High-Dimensional Single-Cell Cartography Reveals Novel Skeletal Muscle-Resident Cell Populations.

Adult tissue repair and regeneration require stem-progenitor cells that can self-renew and generate differentiated progeny. Skeletal muscle regenerative capacity relies on muscle satellite cells (MuSCs) and their interplay with different cell types within the niche. However, our understanding of skeletal muscle tissue cellular composition is limited. Here, using a combined approach of single-cell RNA sequencing and mass cytometry, we precisely mapped 10 different mononuclear cell types in adult mouse muscle. We also characterized gene signatures and determined key discriminating markers of each cell type. We identified two previously understudied cell populations in the interstitial compartment. One expresses the transcription factor scleraxis and generated tenocytes in vitro. The second expresses markers of smooth muscle and mesenchymal cells (SMMCs) and, while distinct from MuSCs, exhibited myogenic potential and promoted MuSC engraftment following transplantation. The blueprint presented here yields crucial insights into muscle-resident cell-type identities and can be exploited to study muscle diseases.

LSD1 controls a nuclear checkpoint in Wnt/ß-Catenin signaling to regulate muscle stem cell self-renewal.

The Wnt/ß-Catenin pathway plays a key role in cell fate determination during development and in adult tissue regeneration by stem cells. These processes involve profound gene expression and epigenome remodeling and linking Wnt/ß-Catenin signaling to chromatin modifications has been a challenge over the past decades. Functional studies of the lysine demethylase LSD1/KDM1A converge to indicate that this epigenetic regulator is a key regulator of cell fate, although the extracellular cues controlling LSD1 action remain largely unknown. Here we show that ß-Catenin is a substrate of LSD1. Demethylation by LSD1 prevents ß-Catenin degradation thereby maintaining its nuclear levels. Consistently, in absence of LSD1, ß-Catenin transcriptional activity is reduced in both MuSCs and ESCs. Moreover, inactivation of LSD1 in mouse muscle stem cells and embryonic stem cells shows that LSD1 promotes mitotic spindle orientation via ß-Catenin protein stabilization. Altogether, by inscribing LSD1 and ß-Catenin in the same molecular cascade linking extracellular factors to gene expression, our results provide a mechanistic explanation to the similarity of action of canonical Wnt/ß-Catenin signaling and LSD1 on stem cell fate.

SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis.

Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.

Myofiber stretch induces tensile and shear deformation of muscle stem cells in their native niche.

Muscle stem cells (MuSCs) are requisite for skeletal muscle regeneration and homeostasis. Proper functioning of MuSCs, including activation, proliferation, and fate decision, is determined by an orchestrated series of events and communication between MuSCs and their niche. A multitude of biochemical stimuli are known to regulate MuSC fate and function. However, in addition to biochemical factors, it is conceivable that MuSCs are subjected to mechanical forces during muscle stretch-shortening cycles because of myofascial connections between MuSCs and myofibers. MuSCs respond to mechanical forces in vitro, but it remains to be proven whether physical forces are also exerted on MuSCs in their native niche and whether they contribute to the functioning and fate of MuSCs. MuSC deformation in their native niche resulting from mechanical loading of ex vivo myofiber bundles was visualized utilizing mT/mG double-fluorescent Cre-reporter mouse and multiphoton microscopy. MuSCs were subjected to 1 h pulsating fluid shear stress (PFSS) with a peak shear stress rate of 6.5 Pa/s. After PFSS treatment, nitric oxide, messenger RNA (mRNA) expression levels of genes involved in regulation of MuSC proliferation and differentiation, ERK 1/2, p38, and AKT activation were determined. Ex vivo stretching of extensor digitorum longus and soleus myofiber bundles caused compression as well as tensile and shear deformation of MuSCs in their niche. MuSCs responded to PFSS in vitro with increased nitric oxide production and an upward trend in iNOS mRNA levels. PFSS enhanced gene expression of c-Fos, Cdk4, and IL-6, whereas expression of Wnt1, MyoD, Myog, Wnt5a, COX2, Rspo1, Vangl2, Wnt10b, and MGF remained unchanged. ERK 1/2 and p38 MAPK signaling were also upregulated after PFSS treatment. We conclude that MuSCs in their native niche are subjected to force-induced deformations due to myofiber stretch-shortening. Moreover, MuSCs are mechanoresponsive, as evidenced by PFSS-mediated expression of factors by MuSCs known to promote proliferation.

