The satellite cell in skeletal muscle: A story of heterogeneity.

Skeletal muscle is a highly represented tissue in mammals and is composed of fibers that are extremely adaptable and capable of regeneration. This characteristic of muscle fibers is made possible by a cell type called satellite cells. Adjacent to the fibers, satellite cells are found in a quiescent state and located between the muscle fibers membrane and the basal lamina. These cells are required for the growth and regeneration of skeletal muscle through myogenesis. This process is known to be tightly sequenced from the activation to the differentiation/fusion of myofibers. However, for the past fifteen years, researchers have been interested in examining satellite cell heterogeneity and have identified different subpopulations displaying distinct characteristics based on localization, quiescence state, stemness capacity, cell-cycle progression or gene expression. A small subset of satellite cells appears to represent multipotent long-term self-renewing muscle stem cells (MuSC). All these distinctions led us to the hypothesis that the characteristics of myogenesis might not be linear and therefore may be more permissive based on the evidence that satellite cells are a heterogeneous population. In this review, we discuss the different subpopulations that exist within the satellite cell pool to highlight the heterogeneity and to gain further understanding of the myogenesis progress. Finally, we discuss the long term self-renewing MuSC subpopulation that is capable of dividing asymmetrically and discuss the molecular mechanisms regulating MuSC polarization during health and disease.

Wnt7a is Required for Regeneration of Dystrophic Skeletal Muscle.

Intramuscular injection of Wnt7a has been shown to accelerate and augment skeletal muscle regeneration and to ameliorate dystrophic progression in mdx muscle, a model for Duchenne muscular dystrophy (DMD). However, loss-of-function studies to investigate the requirement for Wnt7a in muscle regeneration has not been evaluated. Here, we assessed muscle regeneration and function in wild type (WT) and mdx mice where Wnt7a was specifically deleted in muscle using a conditional Wnt7a floxed allele and a Myf5-Cre driver. We found that both WT and mdx mice with deletion of Wnt7a in muscle, exhibited marked deficiencies in muscle regeneration at 21 d following cardiotoxin (CTX) induced injury. Unlike WT, deletion of Wnt7a in mdx resulted in a marked decrease in specific force generation prior to CTX injury. However, both WT and mdx muscle lacking Wnt7a displayed decreased specific force generation following CTX injection. Notably the regeneration deficit observed in mdx mice lacking Wnt7a in muscle was rescued by a single tail vein injection of an extracellular vesicle preparation containing Wnt7a (Wnt7a-EVs). Therefore, we conclude that the regenerative capacity of muscle in mdx mice is due to the upregulation of endogenous Wnt7a following injury, and that systemic delivery of Wnt7a-EVs represents a therapeutic strategy for treating DMD.

Isolation of small extracellular vesicles from regenerating muscle tissue using Tangential Flow Filtration and Size Exclusion Chromatography.

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Wnt binding to Coatomer proteins directs secretion on exosomes independently of palmitoylation.

Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo . Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.

Satellite cell contribution to disease pathology in Duchenne muscular dystrophy.

Progressive muscle weakness and degeneration characterize Duchenne muscular dystrophy (DMD), a lethal, x-linked neuromuscular disorder that affects 1 in 5,000 boys. Loss of dystrophin protein leads to recurrent muscle degeneration, progressive fibrosis, chronic inflammation, and dysfunction of skeletal muscle resident stem cells, called satellite cells. Unfortunately, there is currently no cure for DMD. In this mini review, we discuss how satellite cells in dystrophic muscle are functionally impaired, and how this contributes to the DMD pathology, and the tremendous potential of restoring endogenous satellite cell function as a viable treatment strategy to treat this debilitating and fatal disease.

Quantification of Muscle Satellite Stem Cell Divisions by High-Content Analysis.

High-content screening is commonly performed on 2D cultured cells, which is high throughput but has low biological relevance. In contrast, single myofiber culture assay preserves the satellite cell niche between the basal lamina and sarcolemma and consequently has high biological relevance but is low throughput. We describe here a high-content screening method that utilizes single myofiber culture that addresses the caveats of both techniques. Our method utilizes the transgenic reporter allele Myf5-Cre:R26R-eYFP to differentiate stem and committed cells within a dividing couplet that can be quantified by high-content throughput immunodetection and bioinformatic analysis.

