Quiescent Muscle Stem Cells Research Articles

Myogenin is an essential regulator of adult myofibre growth and muscle stem cell homeostasis.

Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog-/-zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs, we found that myog-/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, Pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog-/-myofibres instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an 'alerted' state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.

Distinct Phases of Postnatal Skeletal Muscle Growth Govern the Progressive Establishment of Muscle Stem Cell Quiescence.

SummaryMuscle stem cells (or muscle satellite cells [MuSCs]) are required for postnatal growth. Yet, the detailed characterization of myogenic progression and establishment of quiescence during this process remains poorly documented. Here, we provide an overview of myogenic cells heterogeneity and dynamic from birth to adulthood using flow cytometry. We demonstrated that PAX7+ cells acquire an increasing ability to progress in the myogenic program from birth to adulthood. We then simultaneously analyzed the cycling state (KI67 expression) of the MuSCs and progenitors (PAX7+) and their progression into myogenic precursors (PAX7−MYOD+) and differentiating cells (MYOG+) in vivo. We identified two distinct peaks of myogenic differentiation between P7–P10 and P21–P28, and showed that the quiescent MuSC pool is established between 7 and 8 weeks of age. Overall our study provides a comprehensive in vivo characterization of myogenic heterogeneity and demonstrates the highly dynamic nature of skeletal muscle postnatal growth process.

Building the case for the calcitonin receptor as a viable target for the treatment of glioblastoma.

Researchers are actively seeking novel targeted therapies for the brain tumour glioblastoma (GBM) as the mean survival is less than 15 months. Here we discuss the proposal that the calcitonin receptor (CT Receptor), expressed in 76–86% of patient biopsies, is expressed by both malignant glioma cells and putative glioma stem cells (GSCs), and therefore represents a potential therapeutic target. Forty-two per cent (42%) of high-grade glioma (HGG; representative of GSCs) cell lines express CT Receptor protein. CT Receptors are widely expressed throughout the life cycle of organisms and in some instances promote apoptosis. Which of the common isoforms of the CT Receptor are predominantly expressed is currently unknown, but a functional response to cell stress of the insert-positive isoform is hypothesised. A model for resistant malignancies is one in which chemotherapy plays a direct role in activating quiescent stem cells for replacement of the tumour tissue hierarchy. The putative role that the CT Receptor plays in maintenance of quiescent cancer stem cells is discussed in view of the activation of the Notch–CT Receptor–collagen V axis in quiescent muscle (satellite) stem cells. The pharmacological CT response profiles of four of the HGG cell lines were reported. Both CT responders and non-responders were sensitive to an immunotoxin based on an anti-CT Receptor antibody. The CALCR mRNA exhibits alternative splicing commonly associated with cancer cells, which could result in the atypical pharmacology exhibited by CT non-responders and an explanation of tumour suppression. Due to the inherent instability of CALCR mRNA, analysis of CT Receptor protein in patient samples will lead to improved data for the expression of CT Receptor in GBM and other cancers, and an understanding of the role and activity of the splice variants. This knowledge will aid the effective targeting of this receptor for treatment of GBM.

Adult Muscle Stem Cells: Exploring the Links Between Systemic and Cellular Metabolism.

Emerging evidence suggests that metabolites are important regulators of skeletal muscle stem cell (MuSC) function and fate. While highly proliferative in early life, MuSCs reside in adult skeletal muscle tissue in a quiescent and metabolically depressed state, but are critical for the homeostatic maintenance and regenerative response of the tissue to damage. It is well established that metabolic activity in MuSC changes with their functional activation, but the spatiotemporal links between physiological metabolism and stem cell metabolism require explicit delineation. The quiescent MuSC is defined by a specific metabolic state, which is controlled by intrinsic and extrinsic factors during physiological and pathological tissue dynamics. However, the extent of tissue and organismal level changes driven by alteration in metabolic state of quiescent MuSC is currently not well defined. In addition to their role as biosynthetic precursors and signaling molecules, metabolites are key regulators of epigenetic mechanisms. Emerging evidence points to metabolic control of epigenetic mechanisms in MuSC and their impact on muscle regenerative capacity. In this review, we explore the links between cell-intrinsic, tissue level, and systemic metabolic state in the context of MuSC metabolic state, quiescence, and tissue homeostasis to highlight unanswered questions.

