Dual inhibition of P38 MAPK and JNK pathways preserves stemness markers and alleviates premature activation of muscle stem cells during isolation.

BACKGROUND: Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored. METHODS: We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources. RESULTS: We monitored PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC. CONCLUSION: Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.

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.

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.

Cellular interactions and microenvironment dynamics in skeletal muscle regeneration and disease.

Skeletal muscle regeneration relies on the intricate interplay of various cell populations within the muscle niche-an environment crucial for regulating the behavior of muscle stem cells (MuSCs) and ensuring postnatal tissue maintenance and regeneration. This review delves into the dynamic interactions among key players of this process, including MuSCs, macrophages (MPs), fibro-adipogenic progenitors (FAPs), endothelial cells (ECs), and pericytes (PCs), each assuming pivotal roles in orchestrating homeostasis and regeneration. Dysfunctions in these interactions can lead not only to pathological conditions but also exacerbate muscular dystrophies. The exploration of cellular and molecular crosstalk among these populations in both physiological and dystrophic conditions provides insights into the multifaceted communication networks governing muscle regeneration. Furthermore, this review discusses emerging strategies to modulate the muscle-regenerating niche, presenting a comprehensive overview of current understanding and innovative approaches.

A 3D-printable gelatin/alginate/ε-poly-l-lysine hydrogel scaffold to enable porcine muscle stem cells expansion and differentiation for cultured meat development.

The mass proliferation of seed cells and imitation of meat structures remain challenging for cell-cultured meat production. With excellent biocompatibility, high water content and porosity, hydrogels are frequently-studied materials for anchorage-dependent cell scaffolds in biotechnology applications. Herein, a scaffold based on gelatin/alginate/ε-Poly-l-lysine (GAL) hydrogel is developed for skeletal muscle cells, which has a great prospect in cell-cultured meat production. In this work, the hydrogel GAL-4:1, composed of gelatin (5 %, w/v), alginate (5 %, w/v) and ε-Poly-l-lysine (molar ratio vs. alginate: 4:1) is selected as cell scaffold based on Young's modulus of 11.29 ± 1.94 kPa, satisfactory shear-thinning property and suitable porous organized structure. The commercially available C2C12 mouse skeletal myoblasts and porcine muscle stem cells (PMuSCs), are cultured in the 3D-printed scaffold. The cells show strong ability of attachment, proliferation and differentiation after induction, showing high biocompatibility. Furthermore, the cellular bioprinting is performed with GAL-4:1 hydrogel and freshly extracted PMuSCs. The extracted PMuSCs exhibit high viability and display early myogenesis (desmin) on the 3D scaffold, suggesting the great potential of GAL hydrogel as 3D cellular constructs scaffolds. Overall, we develop a novel GAL hydrogel as a 3D-printed bioactive platform for cultured meat research.

Laminin as a Key Extracellular Matrix for Proliferation, Differentiation, and Maturation of Porcine Muscle Stem Cell Cultivation.

Extracellular matrix (ECM) proteins play a crucial role in culturing muscle stem cells (MuSCs). However, there is a lack of extensive research on how each of these proteins influences proliferation and differentiation of MuSCs from livestock animals. Therefore, we investigated the effects of various ECM coatings-collagen, fibronectin, gelatin, and laminin-on the proliferation, differentiation, and maturation of porcine MuSCs. Porcine MuSCs, isolated from 14-day-old Berkshire piglets, were cultured on ECM-coated plates, undergoing three days of proliferation followed by three days of differentiation. MuSCs on laminin showed higher proliferation rate than others (p<0.05). There was no significant difference in the mRNA expression levels of PAX7, MYF5, and MYOD among MuSCs on laminin, collagen, and fibronectin (p>0.05). During the differentiation period, MuSCs cultured on laminin exhibited a significantly higher differentiation rate, resulting in thicker myotubes compared to those on other ECMs (p<0.05). Also, MuSCs on laminin showed higher expression of mRNA related with maturated muscle fiber such as MYH1 and MYH4 corresponding to muscle fiber type IIx and muscle fiber type IIb, respectively, compared with MuSCs on other ECM coatings (p<0.05). In summary, our comparison of ECMs revealed that laminin significantly enhances MuSC proliferation and differentiation, outperforming other ECMs. Specifically, muscle fibers cultured on laminin exhibited a more mature phenotype. These findings underscore laminin's potential to advance in vitro muscle research and cultured meat production, highlighting its role in supporting rapid cell proliferation, higher differentiation rates, and the development of mature muscle fibers.

