Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in rainbow trout (Oncorhynchus mykiss).

Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly identified by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope®) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization in rainbow trout. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

Deletion of TECRL promotes skeletal muscle repair by up-regulating EGR2.

Myogenic regeneration relies on the proliferation and differentiation of satellite cells. TECRL (trans-2,3-enoyl-CoA reductase like) is an endoplasmic reticulum protein only expressed in cardiac and skeletal muscle. However, its role in myogenesis remains unknown. We show that TECRL expression is increased in response to injury. Satellite cell-specific deletion of TECRL enhances muscle repair by increasing the expression of EGR2 through the activation of the ERK1/2 signaling pathway, which in turn promotes the expression of PAX7. We further show that TECRL deletion led to the upregulation of the histone acetyltransferase general control nonderepressible 5, which enhances the transcription of EGR2 through acetylation. Importantly, we showed that AAV9-mediated TECRL silencing improved muscle repair in mice. These findings shed light on myogenic regeneration and muscle repair.

Dispase/collagenase cocktail allows for coisolation of satellite cells and fibroadipogenic progenitors from human skeletal muscle.

Satellite cells (SCs) and fibroadipogenic progenitors (FAPs) are progenitor populations found in muscle that form new myofibers postinjury. Muscle development, regeneration, and tissue-engineering experiments require robust progenitor populations, yet their isolation and expansion are difficult given their scarcity in muscle, limited muscle biopsy sizes in humans, and lack of methodological detail in the literature. Here, we investigated whether a dispase and collagenase type 1 and 2 cocktail could allow dual isolation of SCs and FAPs, enabling significantly increased yield from human skeletal muscle. Postdissociation, we found that single cells could be sorted into CD56 + CD31-CD45- (SC) and CD56-CD31-CD45- (FAP) cell populations, expanded in culture, and characterized for lineage-specific marker expression and differentiation capacity; we obtained ∼10% SCs and ∼40% FAPs, with yields twofold better than what is reported in current literature. SCs were PAX7+ and retained CD56 expression and myogenic fusion potential after multiple passages, expanding up to 1012 cells. Conversely, FAPs expressed CD140a and differentiated into either fibroblasts or adipocytes upon induction. This study demonstrates robust isolation of both SCs and FAPs from the same muscle sample with SC recovery more than two times higher than previously reported, which could enable translational studies for muscle injuries.NEW & NOTEWORTHY We demonstrated that a dispase/collagenase cocktail allows for simultaneous isolation of SCs and FAPs with 2× higher SC yield compared with other studies. We provide a thorough characterization of SC and FAP in vitro expansion that other studies have not reported. Following our dissociation, SCs and FAPs were able to expand by up to 1012 cells before reaching senescence and maintained differentiation capacity in vitro demonstrating their efficacy for clinical translation for muscle injury.

Satellite cells in the growth and maintenance of muscle.

Embryonic skeletal muscle growth is contingent upon a population of somite derived satellite cells, however, the contribution of these cells to early postnatal skeletal muscle growth remains relatively high. As prepubertal postnatal development proceeds, the activity and contribution of satellite cells to skeletal muscle growth diminishes. Eventually, at around puberty, a population of satellite cells escapes terminal commitment, continues to express the paired box transcription factor Pax7, and reside in a quiescent state orbiting the myofiber periphery adjacent to the basal lamina. After adolescence, some satellite cell contributions to muscle maintenance and adaptation occur, however, their necessity is reduced relative to embryonic, early postnatal, and prepubertal growth.

The extracellular matrix niche of muscle stem cells.

Preserving the potency of stem cells in adult tissues is very demanding and relies on the concerted action of various cellular and non-cellular elements in a precise stoichiometry. This balanced microenvironment is found in specific anatomical "pockets" within the tissue, known as the stem cell niche. In this review, we explore the interplay between stem cells and their niches, with a primary focus on skeletal muscle stem cells and the extracellular matrix (ECM). Quiescent muscle stem cells, known as satellite cells are active producers of a diverse array of ECM molecules, encompassing major constituents like collagens, laminins, and integrins, some of which are explored in this review. The conventional perception of ECM as merely a structural scaffold is evolving. Collagens can directly interact as ligands with receptors on satellite cells, while other ECM proteins have the capacity to sequester growth factors and regulate their release, especially relevant during satellite cell turnover in homeostasis or activation upon injury. Additionally, we explore an evolutionary perspective on the ECM across a range of multicellular organisms and discuss a model wherein satellite cells are self-sustained by generating their own niche. Considering the prevalence of ECM proteins in the connective tissue of various organs it is not surprising that mutations in ECM genes have pathological implications, including in muscle, where they can lead to myopathies. However, the particular role of certain disease-related ECM proteins in stem cell maintenance highlights the potential contribution of stem cell deregulation to the progression of these disorders.

