Human muscle precursor cells give rise to functional satellite cells in vivo

Mouse satellite cells have been shown to be functional muscle stem cells, in that they are able to regenerate skeletal muscle and to reconstitute the satellite cell pool. Although human muscle precursor cells are able to contribute to skeletal muscle regeneration following transplantation into host mouse muscles, it is uncertain whether they also give rise to functional satellite cells. Here, we transplant human fetal muscle precursor cells into cryodamaged muscles in C(5)-/gamma-chain-/Rag2-host mice. The donor cells gave rise to muscle fibres that persisted for up to 6 months after grafting. Isolated muscle fibres, bearing satellite cells, were prepared from muscles 4 weeks after grafting. When placed in culture, a small proportion of these fibres gave rise to muscle precursor cells of human origin, indicating that the originally grafted cells had formed satellite cells as well as regenerated muscle fibres. These satellite cell-derived human muscle precursor cells were expanded in culture and formed muscle following their transplantation into a second series of host mice. This provides evidence that human, as well as mouse, muscle precursor cells, are capable of forming functional satellite cells in vivo.     

Basal lamina and superfast myosin expression in regenerating cat jaw muscle

We investigated the possible role of extracellular matrix in specifying the expression of superfast myosin during cat jaw muscle regeneration. Equal proportions of muscle tissue from jaw and limb were minced together after killing cellular elements from one source. We allowed the mince to regenerate in the bed of a fast limb muscle. Regenerates were analyzed immunocytochemically at 71 to 294 days after operation. Fibers in control regenerates containing live cells from both sources expressed fast, superfast or slow myosins, or a mixture of these myosins. In regenerates containing only one type of live cells, we detected only myosins appropriate to the live cells. Our results suggest that during regeneration the original extracellular matrix of jaw-closing or limb muscle is unable to specify the expression of superfast or fast myosins, respectively; they point to the cellular elements, probably the satellite cells, as determinants of muscle specificity during regeneration.     

Increase of Cardiotrophin‐1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats

The original report by Pennica et al. on Cardiotrophin‐1 (CT‐1) states that it markedly stimulates hypertrophy in cardiac myocytes both in vitro and in vivo and is predominantly expressed in the early mouse embryonic heart tube. CT‐1 is a member of the interleukin‐6 superfamily and past studies have shown that it exerts trophic effects on neurons, glial cells and their precursors, and is expressed during myogenesis. Thus CT‐1 is associated with physical and pathological changes in skeletal muscle. In this study, we examined whether CT‐1 is expressed in mechanically overloaded, regenerating, and denervated muscles of rats using immunohistochemistry. In the overloaded plantaris muscles at 1 and 3 days postsurgery, CT‐1 immunoreactivity was detected in the mononuclear cells that had infiltrated the extracellular space. CT‐1 immunoreactivity was also observed in the mononuclear cells invading the extracellular space at 2, 4, and 6 days after a bupivacaine injection and in degenerative and necrotic muscle fibers at 2 days postinjection. In the denervated muscles, the CT‐1 immunoreactivity did not change in intensity during the entire period of the denervation (2, 7, and 14 days postsurgery). The cells invading extracellular space and in necrotic muscle fibers possessing CT‐1 immunoreactivity might be muscle precursor cells (satellite cells) or migrating macrophages undergoing phagocytosis. Using double‐immunostainings for anti‐CT‐1/antic‐met, anti‐CT‐1/ anti‐M‐cadherin, and anti‐CT‐1/anti‐ED1, we found that satellite cells and macrophages exhibited CT‐1 immunoreactivity in the damaged muscles after bupivacaine injection. We therefore believe that CT‐1 plays a key role in regeneration and hypertrophy in the skeletal muscle of rats.     

Muscle satellite cells from GRMD dystrophic dogs are not phenotypically distinguishable from wild type satellite cells in ex vivo culture

Duchenne muscular dystrophy is a neuromuscular degenerative disorder caused by the absence of dystrophin protein. It is characterized by progressive muscle weakness and cycles of degeneration/regeneration accompanying chronic muscle damage and repair. Canine models of muscular dystrophy, including the dystrophin-deficient golden retriever muscular dystrophy (GRMD), are the most promising animal models for evaluation of potential therapies, however canine-specific molecular tools are limited. In particular, few immune reagents for extracellular epitopes marking canine satellite cells (muscle stem cells) are available. We generated an antibody to the satellite cell marker syndecan-4 that identifies canine satellite cells. We then characterized isolated satellite cells from GRMD muscle and wildtype muscle by several in vitro metrics, and surprisingly found no significant differences between the two populations. We discuss whether accumulated adverse changes in the muscle environment rather than cell-intrinsic defects may be implicated in the eventual failure of satellite cell efficacy in vivo.     