AXIN1 knockout does not alter AMPK/mTORC1 regulation and glucose metabolism in mouse skeletal muscle.

KEY POINTS: Tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) in mouse does not affect whole-body energy substrate metabolism. AXIN1 imKO does not affect AICAR or insulin-stimulated glucose uptake in adult skeletal muscle. AXIN1 imKO does not affect adult skeletal muscle AMPK or mTORC1 signalling during AICAR/insulin/amino acid incubation, contraction and exercise. During exercise, α2/ß2/γ3AMPK and AMP/ATP ratio show greater increases in AXIN1 imKO than wild-type in gastrocnemius muscle. ABSTRACT: AXIN1 is a scaffold protein known to interact with >20 proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic-anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake-stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signalling or glucose uptake stimulation at rest or in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/ß2/γ3 AMPK activity and AMP/ATP ratio compared to wild-type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2.

BMP signaling regulates satellite cell-dependent postnatal muscle growth.

Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57 In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool.

Muscle satellite cells are functionally impaired in myasthenia gravis: consequences on muscle regeneration.

Myasthenia gravis (MG) is a neuromuscular disease caused in most cases by anti-acetyl-choline receptor (AChR) autoantibodies that impair neuromuscular signal transmission and affect skeletal muscle homeostasis. Myogenesis is carried out by muscle stem cells called satellite cells (SCs). However, myogenesis in MG had never been explored. The aim of this study was to characterise the functional properties of myasthenic SCs as well as their abilities in muscle regeneration. SCs were isolated from muscle biopsies of MG patients and age-matched controls. We first showed that the number of Pax7+ SCs was increased in muscle sections from MG and its experimental autoimmune myasthenia gravis (EAMG) mouse model. Myoblasts isolated from MG muscles proliferate and differentiate more actively than myoblasts from control muscles. MyoD and MyoG were expressed at a higher level in MG myoblasts as well as in MG muscle biopsies compared to controls. We found that treatment of control myoblasts with MG sera or monoclonal anti-AChR antibodies increased the differentiation and MyoG mRNA expression compared to control sera. To investigate the functional ability of SCs from MG muscle to regenerate, we induced muscle regeneration using acute cardiotoxin injury in the EAMG mouse model. We observed a delay in maturation evidenced by a decrease in fibre size and MyoG mRNA expression as well as an increase in fibre number and embryonic myosin heavy-chain mRNA expression. These findings demonstrate for the first time the altered function of SCs from MG compared to control muscles. These alterations could be due to the anti-AChR antibodies via the modulation of myogenic markers resulting in muscle regeneration impairment. In conclusion, the autoimmune attack in MG appears to have unsuspected pathogenic effects on SCs and muscle regeneration, with potential consequences on myogenic signalling pathways, and subsequently on clinical outcome, especially in the case of muscle stress.

Specific pattern of cell cycle during limb fetal myogenesis.

Tight regulation of cell proliferation and differentiation is required to ensure proper growth during development and post-natal life. The source and nature of signals regulating cell proliferation are not well identified in vivo. We investigated the specific pattern of proliferating cells in mouse limbs, using the Fluorescent ubiquitynation-based cell-cycle indicator (Fucci) system, which allowed the visualization of the G1, G1/S transition and S/G2/M phases of the cell cycle in red, yellow or green fluorescent colors, respectively. We also used the retroviral RCAS system to express a Fucci cassette in chick embryos. We performed a comprehensive analysis of the cell cycle state of myogenic cells in fetal limb muscles, adult myoblast primary cultures and isolated muscle fiber cultures using the Fucci transgenic mice. We found that myonuclei of terminally differentiated muscle fibers displayed Fucci red fluorescence during mouse and chick fetal development, in adult isolated muscle fiber (ex vivo) and adult myoblast (in vitro) mouse cultures. This indicated that myonuclei exited from the cell cycle in the G1 phase and are maintained in a blocked G1-like state. We also found that cycling muscle progenitors and myoblasts in G1 phase were not completely covered by the Fucci system. During mouse fetal myogenesis, Pax7+ cells labeled with the Fucci system were observed mostly in S/G2/M phases. Proliferating cells in S/G2/M phases displayed a specific pattern in mouse fetal limbs, delineating individualized muscles. In addition, we observed more Pax7+ cells in S/G2/M phases at muscle tips, compared to the middle of muscles. These results highlight a specific spatial regionalization of cycling cells at the muscle borders and muscle-tendon interface during fetal development.