Time-of-day effects on ex vivo muscle contractility following short-term satellite cell ablation.

Muscle isometric torque fluctuates according to time-of-day with such variation owed to the influence of circadian molecular clock genes. Satellite cells (SCs), the muscle stem cell population, also express molecular clock genes with several contractile-related genes oscillating in a diurnal pattern. Currently, limited evidence exists regarding the relationship between SCs and contractility, although long-term SC ablation alters muscle contractile function. Whether there are acute alterations in contractility following SC ablation and with respect to the time-of-day is unknown. We investigated whether short-term SC ablation affected contractile function at two times of day and whether any such alterations led to different extents of eccentric contraction-induced injury. Using an established mouse model to deplete SCs, we characterized muscle clock gene expression and ex vivo contractility at two times-of-day (morning: 0700 and afternoon: 1500). Morning-SC+ animals demonstrated ∼25%-30% reductions in tetanic/eccentric specific forces and, after eccentric injury, exhibited ∼30% less force-loss and ∼50% less dystrophinnegative fibers versus SC- counterparts; no differences were noted between Afternoon groups (Morning-SC+: -5.63 ± 0.61, Morning-SC-: -7.93 ± 0.61; N/cm2; P < 0.05) (Morning-SC+: 32 ± 2.1, Morning-SC-: 64 ± 10.2; dystrophinnegative fibers; P < 0.05). As Ca++ kinetics underpin force generation, we also evaluated caffeine-induced contracture force as an indirect marker of Ca++ availability and found similar force reductions in Morning-SC+ vs. SC- mice. We conclude that force production is reduced in the presence of SCs in the morning but not in the afternoon, suggesting that SCs may have a time-of-day influence over contractile function.NEW & NOTEWORTHY Muscle isometric torque fluctuates according to time-of-day with such variation owed to molecular clock regulation. Satellite cells (SCs) have recently demonstrated diurnal characteristics related to muscle physiology. In our work, force production was reduced in the presence versus absence of SCs in the morning but, not in the afternoon. Morning-SC+ animals, producing lower force, sustained lesser degrees of injury versus SC- counterparts. One potential mechanism underpinning lower forces produced appears to be lower calcium availability.

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.

Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Skeletal muscle niche, at the crossroad of cell/cell communications.

Skeletal muscle is composed of a variety of tissue and non-tissue resident cells that participate in homeostasis. In particular, the muscle stem cell niche is a dynamic system, requiring direct and indirect communications between cells, involving local and remote cues. Interactions within the niche must happen in a timely manner for the maintenance or recovery of the homeostatic niche. For instance, after an injury, pro-myogenic cues delivered too early will impact on muscle stem cell proliferation, delaying the repair process. Within the niche, myofibers, endothelial cells, perivascular cells (pericytes, smooth muscle cells), fibro-adipogenic progenitors, fibroblasts, and immune cells are in close proximity with each other. Each cell behavior, membrane profile, and secretome can interfere with muscle stem cell fate and skeletal muscle regeneration. On top of that, the muscle stem cell niche can also be modified by extra-muscle (remote) cues, as other tissues may act on muscle regeneration via the production of circulating factors or the delivery of cells. In this review, we highlight recent publications evidencing both local and remote effectors of the muscle stem cell niche.

Muscle stem cell niche dynamics during muscle homeostasis and regeneration.

The process of skeletal muscle regeneration involves a coordinated interplay of specific cellular and molecular interactions within the injury site. This review provides an overview of the cellular and molecular components in regenerating skeletal muscle, focusing on how these cells or molecules in the niche regulate muscle stem cell functions. Dysfunctions of muscle stem cell-to-niche cell communications during aging and disease will also be discussed. A better understanding of how niche cells coordinate with muscle stem cells for muscle repair will greatly aid the development of therapeutic strategies for treating muscle-related disorders.