Regenerative capacity of skeletal muscle

PURPOSE OF REVIEW: The aim of this review is to highlight advances in the field of skeletal muscle regeneration that have been made in the last year. RECENT FINDINGS: Studies have increased our understanding of the activation of satellite cells within their niche on the muscle fibre, the contribution of satellite cell-derived muscle precursor cells to skeletal muscle regeneration and the reduction of satellite cell function in old muscle. Although other stem cells, either bone marrow derived or present within skeletal muscle or other tissues, do contribute to muscle regeneration, recent studies have highlighted that this is at best minimal compared with the ability of satellite cells to regenerate skeletal muscle. The effect of the host muscle environment has been shown to have a profound effect on skeletal muscle regeneration. Age and denervation have a detrimental effect and certain types of muscle injury a positive effect. Work continues on the effect of growth factors on muscle cell lines in vitro and muscle regeneration in vivo. SUMMARY: Recent work has focused on the contribution of satellite-cell derived muscle precursor cells and other stem cells to skeletal muscle regeneration. The muscle environment has a profound effect on the regenerative capacity of resident and implanted cells. Muscle regeneration may be optimized by using the best stem cell population and by modifying the host muscle environment.     

Reduced satellite cell population may lead to contractures in children with cerebral palsy

Aim Satellite cells are the stem cells residing in muscle responsible for skeletal muscle growth and repair. Skeletal muscle in cerebral palsy (CP) has impaired longitudinal growth that results in muscle contractures. We hypothesized that the satellite cell population would be reduced in contractured muscle. Method We compared the satellite cell populations in hamstring muscles from participants with CP contracture (n=8; six males, two females; age range 6–15y; Gross Motor Function Classification System [GMFCS] levels II–V; 4 with hemiplegia, 4 with diplegia) and from typically developing participants (n=8; six males, two females, age range 15–18y). Muscle biopsies were extracted from the gracilis and semitendinosus muscles and mononuclear cells were isolated. Cell surface markers were stained with fluorescently conjugated antibodies to label satellite cells (neural cell adhesion molecule) and inflammatory and endothelial cells (CD34 and CD4 respectively). Cells were analyzed using flow cytometry to determine cell populations. Results After gating for intact cells a mean of 12.8% (SD 2.8%) were determined to be satellite cells in typically developing children, but only 5.3% (SD 2.3%; p<0.05) in children with CP. Hematopoietic and endothelial cell types were equivalent in typically developing children and children with CP (p>0.05) suggesting the isolation procedure was valid. Interpretation A reduced satellite cell population may account for the decreased longitudinal growth of muscles in CP that develop into fixed contractures or the decreased ability to strengthen muscle in CP. This suggests a unique musculoskeletal disease mechanism and provides a potential therapeutic target for debilitating muscle contractures.     

The effect of peripheral nerve injury on immature motor and sensory neurons and on muscle fibres. Possible relation to the histogenesis of Werdnig-Hoffmann disease

Sciatic nerve section in newborn rats causes loss of almost all motoneurons and of two thirds of the sensory neurons. Single motor axons may regenerate but they innervate only few muscle fibres. The nonreinnervated fibres vanish and are replaced by fat cells. The reinnervated muscle fibres are overloaded, hypertrophy, and eventually necrotize. Their satellite cells regenerate new fibres; most of them develop aneurally and then disappear. The muscles, if reinnervated at all, after 1 year consist of a mixture of hypertrophic fibres, non-innervated regenerates, and fat cells, and resemble muscle from patients with infantile spinal muscular atrophy Werdnig-Hoffmann. In immature rat muscles, restricted reinnervation and functional overload of the few innervated fibres maintain a self-enhancing process which eventually leads to the replacement of the entire muscle by fat tissue. It is suggested that Werdnig-Hoffmann disease starts during fetal life and that similar mechanisms are operative. The clinical progression would then depend on the extent of degeneration due to overload and on regeneration and reinnervation in the muscles rather than on the rate of continuous loss of anterior horn cells. Immature motoneurons in rat die when they loose contact with their target; one might therefore speculate that the primary lesion in Werdnig-Hoffmann disease is peripheral rather than central, and that lack of neuronotorophic influences causes the loss of anterior horn cells.     