Skeletal muscle satellite cells and adult myogenesis.

Research focusing on the canonical adult myogenic progenitor, the skeletal muscle satellite cell, is still an ever-growing field 46 years from their initial description. Recent publications revealed numerous new aspects of satellite cell biology, starting from their developmental life to their role as the principal self-renewing myogenic stem cell in adult skeletal muscle and finally their loss during aging. The myogenic potential of satellite cells is under the molecular control of specific paired-box and bHLH transcription factors whose tightly orchestrated balance accounts for an effective skeletal muscle regeneration. New reports also demonstrate satellite cells relationships with blood vessels and the high myogenic potential of stem cell subsets related to both lineages.

Setdb1 protects genome integrity in murine muscle stem cells to allow for regenerative myogenesis and inflammation.

The histone H3 lysine 9 methyltransferase SETDB1 controls transcriptional repression to direct stem cell fate. Here, we show that Setdb1 expression by adult muscle stem cells (MuSCs) is required for skeletal muscle regeneration. We find that SETDB1 represses the expression of endogenous retroviruses (ERVs) in MuSCs. ERV de-repression in Setdb1-null MuSCs prevents their amplification following exit from quiescence and promotes cell death. Multi-omics profiling shows that chromatin decompaction at ERV loci activates the DNA-sensing cGAS-STING pathway, entailing cytokine expression by Setdb1-null MuSCs. This is followed by aberrant infiltration of inflammatory cells, including pathological macrophages. The ensuing histiocytosis is accompanied by myofiber necrosis, which, in addition to progressive MuSCs depletion, completely abolishes tissue repair. In contrast, loss of Setdb1 in fibro-adipogenic progenitors (FAPs) does not impact immune cells. In conclusion, genome maintenance by SETDB1 in an adult somatic stem cell is necessary for both its regenerative potential and adequate reparative inflammation.

SETDB1 modulates the TGFß response in Duchenne muscular dystrophy myotubes.

Overactivation of the transforming growth factor-ß (TGFß) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFß induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFß signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFß target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFß-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.

Satellite cell loss and impaired muscle regeneration in selenoprotein N deficiency.

Selenoprotein N (SelN) deficiency causes a group of inherited neuromuscular disorders termed SEPN1-related myopathies (SEPN1-RM). Although the function of SelN remains unknown, recent data demonstrated that it is dispensable for mouse embryogenesis and suggested its involvement in the regulation of ryanodine receptors and/or cellular redox homeostasis. Here, we investigate the role of SelN in satellite cell (SC) function and muscle regeneration, using the Sepn1(-/-) mouse model. Following cardiotoxin-induced injury, SelN expression was strongly up-regulated in wild-type muscles and, for the first time, we detected its endogenous expression in a subset of mononucleated cells by immunohistochemistry. We show that SelN deficiency results in a reduced basal SC pool in adult skeletal muscles and in an imperfect muscle restoration following a single injury. A dramatic depletion of the SC pool was detected after the first round of degeneration and regeneration that totally prevented subsequent regeneration of Sepn1(-/-) muscles. We demonstrate that SelN deficiency affects SC dynamics on isolated single fibres and increases the proliferation of Sepn1(-/-) muscle precursors in vivo and in vitro. Most importantly, exhaustion of the SC population was specifically identified in muscle biopsies from patients with mutations in the SEPN1 gene. In conclusion, we describe for the first time a major physiological function of SelN in skeletal muscles, as a key regulator of SC function, which likely plays a central role in the pathophysiological mechanism leading to SEPN1-RM.