Decoding the forces that shape muscle stem cell function.

Skeletal muscle is a force-producing organ composed of muscle tissues, connective tissues, blood vessels, and nerves, all working in synergy to enable movement and provide support to the body. While robust biomechanical descriptions of skeletal muscle force production at the body or tissue level exist, little is known about force application on microstructures within the muscles, such as cells. Among various cell types, skeletal muscle stem cells reside in the muscle tissue environment and play a crucial role in driving the self-repair process when muscle damage occurs. Early evidence indicates that the fate and function of skeletal muscle stem cells are controlled by both biophysical and biochemical factors in their microenvironments, but much remains to accomplish in quantitatively describing the biophysical muscle stem cell microenvironment. This book chapter aims to review current knowledge on the influence of biophysical stresses and landscape properties on muscle stem cells in heath, aging, and diseases.

Epigenetic integration of signaling from the regenerative environment.

Skeletal muscle has an extraordinary capacity to regenerate itself after injury due to the presence of tissue-resident muscle stem cells. While these muscle stem cells are the primary contributor to the regenerated myofibers, the process occurs in a regenerative microenvironment where multiple different cell types act in a coordinated manner to clear the damaged myofibers and restore tissue homeostasis. In this regenerative environment, immune cells play a well-characterized role in initiating repair by establishing an inflammatory state that permits the removal of dead cells and necrotic muscle tissue at the injury site. More recently, it has come to be appreciated that the immune cells also play a crucial role in communicating with the stem cells within the regenerative environment to help coordinate the timing of repair events through the secretion of cytokines, chemokines, and growth factors. Evidence also suggests that stem cells can help modulate the extent of the inflammatory response by signaling to the immune cells, demonstrating a cross-talk between the different cells in the regenerative environment. Here, we review the current knowledge on the innate immune response to sterile muscle injury and provide insight into the epigenetic mechanisms used by the cells in the regenerative niche to integrate the cellular cross-talk required for efficient muscle repair.

Long non-coding RNAs and their role in muscle regeneration.

In mammals, most of the genome is transcribed to generate a large and heterogeneous variety of non-protein coding RNAs, that are broadly grouped according to their size. Long noncoding RNAs include a very large and versatile group of molecules. Despite only a minority of them has been functionally characterized, there is emerging evidence indicating long noncoding RNAs as important regulators of expression at multiple levels. Several of them have been shown to be modulated during myogenic differentiation, playing important roles in the regulation of skeletal muscle development, differentiation and homeostasis, and contributing to neuromuscular diseases. In this chapter, we have summarized the current knowledge about long noncoding RNAs in skeletal muscle and discussed specific examples of long noncoding RNAs (lncRNAs and circRNAs) regulating muscle stem cell biology. We have also discussed selected long noncoding RNAs involved in the most common neuromuscular diseases.

Muscle stem cell dysfunction in rhabdomyosarcoma and muscular dystrophy.

Muscle stem cells (MuSCs) are crucial to the repair and homeostasis of mature skeletal muscle. MuSC dysfunction and dysregulation of the myogenic program can contribute to the development of pathology ranging from cancers like rhabdomyosarcoma (RMS) or muscle degenerative diseases such as Duchenne muscular dystrophy (DMD). Both diseases exhibit dysregulation at nearly all steps of myogenesis. For instance, MuSC self-renewal processes are altered. In RMS, this leads to the creation of tumor propagating cells. In DMD, impaired asymmetric stem cell division creates a bias towards producing self-renewing stem cells instead of committing to differentiation. Hyperproliferation of these cells contribute to tumorigenesis in RMS and symmetric expansion of the self-renewing MuSC population in DMD. Both diseases also exhibit a repression of factors involved in terminal differentiation, halting RMS cells in the proliferative stage and thus driving tumor growth. Conversely, the MuSCs in DMD exhibit impaired differentiation and fuse prematurely, affecting myonuclei maturation and the integrity of the dystrophic muscle fiber. Finally, both disease states cause alterations to the MuSC niche. Various elements of the niche such as inflammatory and migratory signaling that impact MuSC behavior are dysregulated. Here we show how these seemingly distantly related diseases indeed have similarities in MuSC dysfunction, underlying the importance of considering MuSCs when studying the pathophysiology of muscle diseases.