Amitosenescence and Pseudomitosenescence: Putative New Players in the Aging Process.

Replicative senescence has initially been defined as a stress reaction of replication-competent cultured cells in vitro, resulting in an ultimate cell cycle arrest at preserved growth and viability. Classically, it has been linked to critical telomere curtailment following repetitive cell divisions, and later described as a response to oncogenes and other stressors. Currently, there are compelling new directions indicating that a comparable state of cellular senescence might be adopted also by postmitotic cell entities, including terminally differentiated neurons. However, the cellular upstream inducers and molecular downstream cues mediating a senescence-like state in neurons (amitosenescence) are ill-defined. Here, we address the phenomenon of abortive atypical cell cycle activity in light of amitosenescence, and discuss why such replicative reprogramming might provide a yet unconsidered source to explain senescence in maturated neurons. We also hypothesize the existence of a G0 subphase as a priming factor for cell cycle re-entry, in analogy to discoveries in quiescent muscle stem cells. In conclusion, we propose a revision of our current view on the process and definition of senescence by encompassing a primarily replication-incompetent state (amitosenescence), which might be expanded by events of atypical cell cycle activity (pseudomitosenescence).

The CalcR-PKA-Yap1 Axis Is Critical for Maintaining Quiescence in Muscle Stem Cells

Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor (CalcR) is critical for sustaining quiescence in muscle satellite (stem) cells(MuSCs). However, the molecular mechanisms by which CalcR signaling regulates quiescence in MuSCs are enigmatic. Here, we demonstrate that transgenic expression of the catalytic domain of protein kinase A (PKA) restores the quiescence of CalcR-mutant MuSCs and delays MuSC activation. Mechanistically, CalcR-activated PKA phosphorylates Lats1/2, the main effector of Hippo signaling, thereby inhibiting the nuclear accumulation of Yap1, which prevents expression of Hippo-target genes, including cell-cycle-related molecules. Importantly, genetic inactivation of Yap1 in CalcR-mutant MuSCs reinstates quiescence in CalcR-mutant MuSCs, indicating that the CalcR-PKA-Lats1/2-Yap1 axis plays a critical role in sustaining MuSC quiescence.

PAX3 Confers Functional Heterogeneity in Skeletal Muscle Stem Cell Responses to Environmental Stress.

Muscle satellite cells (MuSCs) are the quiescent muscle stem cells required for adult skeletal muscle repair. The impact of environmental stress such as pollution on MuSC behavior remains unexplored. We evaluated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, a ubiquitous and highly toxic pollutant, on MuSCs by combining invivo mouse molecular genetic models with exvivo studies. While all MuSCs express the transcription factor PAX7, we show that a subsetalso express PAX3 and exhibit resistance to environmental stress. Upon systemic TCDD treatment, PAX3-negative MuSCs display impaired survival, atypical activation, and sporadic differentiation through xenobiotic arylhydrocarbon receptor signaling. We further show that PAX3-positive MuSCs become sensitized to environmental stress when PAX3 function is impaired and that PAX3-mediated induction of mTORC1 is required for protection. Our study, therefore, identifies a functional heterogeneity of MuSCs in response to environmental stress controlled by PAX3.