Role of microenvironment on muscle stem cell function in health, adaptation, and disease.

The role of the cellular microenvironment has recently gained attention in the context of muscle health, adaption, and disease. Emerging evidence supports major roles for the extracellular matrix (ECM) in regeneration and the dynamic regulation of the satellite cell niche. Satellite cells normally reside in a quiescent state in healthy muscle, but upon muscle injury, they activate, proliferate, and fuse to the damaged fibers to restore muscle function and architecture. This chapter reviews the composition and mechanical properties of skeletal muscle ECM and the role of these factors in contributing to the satellite cell niche that impact muscle regeneration. In addition, the chapter details the effects of satellite cell-matrix interactions and provides evidence that there is bidirectional regulation affecting both the cellular and extracellular microenvironment within skeletal muscle. Lastly, emerging methods to investigate satellite cell-matrix interactions will be presented.

Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-ß-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.

Restoring Mitochondrial Function and Muscle Satellite Cell Signaling: Remedies against Age-Related Sarcopenia.

Sarcopenia has a complex pathophysiology that encompasses metabolic dysregulation and muscle ultrastructural changes. Among the drivers of intracellular and ultrastructural changes of muscle fibers in sarcopenia, mitochondria and their quality control pathways play relevant roles. Mononucleated muscle stem cells/satellite cells (MSCs) have been attributed a critical role in muscle repair after an injury. The involvement of mitochondria in supporting MSC-directed muscle repair is unclear. There is evidence that a reduction in mitochondrial biogenesis blunts muscle repair, thus indicating that the delivery of functional mitochondria to injured muscles can be harnessed to limit muscle fibrosis and enhance restoration of muscle function. Injection of autologous respiration-competent mitochondria from uninjured sites to damaged tissue has been shown to reduce infarct size and enhance cell survival in preclinical models of ischemia-reperfusion. Furthermore, the incorporation of donor mitochondria into MSCs enhances lung and cardiac tissue repair. This strategy has also been tested for regeneration purposes in traumatic muscle injuries. Indeed, the systemic delivery of mitochondria promotes muscle regeneration and restores muscle mass and function while reducing fibrosis during recovery after an injury. In this review, we discuss the contribution of altered MSC function to sarcopenia and illustrate the prospect of harnessing mitochondrial delivery and restoration of MSCs as a therapeutic strategy against age-related sarcopenia.

Regulation of muscle hypertrophy through granulin: Relayed communication among mesenchymal progenitors, macrophages, and satellite cells.

Skeletal muscles exert remarkable regenerative or adaptive capacities in response to injuries or mechanical loads. However, the cellular networks underlying muscle adaptation are poorly understood compared to those underlying muscle regeneration. We employed single-cell RNA sequencing to investigate the gene expression patterns and cellular networks activated in overloaded muscles and compared these results with those observed in regenerating muscles. The cellular composition of the 4-day overloaded muscle, when macrophage infiltration peaked, closely resembled that of the 10-day regenerating muscle. In addition to the mesenchymal progenitor-muscle satellite cell (MuSC) axis, interactome analyses or targeted depletion experiments revealed communications between mesenchymal progenitors-macrophages and macrophages-MuSCs. Furthermore, granulin, a macrophage-derived factor, inhibited MuSC differentiation, and Granulin-knockout mice exhibited blunted muscle hypertrophy due to the premature differentiation of overloaded MuSCs. These findings reveal the critical role of granulin through the relayed communications of mesenchymal progenitors, macrophages, and MuSCs in facilitating efficient muscle hypertrophy.

Lineage tracing reveals a novel PDGFRß+ satellite cell subset that contributes to myo-regeneration of chronically injured rotator cuff muscle.