Immunofluorescence Labeling of Skeletal Muscle in Development, Regeneration, and Disease.

Skeletal muscle is composed of long multinucleated cells, termed myofibers, that are formed through the activation and differentiation of resident muscle stem cells, called satellite cells. In healthy individuals, skeletal muscle enables voluntary locomotion while also playing a role in energy metabolism and thermoregulation. As skeletal muscle is integral to everyday processes, perturbations to skeletal muscle function can have devastating consequences. Here we describe an integral tool in biomedical research of skeletal muscle regeneration and disease, the immunofluorescence staining of myogenic cells. We highlight useful techniques for immunostaining myogenic cells, and we list validated antibodies for the staining of muscle proteins across different species and multiple developmental time points. This includes methods for unmasking antigens following formaldehyde fixation (using myosin heavy chain staining as an example) and practices for preserving endogenous fluorescent proteins by cardiac perfusion fixation.

Carm1 and the Epigenetic Control of Stem Cell Function.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a methyltransferase whose function has been highly studied in the context of nuclear receptor signaling. However, CARM1 is known to epigenetically regulate expression of several myogenic genes involved in differentiation such as Myog and MEF2C. CARM1 also acts to regulate myogenesis through its influence on various cellular processes from embryonic to adult myogenesis. First, CARM1 has a crucial role in establishing polarity-regulated gene expression during an asymmetric satellite cell division by methylating PAX7, leading to the expression of Myf5. Second, satellite cells express the CARM1-FL and CARM1-ΔE15 isoforms. The former has been shown to promote pre-mRNA splicing through its interaction with CA150 and U1C, leading to their methylation and increased activity, while the latter displays a reduction in both metrics, thus, modulating alternative pre-mRNA splice forms in muscle cells. Third, CARM1 is a regulator of autophagy through its positive reinforcement of AMPK activity and gene expression. Autophagy already has known implications in ageing and disease, and CARM1 could follow suite. Thus, CARM1 is a central regulator of several important processes impacting muscle stem cell function and myogenesis.

Transplantation to study satellite cell heterogeneity in skeletal muscle.

Skeletal muscle has a remarkable capacity to regenerate throughout life, which is mediated by its resident muscle stem cells, also called satellite cells. Satellite cells, located periphery to the muscle fibers and underneath the basal lamina, are an indispensable cellular source for muscle regeneration. Satellite cell transplantation into regenerating muscle contributes robustly to muscle repair, thereby indicating that satellite cells indeed function as adult muscle stem cells. Moreover, satellite cells are a heterogenous population in adult tissue, with subpopulations that can be distinguished based on gene expression, cell-cycle progression, ability to self-renew, and bi-potential ability. Transplantation assays provide a powerful tool to better understand satellite cell function in vivo enabling the separation of functionally distinct satellite cell subpopulations. In this review, we focus on transplantation strategies to explore satellite cells' functional heterogeneity, approaches targeting the recipient tissue to improve transplantation efficiency, and common strategies to monitor the behaviour of the transplanted cells. Lastly, we discuss some recent approaches to overcome challenges to enhance the transplantation potential of muscle stem cells.

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.

Acetylation of PAX7 controls muscle stem cell self-renewal and differentiation potential in mice.

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation.

Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

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.

Satellite cells in ageing: use it or lose it.

Individuals that maintain healthy skeletal tissue tend to live healthier, happier lives as proper muscle function enables maintenance of independence and actuation of autonomy. The onset of skeletal muscle decline begins around the age of 30, and muscle atrophy is associated with a number of serious morbidities and mortalities. Satellite cells are responsible for regeneration of skeletal muscle and enter a reversible non-dividing state of quiescence under homeostatic conditions. In response to injury, satellite cells are able to activate and re-enter the cell cycle, creating new cells to repair and create nascent muscle fibres while preserving a small population that can return to quiescence for future regenerative demands. However, in aged muscle, satellite cells that experience prolonged quiescence will undergo programmed cellular senescence, an irreversible non-dividing state that handicaps the regenerative capabilities of muscle. This review examines how periodic activation and cycling of satellite cells through exercise can mitigate senescence acquisition and myogenic decline.

