Quiescent Muscle Stem Cells Research Articles

Enhanced secretion of promyogenic exosomes by quiescent muscle cells.

Signaling interactions are important during skeletal muscle regeneration, where muscle cells in distinct states (quiescent, reactivated, proliferating and differentiated) must coordinate their response to injury. Here, we probed the role of secreted small extracellular vesicles (sEV/exosomes) using a culture model of physiologically relevant cell states seen in muscle regeneration. Unexpectedly, G0 myoblasts exhibited enhanced secretion of sEV (∼150nm) displaying exosome markers (Alix, TSG101, flotillin-1, and CD9), and increased expression of Kibra, a regulator of exosome biogenesis. Perturbation of Kibra levels confirmed a role in controlling sEV secretion rates. Purified sEVs displayed a common exosome marker-enriched proteome in all muscle cell states, as well as state-specific proteins. Exosomes derived from G0 cells showed an antioxidant signature, and were most strongly internalized by differentiated myotubes. Functionally, donor exosomes from all muscle cell states could activate an integrated Wnt reporter in target cells, but only G0-derived exosomes could induce myogenic differentiation in proliferating cells. Taken together, we provide evidence that quiescence in muscle cells is accompanied by enhanced secretion of exosomes with distinct uptake, cargo and signal activating features. Our study suggests the novel possibility that quiescent muscle stem cells in vivo may play a previously under-appreciated signaling role during muscle homeostasis.

RhoA-mediated G12-G13 signaling maintains muscle stem cell quiescence and prevents stem cell loss

Multiple processes control quiescence of muscle stem cells (MuSCs), which is instrumental to guarantee long-term replenishment of the stem cell pool. Here, we describe that the G-proteins G12-G13 integrate signals from different G-protein-coupled receptors (GPCRs) to control MuSC quiescence via activation of RhoA. Comprehensive screening of GPCR ligands identified two MuSC-niche-derived factors, endothelin-3 (ET-3) and neurotensin (NT), which activate G12-G13 signaling in MuSCs. Stimulation with ET-3 or NT prevented MuSC activation, whereas pharmacological inhibition of ET-3 or NT attenuated MuSC quiescence. Inactivation of Gna12-Gna13 or Rhoa but not of Gnaq-Gna11 completely abrogated MuSC quiescence, which depleted the MuSC pool and was associated with accelerated sarcopenia during aging. Expression of constitutively active RhoA prevented exit from quiescence in Gna12-Gna13 mutant MuSCs, inhibiting cell cycle entry and differentiation via Rock and formins without affecting Rac1-dependent MuSC projections, a hallmark of quiescent MuSCs. The study uncovers a critical role of G12-G13 and RhoA signaling for active regulation of MuSC quiescence.

We need to talk-how muscle stem cells communicate.

Skeletal muscle is one of the tissues with the highest ability to regenerate, a finely controlled process which is critically depending on muscle stem cells. Muscle stem cell functionality depends on intrinsic signaling pathways and interaction with their immediate niche. Upon injury quiescent muscle stem cells get activated, proliferate and fuse to form new myofibers, a process involving the interaction of multiple cell types in regenerating skeletal muscle. Receptors in muscle stem cells receive the respective signals through direct cell-cell interaction, signaling via secreted factors or cell-matrix interactions thereby regulating responses of muscle stem cells to external stimuli. Here, we discuss how muscle stem cells interact with their immediate niche focusing on how this controls their quiescence, activation and self-renewal and how these processes are altered in age and disease.

A dual-color PAX7 and MYF5 in vivo reporter to investigate muscle stem cell heterogeneity in regeneration and aging

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5Low MuSCs and skews the stem cell pool toward MYF5High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles.

The E3 ubiquitin ligase Nedd4L preserves skeletal muscle stem cell quiescence by inhibiting their activation

Adult stem cells play a critical role in tissue repair and maintenance. In tissues with slow turnover, including skeletal muscle, these cells are maintained in a mitotically quiescent state yet remain poised to re-enter the cell cycle to replenish themselves and regenerate the tissue. Using a panomics approach we show that the PAX7/NEDD4L axis acts against muscle stem cell activation in homeostatic skeletal muscle. Our findings suggest that PAX7 transcriptionally activates the E3 ubiquitin ligase Nedd4L and that the conditional genetic deletion of Nedd4L impairs muscle stem cell quiescence, with an upregulation of cell cycle and myogenic differentiation genes. Loss of Nedd4L in muscle stem cells results in the expression of doublecortin (DCX), which is exclusively expressed during their invivo activation. Together, these data establish that the ubiquitin proteasome system, mediated by Nedd4L, is a key contributor to the muscle stem cell quiescent state in adult mice.