[Histone methyltransferases and regenerative myogenesis: A focus on SETDB1]. / Histones méthyltransférases et myogenèse régénérative - Un focus sur SETDB1.

Adult skeletal muscle is composed of thousands of fibers (also called myofibers) that contract thus allowing voluntary movements. Following an injury, muscle stem cells, surrounding the myofibers, activate, proliferate, and differentiate to form de novo myofibers. These steps constitute a process called adult (or regenerative) myogenesis. This process is possible thanks to various transcription factors sequentially expressed and regulated by epigenetic factors that modulate the chromatin and therefore lead to the regulation of gene expression. Gene expression changes consequently affect the fate of muscle stem cells. Histone Lysine Methyltransferases methylate some histones involved in the repression of gene expression. We describe here the role of SETDB1 during adult myogenesis, notably its role during muscle stem cell differentiation.

Title: Histones méthyltransférases et myogenèse régénérative - Un focus sur SETDB1. Abstract: Le muscle strié squelettique adulte est composé de milliers de fibres (myofibres) capables de se contracter, permettant ainsi les mouvements volontaires. Après une lésion musculaire, les cellules souches musculaires localisées autour des myofibres s'activent, prolifèrent et se différencient pour former de nouvelles myofibres. Ces différentes étapes forment un processus appelé myogenèse. Cette dernière est rendue possible grâce à l'expression séquentielle de facteurs de transcription régulés par des facteurs épigénétiques qui agissent sur la chromatine afin de moduler l'expression génique et ainsi, le devenir de la cellule souche. Les histones méthyltransférases déposent des marques dites méthyl sur certaines histones afin d'induire la répression génique de régions spécifiques. Nous décrivons ici le rôle de SETDB1 au cours de la myogenèse adulte, et plus spécifiquement pendant la différenciation des cellules souches musculaires.

Single-Cell Transcriptomic Analysis of Mononuclear Cell Populations in Skeletal Muscle.

Skeletal muscle possesses a remarkable regenerative capacity, mainly relying on a population of undifferentiated and unipotent muscle progenitors, called muscle stem cells (MuSCs) or satellite cells, and their interplay with various cell types within the niche. Investigating the cellular composition of skeletal muscle tissues and the heterogeneity among various cell populations is crucial to the unbiased understanding of how cellular networks work in harmony at the population level in the context of skeletal muscle homeostasis, regeneration, aging, and diseases. As opposed to probing the average profile in a cell population, single-cell RNA-seq has unlocked access to the transcriptomic landscape characterization of individual cells in a highly parallel manner. This chapter describes the workflow for single-cell transcriptomic analysis of mononuclear cells in skeletal muscle by taking advantage of the droplet-based single-cell RNA-seq platform, Chromium Single Cell 3' solution from 10x Genomics®. Using this protocol, we can reveal insights into muscle-resident cell-type identities, which can be exploited to study the muscle stem cell niche further.

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies.

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level. Optimized micropatterned substrate design enabled to obtain high culture yields in tightly controlled microenvironments, with myotubes derived from primary human myoblasts displaying spontaneous contractions. Analysis of nuclear morphology confirmed similar myonuclei structure between obtained myotubes and in vivo myofibers, as compared to 2D monolayers. LMNA-related Congenital Muscular Dystrophy (L-CMD) was modeled with successful development of diseased 3D myotubes displaying reduced contraction. The miniaturized myotube technology can thus be used to study contraction characteristics and evaluate how diseases affect muscle organization and force generation. Importantly, it requires significantly fewer starting materials than current systems, which should substantially improve drug screening capability.

Odd skipped-related 1 controls the pro-regenerative response of fibro-adipogenic progenitors.

Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFß signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.

Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression.

Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca(2+) homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.

The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation.

