muscle connective tissue there were GFP+ cells located close to blood vessels that expressed the ScaI or CD34 stem-cell antigens. The rate of settlement of extra- and intralaminal compartments by BM-derived cells was compatible with the view that extralaminal cells constitute a reservoir of satellite cells. We conclude that both muscle satellite cells and stem cell marker-expressing cells located in muscle connective tissue can derive from BM in adulthood.     

Expression and function of heterotypic adhesion molecules during differentiation of human skeletal muscle in culture.

The infiltration of skeletal muscle by leukocytes occurs in a variety of myopathies and frequently accompanies muscle degeneration and regeneration. The latter involves development of new myofibers from precursor myoblasts, and so infiltrating cells may interact with muscle at all stages of differentiation. The authors have investigated the surface expression of ligands for T-cell adhesion during the differentiation of human skeletal muscle in vitro. Myoblasts expressed low levels of ICAM-1 (CD54), which remained constant during muscle cell differentiation and could be induced by cytokines such as gamma-interferon. It is therefore likely that ICAM-1 is involved in the invasive accumulation of lymphocytes during skeletal muscle inflammation. In contrast, LFA-3 (CD58) was expressed at higher levels than ICAM-1 on myoblasts, decreased significantly during myogenesis, and was unaffected by immune mediators. Both ICAM-1 and LFA-3 were able to mediate T cell binding to myoblasts, whereas adhesion to myotubes was independent of the LFA-3 ligand. Although expressed throughout myogenesis, human leukocyte antigen class I and CD44 did not appear to mediate T cell binding. The expression of ligands that facilitate interaction of myogenic cells with lymphocytes may have important implications for myoblast transplantation.     

Production of arrays of cardiac and skeletal muscle myofibers by micropatterning techniques on a soft substrate

Micropatterning and microfabrication techniques have been widely used to pattern cells on surfaces and to have a deeper insight into many processes in cell biology such as cell adhesion and interactions with the surrounding environment. The aim of this study was the development of an easy and versatile technique for the in vitro production of arrays of functional cardiac and skeletal muscle myofibers using micropatterning techniques on soft substrates. Cardiomyocytes were used for the production of oriented cardiac myofibers whereas mouse muscle satellite cells for that of differentiated parallel myotubes. We performed micro-contact printing of extracellular matrix proteins on soft polyacrylamide-based hydrogels photopolymerized onto functionalized glass slides. Our methods proved to be simple, repeatable and effective in obtaining an extremely selective adhesion of both cardiomyocytes and satellite cells onto patterned soft hydrogel surfaces. Cardiomyocytes resulted in aligned cardiac myofibers able to exhibit a synchronous contractile activity after 2 days of culture. We demonstrated for the first time that murine satellite cells, cultured on a soft hydrogel substrate, fuse and form aligned myotubes after 7 days of culture. Immunofluorescence analyses confirmed correct expression of cell phenotype, differentiation markers and sarcomeric organization. These results were obtained in myotubes derived from satellite cells from both wild type and MDX mice which are research models for the study of muscle dystrophy. These arrays of both cardiac and skeletal muscle myofibers could be used as in vitro models for pharmacological screening tests or biological studies at the single fiber level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The development of the facial muscles in man

Morphogenesis and histogenesis of the facial muscles is described in human embryos and fetuses 4.2–360.0 mm (crown-rump length). The microscopic study was performed on 50 specimens that had been variously serially-sectioned and stained. Graphic or wax reconstructions were made from transverse serial sections at 11 representative ages. Three late fetuses and a term infant were studied grossly. Second branchial arch mesenchyme in early embryos (4.2–6.5 mm) becomes increasingly dense, but is not subdivided into distinct premuscle masses. By 8–20 mm, sheet-like collections (laminae) of premyoblasts and early myoblasts extend from the superficial part of the arch into the temporal, occipital, cervical and mandibular regions. Premuscle condensations deep in the arch become the stapedius, posterior digastric and stylohyoid muscles. The infraorbital lamina and the occipital platysma appear by 20–23 mm. The superficial muscles differentiate rapidly between 26 and 37 mm. Most are composed of myoblasts at 41 mm. By 80 mm, all the muscles contain myotubes and are in their definitive positions. At 80–140 mm, the myotubes become young muscle fibers. From 140 mm to 360 mm (term) the muscles increase in size and gain definitive attachments. Superficial muscles differentiate later than the deep and those in the cervicomandibular and occipital regions differentiate earlier than those in the frontal and midfacial regions. The muscle masses form pari passu with the muscular branches of the facial nerve which develop deep to them.     

Circulating human or canine factor IX from retrovirally transduced primary myoblasts and established myoblast cell lines grafted into murine skeletal muscle

We have used retroviral vectors to introduce human or canine factor IX cDNAs into cultured primary murine and canine myoblasts and into the established murine myoblast cell line C2C12. In all cases, the stably infected cells produced biologically active factor IX in culture and secreted detectable amounts into the culture medium both before and after differentiation of the cells into myotubes. Myoblasts and differentiated myotubes are therefore capable of performing all the posttranslational modifications of the coagulation factor required for biological activity. We have grafted the genetically modified myoblasts into skeletal muscles of nude mice and have detected stable levels of circulating human factor IX for up to two months after grafting. We propose that grafting genetically modified primary myoblasts or established myoblast cell lines into skeletal muscle may represent a useful approach to gene therapy for a variety of genetic diseases, including intrinsic muscle disease and defects in circulating proteins as in the hemophilias.