The mitochondrial protein OPA1 regulates the quiescent state of adult muscle stem cells.

Quiescence regulation is essential for adult stem cell maintenance and sustained regeneration. Our studies uncovered that physiological changes in mitochondrial shape regulate the quiescent state of adult muscle stem cells (MuSCs). We show that MuSC mitochondria rapidly fragment upon an activation stimulus, via systemic HGF/mTOR, to drive the exit from deep quiescence. Deletion of the mitochondrial fusion protein OPA1 and mitochondrial fragmentation transitions MuSCs into G-alert quiescence, causing premature activation and depletion upon a stimulus. OPA1 loss activates a glutathione (GSH)-redox signaling pathway promoting cell-cycle progression, myogenic gene expression, and commitment. MuSCs with chronic OPA1 loss, leading to mitochondrial dysfunction, continue to reside in G-alert but acquire severe cell-cycle defects. Additionally, we provide evidence that OPA1 decline and impaired mitochondrial dynamics contribute to age-related MuSC dysfunction. These findings reveal a fundamental role for OPA1 and mitochondrial dynamics in establishing the quiescent state and activation potential of adult stem cells.

GLI3 regulates muscle stem cell entry into GAlert and self-renewal.

Satellite cells are required for the growth, maintenance, and regeneration of skeletal muscle. Quiescent satellite cells possess a primary cilium, a structure that regulates the processing of the GLI family of transcription factors. Here we find that GLI3 processing by the primary cilium plays a critical role for satellite cell function. GLI3 is required to maintain satellite cells in a G0 dormant state. Strikingly, satellite cells lacking GLI3 enter the GAlert state in the absence of injury. Furthermore, GLI3 depletion stimulates expansion of the stem cell pool. As a result, satellite cells lacking GLI3 display rapid cell-cycle entry, increased proliferation and augmented self-renewal, and markedly enhanced regenerative capacity. At the molecular level, we establish that the loss of GLI3 induces mTORC1 signaling activation. Therefore, our results provide a mechanism by which GLI3 controls mTORC1 signaling, consequently regulating muscle stem cell activation and fate.

Analysis of human satellite cell dynamics on cultured adult skeletal muscle myofibers.

BACKGROUND: Maintaining stem cells in physiologically relevant states is necessary to understand cell and context-specific signalling paradigms and to understand complex interfaces between cells in situ. Understanding human stem cell function is largely based on tissue biopsies, cell culture, and transplantation into model organisms. METHODS: Here, we describe a method to isolate post-mortem intact human muscle myofibers and culture muscle stem cells within the niche microenvironment to assay cellular dynamics, stem cell identity, stem cell hierarchy, and differentiation potential. RESULTS: We show human myofiber culture maintains complex cell-cell contacts and extracellular niche composition during culture. Human satellite cells can be cultured at least 8 days, which represents a timepoint of activation, differentiation, and de novo human myofiber formation. We demonstrate that adult human muscle stem cells undergo apicobasal and planar cell divisions and express polarized dystrophin and EGFR. Furthermore, we validate that stimulation of the EGFR pathway stimulates the generation of myogenic progenitors and myogenic differentiation. CONCLUSIONS: This method provides proof of principle evidence for the use of human muscle to evaluate satellite cell dynamics and has applications in pre-clinical evaluation of therapeutics targeting muscle repair.

Isolation of satellite cells and transplantation into mice for lineage tracing in muscle.

Limited methods exist to assay the direct effects of therapeutic intervention on muscle stem cell fate, proliferation or differentiation in an in vivo context. Here we provide an optimized protocol for muscle stem cell isolation and transplantation into mice to deconvolute heterogeneity within isolated stem cell populations. Viable and pure cell populations are isolated within 2 h and can then be used for therapeutic intervention or transplantation to uncover the repopulating and differentiation potential in mice, a physiologically relevant in vivo context. Effects can be assessed 9 d after transplantation. This methodology analyzes cell and sort purity prior to transplantation to improve reproducibility and outlines novel blocking steps to improve tissue staining and analysis. Experience with surgical procedures in mice is recommended before attempting this protocol. Our system is widely applicable for exploring stem cell dynamics within muscle and has already been used to study heterogeneity within muscle stem cell populations and efficacy of therapeutic intervention on isolated stem cell populations.

EGFR-Aurka Signaling Rescues Polarity and Regeneration Defects in Dystrophin-Deficient Muscle Stem Cells by Increasing Asymmetric Divisions.

Loss of dystrophin expression in Duchenne muscular dystrophy (DMD) causes progressive degeneration of skeletal muscle, which is exacerbated by reduced self-renewing asymmetric divisions of muscle satellite cells. This, in turn, affects the production of myogenic precursors and impairs regeneration and suggests that increasing such divisions may be beneficial. Here, through a small-molecule screen, we identified epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurka) as regulators of asymmetric satellite cell divisions. Inhibiting EGFR causes a substantial shift from asymmetric to symmetric division modes, whereas EGF treatment increases asymmetric divisions. EGFR activation acts through Aurka to orient mitotic centrosomes, and inhibiting Aurka blocks EGF stimulation-induced asymmetric division. In vivo EGF treatment markedly activates asymmetric divisions of dystrophin-deficient satellite cells in mdx mice, increasing progenitor numbers, enhancing regeneration, and restoring muscle strength. Therefore, activating an EGFR-dependent polarity pathway promotes functional rescue of dystrophin-deficient satellite cells and enhances muscle force generation.

Orienting Muscle Stem Cells for Regeneration in Homeostasis, Aging, and Disease.

Muscle stem cells, or satellite cells, are required for skeletal muscle maintenance, growth, and repair. Following satellite cell activation, several factors drive asymmetric cell division to generate a stem cell and a proliferative progenitor that forms new muscle. The balance between symmetric self-renewal and asymmetric division significantly impacts the efficiency of regeneration. In this Review, we discuss the relationship of satellite cell heterogeneity and the establishment of polarity to asymmetric division, as well as how these processes are impacted in homeostasis, aging, and disease. We also highlight therapeutic opportunities for targeting satellite cell polarity and self-renewal to stimulate muscle regeneration.

Muscle stem cells.

Feige and Rudnicki introduce muscle stem cells.

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors.

Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types.