PINK1 deficiency alters muscle stem cell fate decision and muscle regenerative capacity

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions.

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.

Epsti1 Regulates the Inflammatory Stage of Early Muscle Regeneration through STAT1-VCP Interaction.

During muscle regeneration, interferon-gamma (IFN-γ) coordinates inflammatory responses critical for activation of quiescent muscle stem cells upon injury via the Janus kinase (JAK) - signal transducer and activator of transcription 1 (STAT1) pathway. Dysregulation of JAK-STAT1 signaling results in impaired muscle regeneration, leading to muscle dysfunction or muscle atrophy. Until now, the underlying molecular mechanism of how JAK-STAT1 signaling resolves during muscle regeneration remains largely elusive. Here, we demonstrate that epithelial-stromal interaction 1 (Epsti1), an interferon response gene, has a crucial role in regulating the IFN-γ-JAK-STAT1 signaling at early stage of muscle regeneration. Epsti1-deficient mice exhibit impaired muscle regeneration with elevated inflammation response. In addition, Epsti1-deficient myoblasts display aberrant interferon responses. Epsti1 interacts with valosin-containing protein (VCP) and mediates the proteasomal degradation of IFN-γ-activated STAT1, likely contributing to dampening STAT1-mediated inflammation. In line with the notion, mice lacking Epsti1 exhibit exacerbated muscle atrophy accompanied by increased inflammatory response in cancer cachexia model. Our study suggests a crucial function of Epsti1 in the resolution of IFN-γ-JAK-STAT1 signaling through interaction with VCP which provides insights into the unexplored mechanism of crosstalk between inflammatory response and muscle regeneration.

Absence of the primary cilia formation gene Talpid3 impairs muscle stem cell function

Skeletal muscle stem cells (MuSC) are crucial for tissue homoeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their requirement for muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Depletion of MuSC after regeneration suggests impaired self-renewal, also consistent with an exacerbated phenotype in TA3iSC-KO mice after repeat injury. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data show that TA3 and primary cilia are important for MuSC self-renewal and pharmacological treatment can efficiently restore muscle regeneration.

Postnatal skeletal muscle myogenesis governed by signal transduction networks: MAPKs and PI3K–Akt control multiple steps

Skeletal muscle myogenesis represents one of the most intensively and extensively examined systems of cell differentiation, tissue formation, and regeneration. Muscle regeneration provides an in vivo model system of postnatal myogenesis. It comprises multiple steps including muscle stem cell (or satellite cell) quiescence, activation, migration, myogenic determination, myoblast proliferation, myocyte differentiation, myofiber maturation, and hypertrophy. A variety of extracellular signaling and subsequent intracellular signal transduction pathways or networks govern the individual steps of postnatal myogenesis. Among them, MAPK pathways (the ERK, JNK, p38 MAPK, and ERK5 pathways) and PI3K–Akt signaling regulate multiple steps of myogenesis. Ca2+, cytokine, and Wnt signaling also participate in several myogenesis steps. These signaling pathways often control cell cycle regulatory proteins or the muscle-specific MyoD family and the MEF2 family of transcription factors. This article comprehensively reviews molecular mechanisms of the individual steps of postnatal skeletal muscle myogenesis by focusing on signal transduction pathways or networks. Nevertheless, no or only a partial signaling molecules or pathways have been identified in some responses during myogenesis. The elucidation of these unidentified signaling molecules and pathways leads to an extensive understanding of the molecular mechanisms of myogenesis.