The CalcR-PKA-Yap1 Axis is Critical for Maintaining Quiescence in Muscle Stem Cells

Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor (CalcR) is critical for sustaining quiescence in muscle satellite (stem) cells (MuSCs). However, the molecular mechanisms, by which CalcR signaling regulates quiescence in MuSCs, are enigmatic. Here, we demonstrate that transgenic expression of the catalytic domain of PKA restores quiescence of CalcR-mutant MuSCs and delays MuSC activation. Mechanistically, CalcR-activated PKA phosphorylates Lats1/2, the main effector of Hippo-signaling, thereby inhibiting nuclear accumulation of Yap1, which prevents expression of Hippo-target genes including cell cycle-related molecules. Importantly, genetic inactivation of Yap1 in CalcR-mutant MuSCs reinstates quiescence in CalcR-mutant MuSCs indicating that the CalcR-PKA-Lats1/2-Yap1 axis plays a critical role for sustaining MuSC quiescence.

The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly in myogenesis

BackgroundMembers of the ZFP36 family of RNA-binding proteins regulate gene expression post-transcriptionally by binding to AU-rich elements in the 3’UTR of mRNA and stimulating mRNA degradation. The proteins within this family target different transcripts in different tissues. In particular, ZFP36 targets myogenic transcripts and may have a role in adult muscle stem cell quiescence. Our study examined the requirement of ZFP36L1 and ZFP36L2 in adult muscle cell fate regulation.MethodsWe generated single and double conditional knockout mice in which Zfp36l1 and/or Zfp36l2 were deleted in Pax7-expressing cells. Immunostained muscle sections were used to analyse resting skeletal muscle, and a cardiotoxin-induced injury model was used to determine the regenerative capacity of muscle.ResultsWe show that ZFP36L1 and ZFP36L2 proteins are expressed in satellite cells. Mice lacking the two proteins in Pax7-expressing cells have reduced body weight and have reduced skeletal muscle mass. Furthermore, the number of satellite cells is reduced in adult skeletal muscle and the capacity of this muscle to regenerate following muscle injury is diminished.ConclusionZFP36L1 and ZFP36L2 act redundantly in myogenesis. These findings add further intricacy to the regulation of the cell fate of Pax7-expressing cells in skeletal muscle by RNA-binding proteins.

Oncogenic Amplification of Zygotic Dux Factors in Regenerating p53-Deficient Muscle Stem Cells Defines a Molecular Cancer Subtype

The identity of tumor-initiating cells in many cancer types is unknown. Tumors often express genes associated with embryonic development, although the contributions of zygotic programs to tumor initiation and formation are poorly understood. Here, we show that regeneration-induced loss of quiescence in p53-deficient muscle stem cells (MuSCs) results in rhabdomyosarcoma formation with 100% penetrance. Genomic analyses of purified tumor cells revealed spontaneous and discrete oncogenic amplifications in MuSCs that drive tumorigenesis, including, but not limited to, the amplification of the cleavage-stage Dux transcription factor (TF) Duxbl. We further found that Dux factors drive an early embryonic gene signature that defines a molecular subtype across a broad range of human cancers. Duxbl initiates tumorigenesis by enforcing a mesenchymal-to-epithelial transition, and targeted inactivation of Duxbl specifically in Duxbl-expressing tumor cells abolishes their expansion. These findings reveal how regeneration and genomic instability can interact to activate zygotic genes that drive tumor initiation and growth.

Cellular localization of the cell cycle inhibitor Cdkn1c controls growth arrest of adult skeletal muscle stem cells.

Adult skeletal muscle maintenance and regeneration depend on efficient muscle stem cell (MuSC) functions. The mechanisms coordinating cell cycle with activation, renewal, and differentiation of MuSCs remain poorly understood. Here, we investigated how adult MuSCs are regulated by CDKN1c (p57kip2), a cyclin-dependent kinase inhibitor, using mouse molecular genetics. In the absence of CDKN1c, skeletal muscle repair is severely impaired after injury. We show that CDKN1c is not expressed in quiescent MuSCs, while being induced in activated and proliferating myoblasts and maintained in differentiating myogenic cells. In agreement, isolated Cdkn1c-deficient primary myoblasts display differentiation defects and increased proliferation. We further show that the subcellular localization of CDKN1c is dynamic; while CDKN1c is initially localized to the cytoplasm of activated/proliferating myoblasts, progressive nuclear translocation leads to growth arrest during differentiation. We propose that CDKN1c activity is restricted to differentiating myoblasts by regulated cyto-nuclear relocalization, coordinating the balance between proliferation and growth arrest.

