This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.

With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs, isolated from young wild-type mice, to progeroid mice confer significant lifespan and healthspan extension. The transplanted MDSPCs improve degenerative changes and vascularization in tissues where donor cells are not detected, suggesting that their therapeutic effect may be mediated by secreted factor(s). Indeed, young wild-type-MDSPCs rescue proliferation and differentiation defects of aged MDSPCs when co-cultured. These results establish that adult stem/progenitor cell dysfunction contributes to ageing-related degeneration and suggests a therapeutic potential of post-natal stem cells to extend health.

We have isolated a population of muscle-derived stem cells (MDSCs) that, when compared with myoblasts, display an improved regeneration capacity, exhibit better cell survival, and improve myogenesis and angiogenesis. In addition, we and others have observed that the origin of the MDSCs may reside within the blood vessel walls (endothelial cells and pericytes). Here, we investigated the role of vascular endothelial growth factor (VEGF)-mediated angiogenesis in MDSC transplantation-based skeletal muscle regeneration in mdx mice (an animal model of muscular dystrophy). We studied MDSC and MDSC transduced to overexpress VEGF; no differences were observed in vitro in terms of phenotype or myogenic differentiation. However, after in vivo transplantation, we observe an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in muscle fibrosis in muscles transplanted with VEGF-expressing cells when compared to control cells. In contrast, we observe a significant decrease in vascularization and an increase in fibrosis in the muscles transplanted with MDSCs expressing soluble forms-like tyrosine kinase 1 (sFlt1) (VEGF-specific antagonist) when compared to control MDSCs. Our results indicate that VEGF-expressing cells do not increase the number of dystrophin-positive fibers in the injected mdx muscle, when compared to the control MDSCs. Together the results suggest that the transplantation of VEGF-expressing MDSCs improved skeletal muscle repair through modulation of angiogenesis, regeneration and fibrosis in the injected mdx skeletal muscle.