The ontogeny of soleus muscles in mdx and wild type mice

The satellite cell, the organotypic muscle stem cell, is the key element in ontogenetic and load induced muscle fibre growth and repair. It is therefore possible that the satellite pool becomes exhausted with age, especially in mdx mice where dystrophin deficiency results in skeletal muscle degeneration. We compared structural criteria and satellite cell frequencies in soleus muscles of 26 mdx and 23 wild type mice aged between 26 and 720 days. The total number of muscle fibres was similar in both groups and remained stable throughout life, except for an early increase in wild type mice. However, in mdx muscles there was always a proportion of small-diameter fibres which resulted in a reduction in the effective myogenic area on cross-section, whereas total cross-sectional area and muscle weights were increased relative to controls throughout life. In adult animals, the frequency and numbers of satellite cells remained stable with age and were similar in both animal groups. Satellite cell numbers showed some considerable variation between individual animals, although with a markedly smaller variability between results of the same animal, pointing to the satellite cell pool being an individual variant.     

Satellite cells in slow and fast rat muscles differ in respect to acetylcholinesterase regulation mechanisms they convey to their descendant myofibers during regeneration

The hypothesis of satellite cell diversity in slow and fast mammalian muscles was tested by examining acetylcholinesterase (AChE) regulation in muscles regenerating (1) under conditions of muscle disuse (tenotomy, leg immobilization) in which the pattern of neural stimulation is changed, and (2) after cross-transplantation when the regenerating muscle develops under a foreign neural stimulation pattern. Soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were allowed to regenerate after ischemic-toxic injury either in their own sites or had been cross-transplanted to the site of the other muscle. Molecular forms of AChE in regenerating muscles were analyzed by velocity sedimentation in linear sucrose gradients. Neither tenotomy nor limb immobilization significantly affected the characteristic pattern of AChE molecular forms in regenerating SOL muscles, suggesting that the neural stimulation pattern is probably not decisive for its induction. During an early phase of regeneration, the general pattern of AChE molecular forms in the cross-transplanted regenerating muscle was predominantly determined by the type of its muscle of origin, and much less by the innervating nerve which exerted only a modest modifying effect. However, alkali-resistant myofibrillar ATPase activity on which the separation of muscle fibers into type I and type II is based, was determined predominantly by the motor nerve innervating the regenerating muscle. Mature regenerated EDL muscles (13 weeks after injury) which had been innervated by the SOL nerve became virtually indistinguishable from the SOL muscles in regard to their pattern of AChE molecular forms. However, AChE patterns of mature regenerated SOL muscles that had been innervated by the EDL nerve still displayed some features of the SOL pattern. In regard to AChE regulation, muscle satellite cells from slow or fast rat muscles convey to their descendant myotubes the information shifting their initial development in the direction of either slow or fast muscle, respectively. The satellite cells in fast or slow muscles are, therefore, intrinsically different. Intrinsic information is expressed mostly during an early phase of regeneration whereas later on the regulatory influence of the motor nerve more or less predominates. © 1994 Wiley-Liss, Inc.     

Continuous myonuclear addition to single extraocular myofibers in uninjured adult rabbits

Extraocular muscles (EOM) are unique among mammalian skeletal muscles in that they normally express molecules associated with muscle development and regeneration. In this study we show that satellite cells of EOM, unlike those of other skeletal muscles, continually divide in the normal, uninjured adult. Adult EOM contained activated satellite cells positive for the myogenic regulatory factor MyoD. EOM satellite cells did not require a prolonged activation period prior to onset of cell division and differentiation in vitro. EOM satellite cells incorporated bromodeoxyuridine (brdU), a marker for cell division, and with longer postlabeling survival, brdU-labeled nuclei populated EOM myofibers. This was not seen with leg muscle. These findings suggest the possibility that continual division of satellite cells and fusion of their daughter myocytes with existing adult EOM myofibers contribute to the unique sparing or susceptibility of EOM to certain muscle diseases.     

Muscle regeneration after imposed injury is better in younger than older mdx dystrophic mice

The ability of a fast-twitch dystrophic muscle to regenerate was compared at two ages to control muscle regeneration. Myofiber growth, and the distribution of nuclei in fibers were used to characterize the muscle regeneration 3 and 6 weeks after injury. In control and mdx muscles, myosatellite cell proliferation was completed by 3 weeks after injury. Mdx muscle regenerated as well as controls, based on similar distribution of myofiber cross sectional area, and the percent of centronucleation, typical of regenerated fibers. In addition, muscle from the younger dystrophic mdx mice grew to unoperated levels with no net change in fiber area distribution, while older muscles did not regenerate as well. There were also more peripheral (satellite cell) nuclei observed in younger mdx muscle than in older muscles, after the most active phase of dystrophy.     

Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1

Aims: Myotonic dystrophy type 1 (DM1), one of the most common forms of inherited neuromuscular disorders in the adult, is characterized by progressive muscle weakness and wasting leading to distal muscle atrophy whereas proximal muscles of the same patients are spared during the early phase of the disease. In this report, the role of satellite cell dysfunction in the progressive muscular atrophy has been investigated. Methods: Biopsies were obtained from distal and proximal muscles of the same DM1 patients. Histological and immunohistological analyses were carried out and the past regenerative history of the muscle was evaluated. Satellite cell number was quantified in vivo and proliferative capacity was determined in vitro. Results: The size of the CTG expansion was positively correlated with the severity of the symptoms and the degree of muscle histopathology. Marked atrophy associated with typical DM1 features was observed in distal muscles of severely affected patients whereas proximal muscles were relatively spared. The number of satellite cells was significantly increased (twofold) in the distal muscles whereas very little regeneration was observed as confirmed by telomere analyses and developmental MyHC staining (0.3–3%). The satellite cells isolated from the DM1 distal muscles had a reduced proliferative capacity (36%) and stopped growing prematurely with telomeres longer than control cells (8.4 vs. 7.1 kb), indicating that the behaviour of these precursor cells was modified. Conclusions: Our results indicate that alterations in the basic functions of the satellite cells progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.     

Activating muscle stem cells: therapeutic potential in muscle diseases

PURPOSE OF REVIEW: The satellite cell is the principal muscle stem cell. Recent research, however, has highlighted new stem cell sources that, once activated in the muscle tissue, can participate in muscle regeneration. This article reviews the state of research on stem cell populations that have potential for treatment of muscular dystrophies. RECENT FINDINGS: Despite recent findings about the stem cell character of satellite cells and their in-vivo myogenic potential, limitations related to muscle precursor cell transfer therapy have encouraged the investigation of stem cell sources other than satellite cells. Current research is focused on identifying the best stem cell in the endothelial compartment, which is able to be systemically delivered to reach all the muscles and to contribute to widespread muscle regeneration within these muscles. SUMMARY: Current results highlight many possible stem cell sources for stem cell therapy of muscle diseases, and work is ongoing to identify the most effective candidate that is able to robustly regenerate muscle tissue and to functionally repopulate the muscle stem cell compartment.     

Transplantation of primary satellite cells improves properties of reinnervated skeletal muscles

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. We tested the hypothesis that transplantation of satellite cells into reinnervated rabbit tibialis anterior (TA) muscles improves their properties. Adult rabbits underwent transection and immediate suture of the common peroneal nerve. In order to provide an environment favorable for cell transplantation, TA were then made to degenerate by cardiotoxin injection, either immediately or after a 2-month delay, which is sufficient for muscle reinnervation. In both cases, the injured TA were transplanted with cultured satellite cells 5 days after induction of muscle degeneration. When cells were transferred immediately after nerve repair, drastic morphological and functional muscle alterations were observed. However, when the muscles were allowed to become reinnervated before cell transplantation, muscles were heavier and developed a significantly higher maximal force compared to denervated-reinnervated muscles. Thus, application of the cell therapy protocol improved properties of denervated muscles only when they were allowed to become innervated. These results, which represent the application of cell therapy to improve force recovery of reinnervated muscles, will be of significant interest in certain clinical contexts, particularly after immediate or delayed muscle reinnervation.     

Shorter telomeres in dystrophic muscle consistent with extensive regeneration in young children

Muscular dystrophies are characterised by continuous cycles of degeneration and regeneration resulting in an eventual diminution of the muscle mass and extensive fibrosis. In somatic cells chromosomal telomeres shorten with each round of cell division and telomere length is considered to be a biomarker of the replicative history of the cell. We have previously shown that human myoblasts have a limited proliferative capacity, and that normal skeletal muscle has a very low level of nuclear turnover. However, in patients suffering from muscular dystrophy the satellite cells will be forced to make repeated rounds of cell division, driving the cells towards senescence. In this study we have used the telomere length to quantify the intensity of the muscle cell turnover in biopsies from dystrophic patients of different ages. Our results show that as soon as the first clinical symptoms become apparent the muscle has already undergone extensive regeneration and the rate of telomere loss is 14 times greater than that observed in controls. This confirms that the decline in regenerative capacity is due to the premature senescence of the satellite cells induced by their excessive proliferation during muscle repair.     