Adult skeletal muscle harbours a population of muscle stem cells (MuSCs) that are required for repair after tissue injury. In youth, MuSCs return to a reversible state of cell-cycle arrest termed 'quiescence' after injury resolution. Conversely, some MuSCs in aged muscle remain semi-activated, causing a premature response to injuries that results in incomplete repair and eventual stem cell depletion. Regulating this balance between MuSC quiescence and activation may hold the key to restoring tissue homeostasis with age, but is incompletely understood. To fill this gap, we developed a simple and tractable in vitro method, to rapidly inactivate MuSCs freshly isolated from young murine skeletal muscle, and return them to a quiescent-like state for at least 1-week, which we name mini-IDLE (Inactivation and Dormancy LEveraged in vitro). This was achieved by introducing MuSCs into a 3D bioartificial niche comprised of a thin sheet of mouse myotubes, which we demonstrate provides the minimal cues necessary to induce quiescence. With different starting numbers of MuSCs, the assay revealed cellular heterogeneity and population-level adaptations that converged on a common niche repopulation density; behaviours previously observed only in vivo. Quiescence-associated hallmarks included a Pax7+CalcR+DDX6+MyoD-c-FOS- signature, quiescent-like morphologies, and polarized niche markers. Leveraging high-content bioimaging pipelines, we demonstrate a relationship between morphology and cell fate signatures for possible real-time morphology-based screening. When using MuSCs from aged muscle, they displayed aberrant proliferative activities and delayed inactivation kinetics, among other quiescence-associated defects that we show are partially rescued by wortmannin treatment. Thus, the assay offers an unprecedented opportunity to systematically investigate long-standing queries in areas such as regulation of pool size and functional heterogeneity within the MuSC population, and to uncover quiescence regulators in youth and age.

When our muscles are injured, stem cells in the tissue are activated to start the repair process. However, when there is no damage, these cells tend to stay in a protective, dormant state known as quiescence. If quiescence is not maintained, the stem cells cannot properly repair when the muscle is damaged. This happens in old age, when a proportion of the cells remain semi-activated, and become depleted. However, researchers still do not fully understand how quiescence is regulated. This is partly because in order to study quiescence, live animals must be used, because muscle stem cells immediately come out of quiescence when they are removed from muscle tissue. To overcome this experimental limitation, Jacques et al. developed a new method to study muscle stem cells by transferring them from mice into three-dimensional engineered muscle tissue grown in the lab. This tissue is made by infiltrating the pores of teabag paper with muscle progenitor cells, which then fuse with one another to make a thin muscle that contains three layers of contractile muscle cells. Introducing muscle stem cells from young healthy animals into this engineered muscle tissue allowed them to return to a quiescent-like state and to remain in that state for at least a week. Cells from older animals could also be returned to dormancy if they were chemically treated after placing them in the engineered muscle tissue. The approach works in a miniaturized fashion, with each engineered tissue requiring less than one per cent of the muscle stem cells collected from each mouse. This allows 100 times as many experiments compared to the current methods using live animals. This system could help researchers to study the genetic and chemical influences on muscle stem cell quiescence. Further understanding in this area could lead to treatments that restore healing abilities in older muscle tissue.

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.

Reduced growth rate of aged muscle stem cells is associated with impaired mechanosensitivity.

Aging-associated muscle wasting and impaired regeneration are caused by deficiencies in muscle stem cell (MuSC) number and function. We postulated that aged MuSCs are intrinsically impaired in their responsiveness to omnipresent mechanical cues through alterations in MuSC morphology, mechanical properties, and number of integrins, culminating in impaired proliferative capacity. Here we show that aged MuSCs exhibited significantly lower growth rate and reduced integrin-α7 expression as well as lower number of phospho-paxillin clusters than young MuSCs. Moreover, aged MuSCs were less firmly attached to matrigel-coated glass substrates compared to young MuSCs, as 43% of the cells detached in response to pulsating fluid shear stress (1 Pa). YAP nuclear localization was 59% higher than in young MuSCs, yet YAP target genes Cyr61 and Ctgf were substantially downregulated. When subjected to pulsating fluid shear stress, aged MuSCs exhibited reduced upregulation of proliferation-related genes. Together these results indicate that aged MuSCs exhibit impaired mechanosensitivity and growth potential, accompanied by altered morphology and mechanical properties as well as reduced integrin-α7 expression. Aging-associated impaired muscle regenerative capacity and muscle wasting is likely due to aging-induced intrinsic MuSC alterations and dysfunctional mechanosensitivity.