Measurement of Myonuclear Accretion In Vitro and In Vivo.

Adult skeletal muscle stem cells (MuSC) are the regenerative precursors of myofibers and also have an important role in myofiber growth, adaptation, and maintenance by fusing to the myofibers-a process referred to as "myonuclear accretion." Due to a focus on MuSC function during regeneration, myofibers remain a largely overlooked component of the MuSC niche influencing MuSC fate. Here, we describe a method to directly measure the rate of myonuclear accretion in vitro and in vivo using ethynyl-2'-deoxyuridine (EdU)-based tracing of MuSC progeny. This method supports the dissection of MuSC intrinsic and myofiber-derived factors influencing myonuclear accretion as an alternative fate of MuSCs supporting myofiber homeostasis and plasticity.

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.

Psat1-generated α-ketoglutarate and glutamine promote muscle stem cell activation and regeneration.

By satisfying bioenergetic demands, generating biomass, and providing metabolites serving as cofactors for chromatin modifiers, metabolism regulates adult stem cell biology. Here, we report that a branch of glycolysis, the serine biosynthesis pathway (SBP), is activated in regenerating muscle stem cells (MuSCs). Gene inactivation and metabolomics revealed that Psat1, one of the three SBP enzymes, controls MuSC activation and expansion of myogenic progenitors through production of the metabolite α-ketoglutarate (α-KG) and α-KG-generated glutamine. Psat1 ablation resulted in defective expansion of MuSCs and impaired regeneration. Psat1, α-KG, and glutamine were reduced in MuSCs of old mice. α-KG or glutamine re-established appropriate muscle regeneration of adult conditional Psat1 -/- mice and of old mice. These findings contribute insights into the metabolic role of Psat1 during muscle regeneration and suggest α-KG and glutamine as potential therapeutic interventions to ameliorate muscle regeneration during aging.

A serine metabolic enzyme is flexing its muscle to help repair skeletal muscle.

Metabolic reprogramming of stem cells is a targetable pathway to control regeneration. Activation of stem cells results in down-regulation of oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) and turns on glycolysis to provide fuel for proliferation and specific signaling events. How cell type-specific events are regulated is unknown. In this issue of Genes & Development Ciuffoli and colleagues (pp. 151-167) use metabolomic, gene inactivation, and functional approaches to show that phosphoserine aminotransferase (Psat1), an enzyme in serine biosynthesis, is activated in muscle stem cells and contributes to cell expansion and skeletal muscle regeneration via the production of α-ketoglutarate and glutamine.

In vivo PSC differentiation as a platform to identify factors for improving the engraftability of cultured muscle stem cells.

Producing an adequate number of muscle stem cells (MuSCs) with robust regenerative potential is essential for the successful cell therapy of muscle-wasting disorders. We have recently developed a method to produce skeletal myogenic cells with exceptional engraftability and expandability through an in vivo pluripotent stem cell (PSC) differentiation approach. We have subsequently mapped engraftment and gene expression and found that leukemia inhibitory factor receptor (Lifr) expression is positively correlated with engraftability. We therefore investigated the effect of LIF, the endogenous ligand of LIFR, on cultured MuSCs and examined their engraftment potential. We found that LIF-treated MuSCs exhibited elevated expression of PAX7, formed larger colonies from single cells, and favored the retention of PAX7+ "reserve cells" upon myogenic differentiation. This suggested that LIF promoted the maintenance of cultured MuSCs at a stem cell stage. Moreover, LIF enhanced the engraftment capability of MuSCs that had been expanded in vitro for 12 days by 5-fold and increased the number of MuSCs that repopulated the stem cell pool post-transplantation. These results thereby demonstrated the effectiveness of our in vivo PSC differentiation platform to identify positive regulators of the engraftability of cultured MuSCs.