Massive rotator cuff (RC) tendon tears are associated with progressive fibro-adipogenesis and muscle atrophy that altogether cause shoulder muscle wasting. Platelet derived growth factor ß (PDGFRß) lineage cells, that co-express PDGFRα have previously been shown to directly contribute to scar formation and fat accumulation in a mouse model of irreversible tendon and nerve transection (TTDN). Conversely, PDGFRß+ lineage cells have also been  shown to be myogenic in cultures and in other models of skeletal muscle injury. We therefore hypothesized that PDGFRß demarcates two distinct RC residing subpopulations, fibro-adipogenic and myogenic, and aimed to elucidate the identity of the PDGFRß myogenic precursors and evaluate their contribution, if any, to RC myo-regeneration. Lineage tracing revealed increasing contribution of PDGFRß+ myo-progenitors to the formation of GFP+ myofibers, which were the most abundant myofiber type in regenerated muscle at 2 weeks post-TTDN. Muscle regeneration preceded muscle atrophy and both advanced from the lateral site of tendon transection to the farthest medial region. GFP+/PDGFRß+Sca-1-lin-CXCR4+Integrin-ß1+ marked a novel subset of satellite cells with confirmed myogenic properties. Further studies are warranted to identify the existence of PDGFRß+ satellite cells in human and other mouse muscles and to define their myo-regenerative potential following acute and chronic muscle injury.

Vitamin C regulates skeletal muscle post-injury regeneration by promoting myoblast proliferation through its direct interaction with the Pax7 protein.

Previous studies have shown that vitamin C (VC), an essential vitamin for the human body, can promote the differentiation of muscle satellite cells (MuSCs) in vitro and play an important role in skeletal muscle post-injury regeneration. However, the molecular mechanism of VC regulating MuSC proliferation has not been elucidated. In this study, the role of VC in promoting MuSC proliferation and its molecular mechanism were explored using cell molecular biology and animal experiments. The results showed that VC accelerates the progress of skeletal muscle post-injury regeneration by promoting MuSC proliferation in vivo. VC can also promote skeletal muscle regeneration in the case of atrophy. Using the C2C12 myoblast murine cell line, we observed that VC also stimulated cell proliferation. In addition, after an in vitro study establishing the occurrence of a physical interaction between VC and Pax7, we observed that VC also upregulated the total and nuclear Pax7 protein levels. This mechanism increased the expression of Myf5 (Myogenic Factor 5), a Pax7 target gene. This study establishes a theoretical foundation for understanding the regulatory mechanisms underlying VC-mediated MuSC proliferation and skeletal muscle regeneration. Moreover, it develops the application of VC in animal muscle nutritional supplements and treatment of skeletal muscle-related diseases.

MyoD Over-Expression Rescues GST-bFGF Repressed Myogenesis.

During embryogenesis, basic fibroblast growth factor (bFGF) is released from neural tube and myotome to promote myogenic fate in the somite, and is routinely used for the culture of adult skeletal muscle (SKM) stem cells (MuSC, called satellite cells). However, the mechanism employed by bFGF to promote SKM lineage and MuSC proliferation has not been analyzed in detail. Furthermore, the question of if the post-translational modification (PTM) of bFGF is important to its stemness-promoting effect has not been answered. In this study, GST-bFGF was expressed and purified from E.coli, which lacks the PTM system in eukaryotes. We found that both GST-bFGF and commercially available bFGF activated the Akt-Erk pathway and had strong cell proliferation effect on C2C12 myoblasts and MuSC. GST-bFGF reversibly compromised the myogenesis of C2C12 myoblasts and MuSC, and it increased the expression of Myf5, Pax3/7, and Cyclin D1 but strongly repressed that of MyoD, suggesting the maintenance of myogenic stemness amid repressed MyoD expression. The proliferation effect of GST-bFGF was conserved in C2C12 over-expressed with MyoD (C2C12-tTA-MyoD), implying its independence of the down-regulation of MyoD. In addition, the repressive effect of GST-bFGF on myogenic differentiation was almost totally rescued by the over-expression of MyoD. Together, these evidences suggest that (1) GST-bFGF and bFGF have similar effects on myogenic cell proliferation and differentiation, and (2) GST-bFGF can promote MuSC stemness and proliferation by differentially regulating MRFs and Pax3/7, (3) MyoD repression by GST-bFGF is reversible and independent of the proliferation effect, and (4) GST-bFGF can be a good substitute for bFGF in sustaining MuSC stemness and proliferation.

Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7+satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies.

Muscle stem cells as immunomodulator during regeneration.

The skeletal muscle is well known for its remarkable ability to regenerate after injuries. The regeneration is a complex and dynamic process that involves muscle stem cells (also called muscle satellite cells, MuSCs), fibro-adipogenic progenitors (FAPs), immune cells, and other muscle-resident cell populations. The MuSCs are the myogenic cell populaiton that contribute nuclei directly to the regenerated myofibers, while the other cell types collaboratively establish a microenvironment that facilitates myogenesis of MuSCs. The myogenic process includes activation, proliferation and differentiationof MuSCs, and subsequent fusion their descendent mononuclear myocytes into multinuclear myotubes. While the contributions of FAPs and immune cells to this microenvironment have been well studied, the influence of MuSCs on other cell types remains poorly understood. This review explores recent evidence supporting the potential role of MuSCs as immunomodulators during muscle regeneration, either through cytokine production or ligand-receptor interactions.