The transcription factor Lef1 switches partners from β-catenin to Smad3 during muscle stem cell quiescence.

Skeletal muscle stem cells (MuSCs), also known as satellite cells, persist in adult mammals by entering a state of quiescence (G0) during the early postnatal period. Quiescence is reversed during damage-induced regeneration and re-established after regeneration. Entry of cultured myoblasts into G0 is associated with a specific, reversible induction of Wnt target genes, thus implicating members of the Tcf and Lef1 (Tcf/Lef) transcription factor family, which mediate transcriptional responses to Wnt signaling, in the initiation of quiescence. We found that the canonical Wnt effector β-catenin, which cooperates with Tcf/Lef, was dispensable for myoblasts to enter quiescence. Using pharmacological and genetic approaches in cultured C2C12 myoblasts and in MuSCs, we demonstrated that Tcf/Lef activity during quiescence depended not on β-catenin but on the transforming growth factor-β (TGF-β) effector and transcriptional coactivator Smad3, which colocalized with Lef1 at canonical Wnt-responsive elements and directly interacted with Lef1 specifically in G0 Depletion of Smad3, but not β-catenin, reduced Lef1 occupancy at target promoters, Tcf/Lef target gene expression, and self-renewal of myoblasts. In vivo, MuSCs underwent a switch from β-catenin-Lef1 to Smad3-Lef1 interactions during the postnatal switch from proliferation to quiescence, with β-catenin-Lef1 interactions recurring during damage-induced reactivation. Our findings suggest that the interplay of Wnt-Tcf/Lef and TGF-β-Smad3 signaling activates canonical Wnt target promoters in a manner that depends on β-catenin during myoblast proliferation but is independent of β-catenin during MuSC quiescence.

Induction of muscle stem cell quiescence by the secreted niche factor Oncostatin M

The balance between stem cell quiescence and proliferation in skeletal muscle is tightly controlled, but perturbed in a variety of disease states. Despite progress in identifying activators of stem cell proliferation, the niche factor(s) responsible for quiescence induction remain unclear. Here we report an in vivo imaging-based screen which identifies Oncostatin M (OSM), a member of the interleukin-6 family of cytokines, as a potent inducer of muscle stem cell (MuSC, satellite cell) quiescence. OSM is produced by muscle fibers, induces reversible MuSC cell cycle exit, and maintains stem cell regenerative capacity as judged by serial transplantation. Conditional OSM receptor deletion in satellite cells leads to stem cell depletion and impaired regeneration following injury. These results identify Oncostatin M as a secreted niche factor responsible for quiescence induction, and for the first time establish a direct connection between induction of quiescence, stemness, and transplantation potential in solid organ stem cells.

Loss of heterogeneity, quiescence, and differentiation in muscle stem cells.

Skeletal muscle stem cells in the adult display heterogeneity that has been functionally linked to their behavior, self-renewal capacity, and resistance to stress in hostile environments. Behavioral heterogeneity emerges also during developmental myogenesis. Muscle stem cell diversity may be functionally linked to the changing needs of skeletal muscle regeneration. Intriguingly, dramatic reduction of stem cell diversity, the "clonal drift", that implies loss of stem cells and related expansion of clonally related stem cells has been reported for tissue replacement in several adult tissues and suggested in the zebrafish embryo. A recent study shows clonal drift of muscle stem cells in the zebrafish embryo caused by inhibition of the cell cycle and directed by the homeobox protein Meox1. Although stem cell quiescence is associated with inhibition of the transition phase G0/G1 of the cell cycle, Meox1 triggers the muscle stem cell fate by an arrest in G2 phase. Why efficient muscle growth in the zebrafish embryo requires sacrificing stem cell heterogeneity in favor of a small number of dominant clones has not been elucidated. The significance of G2-halted stem cells, which are generally associated with robust regeneration capacity, is also intriguing. These processes are relevant for understanding organ growth and the mechanisms that govern stem cell quiescence.

Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence

Tissue regeneration depends on the timely activation of adult stem cells. In skeletal muscle, the adult stem cells maintain a quiescent state and proliferate upon injury. We show that muscle stem cells (MuSCs) use direct translational repression to maintain the quiescent state. High-resolution single-molecule and single-cell analyses demonstrate that quiescent MuSCs express high levels of Myogenic Differentiation 1 (MyoD) transcript in vivo, whereas MyoD protein is absent. RNA pulldowns and costainings show that MyoD mRNA interacts with Staufen1, a potent regulator of mRNA localization, translation, and stability. Staufen1 prevents MyoD translation through its interaction with the MyoD 3'-UTR. MuSCs from Staufen1 heterozygous (Staufen1+/-) mice have increased MyoD protein expression, exit quiescence, and begin proliferating. Conversely, blocking MyoD translation maintains the quiescent phenotype. Collectively, our data show that MuSCs express MyoD mRNA and actively repress its translation to remain quiescent yet primed for activation.

Translational Control of the Myogenic Program in Developing, Regenerating, and Diseased Skeletal Muscle.

Translational control of genes that code for protein allows a cell to rapidly respond to changes in its environment, in part because translational control of gene expression does not depend on upstream events required to produce an mRNA molecule. The importance of translational control has been highlighted by studies concerning muscle development, regeneration, and disease. Translational control of specific mRNAs is achieved by microRNAs and RNA-binding proteins, which are particularly relevant to developmental myogenesis, where they ensure the stepwise differentiation of multipotent progenitors to committed myogenic progenitors that ultimately fuse into slow- or fast-type myofibers that make up skeletal muscle. The importance of translational control is also illustrated in muscle disease, where deregulated microRNA expression accelerates or delays progression of disease. Skeletal muscle is also unique for its remarkable capacity to regenerate after injury, which requires the activity of quiescent muscle stem cells, named satellite cells for their position underneath the basal lamina of the myofiber. Mitotically quiescent satellite cells are primed to activate the cell cycle and myogenic program, a unique feature that requires specific regulation of mRNA translation converging with pathways that regulate global protein synthesis. Emerging concepts in translational control of gene expression have shed light on multiple layers of control over the myogenic program. In parallel, the development and regeneration of skeletal muscle represents a unique, relevant, and highly defined context within which new concepts in translational control of gene expression should emerge.

Fragile X mental retardation protein regulates skeletal muscle stem cell activity by regulating the stability of Myf5 mRNA

BackgroundRegeneration of adult tissues relies on adult stem cells that are primed to enter a differentiation program, while typically remaining quiescent. In mouse skeletal muscle, these features are reconciled by multiple translational control mechanisms that ensure primed muscle stem cells (MuSCs) are not activated. In quiescent MuSCs, this concept is illustrated by reversible microRNA silencing of Myf5 translation, mediated by microRNA-31 and fragile X mental retardation protein (FMRP).MethodsIn this work, we take advantage of FMRP knockout (Fmr1−/−) mice to support the role for FMRP in maintaining stem cell properties of the MuSC. We compare the activity of MuSCs in vivo after acute injury and engraftment, as well as ex vivo during culture. We use RNA immunoprecipitation and 3’UTR poly-adenine (poly(A)) length assays to assess the impact of FMRP on the stability of transcripts for myogenic regulatory factors.ResultsWe show that RNA-binding FMRP is required to maintain the MuSC pool. More specifically, FMRP is required for stem cell properties of muscle stem cells, which include MuSC capacity to prime the myogenic program, their self-renewal, and their capacity to efficiently regenerate muscle. We provide evidence that FMRP regulation of MuSC activity occurs in part by the capacity of FMRP to directly bind Myf5 transcripts and impact rates of Myf5 deadenylation.ConclusionsOur results provide further evidence supporting a role for post-transcriptional silencing platforms by RNA-binding proteins in maintaining stemness properties of adult stem cells. In addition, deregulated MuSC activity in the absence of Fmr1 may have implications for fragile X syndrome, which is associated with muscle hypotonia during infancy.

