Cellular dynamics in the muscle satellite cell niche

Satellite cells, the quintessential skeletal muscle stem cells, reside in a specialized local environment whose anatomy changes dynamically during tissue regeneration. The plasticity of this niche is attributable to regulation by the stem cells themselves and to a multitude of functionally diverse cell types. In particular, immune cells, fibrogenic cells, vessel‐associated cells and committed and differentiated cells of the myogenic lineage have emerged as important constituents of the satellite cell niche. Here, we discuss the cellular dynamics during muscle regeneration and how disease can lead to perturbation of these mechanisms. To define the role of cellular components in the muscle stem cell niche is imperative for the development of cell‐based therapies, as well as to better understand the pathobiology of degenerative conditions of the skeletal musculature.     

Precocious in vitro development of satellite cells from dystrophic chicken muscle

Summary Satellite cells, liberated from pectoral muscle of juvenile dystrophic chickens by sequential treatment with collagenase, hyaluronidase, and trypsin and preplated to remove fibroblasts and cultured on gelatin proliferated rapidly, fused and formed confluent muscle cultures within 6 d in vitro with minimal contamination by fibroblasts. When identical isolation and culturing techniques were applied to muscle from age-mateched normal chickens proliferation and differentiation were slower, contamination with fibroblasts was much greater, and only a small number of myotubes were formed. After injection of the myotoxic anesthetic marcaine into normal pectoral muscle for 5 consecutive days, myotube formation was accelerated in satellite cell cultures, but the rate of differentiation was not as rapid as that occurring in cells from dystrophic muscle. This research was supported by a grant from the Muscular Dystrophy Association of Canada.     

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2′-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7^highMyoD^lowEdU^pos) and activated satellite cells (Pax7^highMyoD^highEdU^pos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.     

盐酸布比卡因、透明质酸酶对大鼠在体肌卫星细胞增殖的影响

目的 :研究盐酸布比卡因和透明质酸酶对成年大鼠肌卫星细胞在体增殖的影响。方法 :免疫组化法 ,H .E染色法 ,光镜和电镜观察。结果 :①正常对照组和生理盐水组肌纤维完整 ,有少量Desmin阳性肌卫星细胞 ,面密度值为 0 .66%± 0 .57%和 2 .48%± 1.13 %。生理盐水组较正常对照组无显著差异 (P >0 .0 5)。②透明质酸酶组肌纤维完整 ,Desmin阳性肌卫星细胞数量增加 ,面密度值为 2 .52 %± 1.41% ,较生理盐水组和正常对照组无显著差异(P >0 .0 5)。③盐酸布比卡因组和盐酸布比卡因 +透明质酸酶混合液组均可见大量坏死和溶解的肌纤维 ,并伴有肌卫星细胞的激活、增殖 ,Desmin阳性肌卫星细胞显著增加 ,并有部分融合形成小肌管。面密度值分别为 19.0 1%± 4.74%和 2 2 .41%± 7.64% ,较生理盐水组显著增加 (P <0 .0 1)。结论 :局麻药盐酸布比卡因能引起在体肌卫星细胞的活化、增殖并形成肌管 ,单独透明质酸酶溶液在本实验条件下对在体肌卫星细胞无明显作用     

Engineering skeletal muscle tissue--new perspectives in vitro and in vivo

Muscle tissue engineering (TE) has not yet been clinically applied because of several problems. However, the field of skeletal muscle TE has been developing tremendously and new approaches and techniques have emerged. This review will highlight recent developments in the field of nanotechnology, especially electrospun nanofibre matrices, as well as potential cell sources for muscle TE. Important developments in cardiac muscle TE and clinical studies on Duchenne muscular dystrophy (DMD) will be included to show their implications on skeletal muscle TE.     

Muscle origin of porcine satellite cells affects in vitro differentiation potential

Post‐natal muscle regeneration relies on the activation of tissue stem cells known as satellite cells, to repair damage following exercise trauma and disease. Satellite cells from individual muscles are known to be heterogeneous with regard to proliferation, fusion and transplantation abilities, although the muscle origin has rarely been considered pertinent to their differentiation capabilities. In this study we compared the potential of two functionally distinct skeletal muscle satellite cell populations from porcine diaphragm and hind‐limb semi‐membranosus muscles. These two muscles were chosen primarily for differences in metabolic and contractile properties: the diaphragm is more continuously active and has a greater oxidative capacity. Cells were induced to differentiate towards myogenic and adipogenic lineages, and here we have shown that cells from diaphragm exhibit a significantly greater degree of myogenesis compared with those from semi‐membranosus, while the converse was true for adipogenesis. Unexpectedly, both conditions generated small numbers of cells with neuronal characteristics for both muscle types, although more so in cells derived from the diaphragm. With increased interest in muscle adiposity with age and disease, these findings suggest that muscle origin of satellite cells does affect lineage fate, however whether differences in developmental origin or metabolic activity of the parent tissue govern this, remains to be determined. Copyright © 2010 John Wiley & Sons, Ltd.     

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant‐derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast‐CD56⁺ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56‐purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non‐human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H₂O₂)‐induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non‐toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.     

Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy

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Combination of inflammation-related cytokines promotes long-term muscle stem cell expansion

Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.     

Quantitative and qualitative in vitro analysis of the stem cell potential of hematopoietic cells purified from murine skeletal muscle

The murine skeletal muscle contains hematopoietic stem cells, but this potential has so far not been studied quantitatively or qualitatively in vitro. To quantify the hematopoietic stem cell potential, we have used highly purified SP/CD45^＋ cells in long-term culture initiating cell （LTC-IC） assays. The SP/CD45^＋ cell population purified from murine muscle was found to have significant stem cell activity with an LTC-IC frequency of 1/640. Single-cell-sorted SP/CD45^＋ cells from muscle exhibited robust proliferative activity in vitro at day 16 （380-fold amplification）, especially after culture with OP-9 layers that also support embryonic stem cells. Amplified cell populations originating from single cells exhibited multilineage differentiation ability with evidence of myeloid, lymphoid and NK cell markers. Thus, our results demonstrate that hematopoietic stem cells that can be quantified by LTC-IC assays exist in the murine skeletal muscle and show also for the first time, at the single-cell level, that these cells exhibit multilineage differentiation ability and major proliferative potential.     

Roles of phosphatase and tensin homolog in skeletal muscle

The phosphatase and tensin homolog (PTEN), originally identified as a tumor suppressor, is an important regulator of the PI3K–Akt pathway. PTEN plays crucial roles in various cellular processes, including cell survival, cell growth, cell proliferation, cell differentiation, and cell metabolism. In metabolic tissues, PTEN expression affects insulin sensitivity and glucose homeostasis. In skeletal muscle, the deletion of PTEN regulates muscle development and protects the mutant mice from insulin resistance and diabetes. Notably, the regulatory role of PTEN in skeletal muscle stem cells has been recently reported. In this review, we mainly discuss the role of PTEN in regulating the development, glucose metabolism, stem cell fate decision, and regeneration of skeletal muscle.