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.

Navigating translational control of gene expression in satellite cells.

Satellite cells, named for their satellite position around the sarcolemma of the myofibre, are responsible for skeletal muscle regeneration. Satellite cells normally reside in a quiescent state, but rapidly activate the myogenic program and the cell cycle in response to injury. Translational control of gene expression has emerged as an important regulator of satellite cell activity. Quiescent satellite cells maintain low levels of protein synthesis and selectively translate specific mRNAs to conserve limited energy. Activated satellite cells rapidly restore global protein synthesis to meet the demands of proliferating myogenic progenitors that participate in muscle repair. We propose a model by which translational control enables rapid protein level changes in response to injury-induced environmental shifts, serving as both a brake mechanism during quiescence and an accelerator for injury response. In this Chapter, we navigate the processing, translation and metabolism of newly transcribed mRNAs. We review the modifications of mRNA that occur during mRNA processing in the nucleus of satellite cells, and illustrate how these modifications impact the translation and stability of mRNAs. In the cytoplasm, we review how pathways work in concert to regulate protein synthesis globally, while trans acting microRNAs and RNA binding proteins modify specific mRNA translation within a context of tightly regulated protein synthesis. While navigating translational control of gene expression in satellite cells, this chapter reveals that despite significant progress, the field remains nascent in the broader scope of translational control in cell biology. We propose that future investigations will benefit from incorporating emerging global analyses to study translational control of gene expression in rare satellite cells, and we pose unanswered questions that warrant future exploration.

3D organization of enhancers in MuSCs.

Skeletal muscle stem cells (MuSCs), also known as satellite cells, are essential for muscle growth and injury induced regeneration. In healthy adult muscle, MuSCs remain in a quiescent state located in a specialized niche beneath the basal lamina. Upon injury, these dormant MuSCs can quickly activate to re-enter the cell cycle and differentiate into new myofibers, while a subset undergoes self-renewal and returns to quiescence to restore the stem cell pool. The myogenic lineage progression is intricately controlled by complex intrinsic and extrinsic cues and coupled with dynamic transcriptional programs. In transcriptional regulation, enhancers are key regulatory elements controlling spatiotemporal gene expression through physical contacting promoters of target genes. The three-dimensional (3D) chromatin architecture is known to orchestrate the establishment of proper enhancer-promoter interactions throughout development and aging. However, studies dissecting the 3D organization of enhancers in MuSCs are just emerging. Here, we provide an overview of the general properties of enhancers and newly developed methods for assessing their activity. In particular, we summarize recent discoveries regarding the 3D rewiring of enhancers during MuSC specification, lineage progression as well as aging.

Circadian timing of satellite cell function and muscle regeneration.

Recent research has highlighted an important role for the molecular circadian machinery in the regulation of tissue-specific function and stress responses. Indeed, disruption of circadian function, which is pervasive in modern society, is linked to accelerated aging, obesity, and type 2 diabetes. Furthermore, evidence supporting the importance of the circadian clock within both the mature muscle tissue and satellite cells to regulate the maintenance of muscle mass and repair capacity in response injury has recently emerged. Here, we review the discovery of circadian clocks within the satellite cell (a.k.a. adult muscle stem cell) and how they act to regulate metabolism, epigenetics, and myogenesis during both healthy and diseased states.

Chromatin organization of muscle stem cell.

The proper functioning of skeletal muscles is essential throughout life. A crucial crosstalk between the environment and several cellular mechanisms allows striated muscles to perform successfully. Notably, the skeletal muscle tissue reacts to an injury producing a completely functioning tissue. The muscle's robust regenerative capacity relies on the fine coordination between muscle stem cells (MuSCs or "satellite cells") and their specific microenvironment that dictates stem cells' activation, differentiation, and self-renewal. Critical for the muscle stem cell pool is a fine regulation of chromatin organization and gene expression. Acquiring a lineage-specific 3D genome architecture constitutes a crucial modulator of muscle stem cell function during development, in the adult stage, in physiological and pathological conditions. The context-dependent relationship between genome structure, such as accessibility and chromatin compartmentalization, and their functional effects will be analysed considering the improved 3D epigenome knowledge, underlining the intimate liaison between environmental encounters and epigenetics.