Quiescence of human muscle stem cells is favored by culture on natural biopolymeric films

BackgroundSatellite cells are quiescent resident muscle stem cells that present an important potential to regenerate damaged tissue. However, this potential is diminished once they are removed from their niche environment in vivo, prohibiting the long-term study and genetic investigation of these cells. This study therefore aimed to provide a novel biomaterial platform for the in-vitro culture of human satellite cells that maintains their stem-like quiescent state, an important step for cell therapeutic studies.MethodsHuman muscle satellite cells were isolated from two donors and cultured on soft biopolymeric films of controlled stiffness. Cell adhesive phenotype, maintenance of satellite cell quiescence and capacity for gene manipulation were investigated using FACS, western blotting, fluorescence microscopy and electron microscopy.ResultsAbout 85% of satellite cells cultured in vitro on soft biopolymer films for 3 days maintained expression of the quiescence marker Pax7, as compared with 60% on stiffer films and 50% on tissue culture plastic. The soft biopolymeric films allowed satellite cell culture for up to 6 days without renewing the media. These cells retained their stem-like properties, as evidenced by the expression of stem cell markers and reduced expression of differentiated markers. In addition, 95% of cells grown on these soft biopolymeric films were in the G0/G1 stage of the cell cycle, as opposed to those grown on plastic that became activated and began to proliferate and differentiate.ConclusionsOur study identifies a new biomaterial made of a biopolymer thin film for the maintenance of the quiescence state of muscle satellite cells. These cells could be activated at any point simply by replating them onto a plastic culture dish. Furthermore, these cells could be genetically manipulated by viral transduction, showing that this biomaterial may be further used for therapeutic strategies.

Pten is necessary for the quiescence and maintenance of adult muscle stem cells

Satellite cells (SCs) are myogenic stem cells required for regeneration of adult skeletal muscles. A proper balance among quiescence, activation and differentiation is essential for long-term maintenance of SCs and their regenerative function. Here we show a function of Pten (phosphatase and tensin homologue) in quiescent SCs. Deletion of Pten in quiescent SCs leads to their spontaneous activation and premature differentiation without proliferation, resulting in depletion of SC pool and regenerative failure. However, prior to depletion, Pten-null activated SCs can transiently proliferate upon injury and regenerate injured muscles, but continually decline during regeneration, suggesting an inability to return to quiescence. Mechanistically, Pten deletion increases Akt phosphorylation, which induces cytoplasmic translocation of FoxO1 and suppression of Notch signalling. Accordingly, constitutive activation of Notch1 prevents SC depletion despite Pten deletion. Our findings delineate a critical function of Pten in maintaining SC quiescence and reveal an interaction between Pten and Notch signalling.

Simultaneous Measurement of Mitochondrial and Glycolytic Activity in Quiescent Muscle Stem Cells.

Cellular metabolism has recently been identified as an important regulator of cell identity, with several adult stem cell populations having been observed to undergo a shift in metabolism during important changes in cell state, such as during the shift from quiescence to proliferation. In this chapter, a method to characterize the metabolism of quiescent skeletal muscle stem cells is presented. This technique will allow for the comparison of quiescent muscle stem cells isolated from two or more different mouse models.