Single-cell analyses uncover granularity of muscle stem cells.

Satellite cells are the main muscle-resident cells responsible for muscle regeneration. Much research has described this population as being heterogeneous, but little is known about the different roles each subpopulation plays. Recent advances in the field have utilized the power of single-cell analysis to better describe and functionally characterize subpopulations of satellite cells as well as other cell groups comprising the muscle tissue. Furthermore, emerging technologies are opening the door to answering as-yet-unresolved questions pertaining to satellite cell heterogeneity and cell fate decisions.

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.

MLL1 is required for PAX7 expression and satellite cell self-renewal in mice.

PAX7 is a paired-homeobox transcription factor that specifies the myogenic identity of muscle stem cells and acts as a nodal factor by stimulating proliferation while inhibiting differentiation. We previously found that PAX7 recruits the H3K4 methyltransferases MLL1/2 to epigenetically activate target genes. Here we report that in the absence of Mll1, myoblasts exhibit reduced H3K4me3 at both Pax7 and Myf5 promoters and reduced Pax7 and Myf5 expression. Mll1-deficient myoblasts fail to proliferate but retain their differentiation potential, while deletion of Mll2 had no discernable effect. Re-expression of PAX7 in committed Mll1 cKO myoblasts restored H3K4me3 enrichment at the Myf5 promoter and Myf5 expression. Deletion of Mll1 in satellite cells reduced satellite cell proliferation and self-renewal, and significantly impaired skeletal muscle regeneration. Pax7 expression was unaffected in quiescent satellite cells but was markedly downregulated following satellite cell activation. Therefore, MLL1 is required for PAX7 expression and satellite cell function in vivo. Furthermore, PAX7, but not MLL1, is required for Myf5 transcriptional activation in committed myoblasts.

Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression.

Human embryonic stem cell (hESC)-derived skeletal muscle progenitors (SMP)-defined as PAX7-expressing cells with myogenic potential-can provide an abundant source of donor material for muscle stem cell therapy. As in vitro myogenesis is decoupled from in vivo timing and 3D-embryo structure, it is important to characterize what stage or type of muscle is modeled in culture. Here, gene expression profiling is analyzed in hESCs over a 50 day skeletal myogenesis protocol and compared to datasets of other hESC-derived skeletal muscle and adult murine satellite cells. Furthermore, day 2 cultures differentiated with high or lower concentrations of CHIR99021, a GSK3A/GSK3B inhibitor, were contrasted. Expression profiling of the 50 day time course identified successively expressed gene subsets involved in mesoderm/paraxial mesoderm induction, somitogenesis, and skeletal muscle commitment/formation which could be regulated by a putative cascade of transcription factors. Initiating differentiation with higher CHIR99021 concentrations significantly increased expression of MSGN1 and TGFB-superfamily genes, notably NODAL, resulting in enhanced paraxial mesoderm and reduced ectoderm/neuronal gene expression. Comparison to adult satellite cells revealed that genes expressed in 50-day cultures correlated better with those expressed by quiescent or early activated satellite cells, which have the greatest therapeutic potential. Day 50 cultures were similar to other hESC-derived skeletal muscle and both expressed known and novel SMP surface proteins. Overall, a putative cascade of transcription factors has been identified which regulates four stages of myogenesis. Subsets of these factors were upregulated by high CHIR99021 or their binding sites were significantly over-represented during SMP activation, ranging from quiescent to late-activated stages. This analysis serves as a resource to further study the progression of in vitro skeletal myogenesis and could be mined to identify novel markers of pluripotent-derived SMPs or regulatory transcription/growth factors. Finally, 50-day hESC-derived SMPs appear similar to quiescent/early activated satellite cells, suggesting they possess therapeutic potential.