Factors inducing mast cell accumulation in skeletal muscle

It has been suggested that mast cells contribute to the phenotype of dystrophinopathies, but the mechanisms of their recruitment into the skeletal muscle remain hypothetical. The aim of this study is to quantify the presence of mast cells in muscle during the cellular events of myofibre degeneration and regeneration. For this purpose, we compare the mast cell profile in dystrophin-deficient mdx mice in which muscles exhibit spontaneous cycles of degeneration-regeneration from 3 weeks of age, with that in Swiss mice in which muscles were injured either by ischaemia or by notexin injection. Notexin is an A2-type phospholipase that rapidly disrupts myofibre plasma membranes, while ischaemia results in a slower process of degeneration. Both lesions are followed by a successful regeneration. In intact muscles, mast cell counts (mean±SEM/mm²) range from 1.8± 1 to 4.3 ± 1.6. The injection of notexin is far more potent in recruiting mast cells into damaged muscle than is ischaemia (118.5±13.0 vs 12.3 ± 1.8/mm²). Thus we conclude that the early disruption of the myofibre membrane could elicit mast cell accumulation in skeletal muscle. This may explain the elevated number of mast cells observed in mdx muscles, as dystrophin deficiency is thought to induce myofibre membrane leakage. On the other hand, mast cells are more numerous in muscles of young and adult mdx mice that are allowed to regenerate, than in muscles of older animals in which there is little regeneration and fibrosis develops. In injured muscles, the peak of mast cell number is at the onset of regeneration (by day 3 after notexin injection, and by day 11 after ischaemia), rather than during the phase of myofibre necrosis. Therefore, we suggest that the mast cells, through the effects of released mediators, could contribute to muscle regeneration.     

Oxidative metabolism of hypertrophic skeletal muscle in the rat.

The object of this study was to determine whether skeletal muscle adjusts its oxidative metabolism in response to compensatory hypertrophy. We therefore, measured the production of ¹⁴CO₂ from glucose-6-¹⁴C and β-hydroxybutyrate-3-¹⁴C by homogenates of rat plantaris and soleus muscles undergoing compensatory hypertrophy produced by elimination of synergists. There was a decrease in substrate oxidation by hypertrophic muscles. These results are in contrast to the increased oxidative capacity observed in skeletal muscle following endurance training.     

Role of persisting basement membrane in the reorganization of myofibres originating from myogenic cell grafts in the rat.

Satellite cells grafted at the site of an irreversible muscle injury regenerate normal myofibres that become organized in fascicles. The role of the basement membrane in organization of the newly formed muscle fibres was investigated using polyclonal antibodies against laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan. In ungrafted muscles, original basement membranes were reactive to these antibodies at 7, 14 and 45 days after injury. Labelling of satellite cells with FITC-latex beads showed the labelled myoblasts and new myofibres within the remnants of old basement membranes at 7 days after cell implantation and thereafter. Electron microscopy of injured-ungrafted muscles showed persistence of electron dense material corresponding to thin layers of old basal laminae partially interrupted. After cell grafting, myotubes developed within these structures and were surrounded by redundant basal laminae. These results suggest that grafted cells are able to migrate inside the basement membranes which serve as scaffolding for their development.     

Active hepatocyte growth factor is present in skeletal muscle extracellular matrix

When skeletal muscle is stretched or injured, satellite cells are activated to proliferate, and this process can be mediated by release of the active form of hepatocyte growth factor (HGF) from the extracellular matrix. The objective of these experiments was to determine whether the mechanism of release includes proteolytic activation of pro-HGF. Extracellular HGF in uninjured adult rat extensor digitorum longus muscle was extracted by treatment with 1 M NaCl or heparinases I and III in the presence of a cocktail of serine protease inhibitors. Active HGF heterodimer was the predominant form present, but both pro-HGF and active HGF were extracted when muscle was incubated with Triton X-100 or crush-injured. Incubation of exogenous pro-HGF with uninjured or crush-injured skeletal muscle resulted in cleavage to the active form, indicating that endogenous extracellular proteases are present and capable of rapidly converting pro-HGF to active HGF. Finally, treatment with sodium nitroprusside, a nitric oxide (NO) donor, resulted in liberation of active HGF. These experiments indicate that the active form of HGF is present in the extracellular compartment of uninjured skeletal muscle; therefore, the mechanism of HGF release in response to stretch and NO does not require proteolytic activation of pro-HGF.