Human muscle cells sensitivity to chikungunya virus infection relies on their glycolysis activity and differentiation stage

Changes to our environment have led to the emergence of human pathogens such as chikungunya virus. Chikungunya virus infection is today a major public health concern. It is a debilitating chronic disease impeding patients' mobility, affecting millions of people. Disease development relies on skeletal muscle infection. The importance of skeletal muscle in chikungunya virus infection led to the hypothesis that it could serve as a viral reservoir and could participate to virus persistence. Here we questioned the interconnection between skeletal muscle cells metabolism, their differentiation stage and the infectivity of the chikungunya virus. We infected human skeletal muscle stem cells at different stages of differentiation with chikungunya virus to study the impact of their metabolism on virus production and inversely the impact of virus on cell metabolism. We observed that chikungunya virus infectivity is cell differentiation and metabolism-dependent. Chikungunya virus interferes with the cellular metabolism in quiescent undifferentiated and proliferative muscle cells. Moreover, activation of chikungunya infected quiescent muscle stem cells, induces their proliferation, increases glycolysis and amplifies virus production. Therefore, our results showed that Chikungunya virus infectivity and the antiviral response of skeletal muscle cells relies on their energetic metabolism and their differentiation stage. Then, muscle stem cells could serve as viral reservoir producing virus after their activation.

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

BackgroundThe capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.MethodsIn the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.ResultsWe observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.ConclusionsOverall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.

Sugt1 loss in skeletal muscle stem cells impairs muscle regeneration and causes premature muscle aging

Abstract Adult skeletal muscle stem cells (MuSCs) are essential for muscle homeostasis and regeneration. During aging, the number of MuSCs and their regenerative capacity gradually decline but the underlying mechanisms remain elusive. Here, we identify Sugt1 (suppressor of G2 allele of SKP1 homolog), which is a chaperone for kinetochore function during mitosis and is essential for muscle regeneration by regulating MuSC proliferation. Sugt1 expression level is low in quiescent MuSCs but highly induced when the cells become activated and expand as proliferating myoblasts. Inducible inactivation of Sugt1 in MuSCs causes impaired muscle regeneration upon acute injury by impairing MuSC proliferation. Furthermore, loss of Sugt1 leads to cell cycle arrest in the G2/M phase and cellular senescence. Moreover, long-term loss of Sugt1 in MuSCs results in precocious muscle aging by inhibiting MuSC cell proliferation and promoting cellular senescence. Mechanistically, we identify a cytosolic E3 ubiquitin-ligase, Trim21 as a protein interacting partner for Sugt1 in myoblast cells. We further demonstrate that Sugt1 promotes the ubiquitination of p21 via Trim21; and Sugt1 loss causes p21 accumulation to inhibit cell cycle progression and stimulates cellular senescence. Collectively, our findings uncover that Sugt1 is an essential regulator for MuSC regenerative function during muscle regeneration and aging.

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Emerging links between metabolism and epigenetic regulation of muscle stem cells

Muscle regeneration in response to injury or exercise relies on the ability of muscle stem cells to proliferate and differentiate to repair the damage. In the absence of damage, muscle stem cells are quiescent: they do not proliferate and have a very low metabolism. Recent studies have linked the metabolic state of the adult muscle stem cell to its epigenetic regulation. This article synthesizes the known concepts about histone modifications and metabolic pathways found in quiescent muscle stem cells, as well as the metabolic and epigenetic changes leading to muscle stem cell activation in response to injury. Here, we discuss the heterogeneity in quiescent stem cell metabolism and compare the metabolism of quiescent and activated muscle stem cells, and describe the epigenetic changes related to their activation. We also discuss the involvement of SIRT1, an important effector of muscle stem cells metabolism, together with the effects of aging and caloric restriction.

Polycomb Ezh1 maintains murine muscle stem cell quiescence through non-canonical regulation of Notch signaling.

Organismal homeostasis and regeneration are predicated on committed stem cells that can reside for long periods in a mitotically dormant but reversible cell-cycle arrest state defined as quiescence. Premature escape from quiescence is detrimental, as it results in stem cell depletion, with consequent defective tissue homeostasis and regeneration. Here, we report that Polycomb Ezh1 confers quiescence to murine muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence. Following repeated injuries, the MuSC pool is progressively depleted, resulting in failure to sustain proper muscle regeneration. Rather than regulating repressive histone H3K27 methylation, Ezh1 maintains gene expression of the Notch signaling pathway in MuSCs. Selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs.

ApoE isoform does not influence skeletal muscle regeneration in adult mice.

Introduction: Apolipoprotein E (ApoE) has been shown to be necessary for proper skeletal muscle regeneration. Consistent with this finding, single-cell RNA-sequencing analyses of skeletal muscle stem cells (MuSCs) revealed that Apoe is a top marker of quiescent MuSCs that is downregulated upon activation. The purpose of this study was to determine if muscle regeneration is altered in mice which harbor one of the three common human ApoE isoforms, referred to as ApoE2, E3 and E4. Methods: Histomorphometric analyses were employed to assess muscle regeneration in ApoE2, E3, and E4 mice after 14days of recovery from barium chloride-induced muscle damage in vivo, and primary MuSCs were isolated to assess proliferation and differentiation of ApoE2, E3, and E4 MuSCs in vitro. Results: There was no difference in the basal skeletal muscle phenotype of ApoE isoforms as evaluated by section area, myofiber cross-sectional area (CSA), and myonuclear and MuSC abundance per fiber. Although there were no differences in fiber-type frequency in the soleus, Type IIa relative frequency was significantly lower in plantaris muscles of ApoE4 mice compared to ApoE3. Moreover, ApoE isoform did not influence muscle regeneration as assessed by fiber frequency, fiber CSA, and myonuclear and MuSC abundance. Finally, there were no differences in the proliferative capacity or myogenic differentiation potential of MuSCs between any ApoE isoform. Discussion: Collectively, these data indicate nominal effects of ApoE isoform on the ability of skeletal muscle to regenerate following injury or the in vitro MuSC phenotype.

Visualization of RNA Transcripts in Muscle Stem Cells Using Single-Molecule Fluorescence In Situ Hybridization.

Uncovering the transcriptomic signatures of quiescent muscle stem cells elicits the regulatory networks on stem cell quiescence. However, the spatial clues of the transcripts are missing in the commonly used quantitative analysis such as qPCR and RNA-seq. Visualization of RNA transcripts using single-molecule in situ hybridization provides additional subcellular localization clues to understanding gene expression signatures. Here, we provide an optimized protocol of smFISH analysis on Fluorescence-Activated Cell Sorting isolated muscle stem cells to visualize low-abundance transcripts.

Heterogeneous levels of delta-like 4 within a multinucleated niche cell maintains muscle stem cell diversity.

The quiescent muscle stem cell (QSC) pool is heterogeneous and generally characterized by the presence and levels of intrinsic myogenic transcription factors. Whether extrinsic factors maintain the diversity of states across the QSC pool remains unknown. The muscle fiber is a multinucleated syncytium that serves as a niche to QSCs, raising the possibility that the muscle fiber regulates the diversity of states across the QSC pool. Here, we show that the muscle fiber maintains a continuum of quiescent states, through a gradient of Notch ligand, Dll4, produced by the fiber and captured by QSCs. The abundance of Dll4 captured by the QSC correlates with the protein levels of the stem cell (SC) identity marker, Pax7. Niche-specific loss of Dll4 decreases QSC diversity and shifts the continuum to cell states that are biased toward more proliferative and committed fates. We reveal that fiber-derived Mindbomb1 (Mib1), an E3 ubiquitin ligase activates Dll4 and controls the heterogeneous levels of Dll4. In response to injury, with a Dll4-replenished niche, the normal continuum and diversity of the SC pool is restored, demonstrating bidirectionality within the SC continuum. Our data show that a post-translational mechanism controls heterogeneity of Notch ligands in a multinucleated niche cell to maintain a continuum of metastable states within the SC pool during tissue homeostasis.

The adhesion G-protein-coupled receptor Gpr116 is essential to maintain the skeletal muscle stem cell pool.

Skeletal muscle is populated with a reservoir of quiescent muscle stem cells (MuSCs), which regenerate the tissue after injury. Here, we show that the adhesion G-protein-coupled receptor Gpr116 is essential for long-term maintenance of the MuSC pool. Quiescent MuSCs express high levels of Gpr116, which is rapidly downregulated upon MuSC activation. MuSCs deficient for Gpr116 exhibit progressive depletion over time and are defective in self-renewal. Adhesion G-protein-coupled receptors contain an agonistic peptide sequence, called the "Stachel" sequence, within their long N-terminal ectodomains. Stimulation of MuSCs with the GPR116 Stachel peptide delays MuSC activation and differentiation. Stachel peptide stimulation of GPR116 leads to strong interaction with β-arrestins. Stimulation of GPR116 increases the nuclear localization of β-arrestin1, where it interacts with cAMP response element binding protein to regulate gene expression. Altogether, we propose a model by which GPR116 maintains the MuSC pool via nuclear functions of β-arrestin1.