Muscle Stem Cell Differentiation Research Articles

Nicotinamide and pyridoxine stimulate muscle stem cell expansion and enhance regenerative capacity during aging.

Skeletal muscle relies on resident muscle stem cells (MuSCs) for growth and repair. Aging and muscle diseases impair MuSC function, leading to stem cell exhaustion and regenerative decline that contribute to the progressive loss of skeletal muscle mass and strength. In the absence of clinically available nutritional solutions specifically targeting MuSCs, we used a human myogenic progenitor (hMP) high-content imaging screen of natural molecules from food to identify nicotinamide (NAM) and pyridoxine (PN) as bioactive nutrients that stimulate MuSCs and have history of safe human use. NAM and PN synergize via CK1-mediated cytoplasmic β-catenin activation and AKT signaling to promote amplification and differentiation of MuSCs. Oral treatment with a combination of NAM/PN accelerates muscle regeneration in vivo by stimulating MuSCs, increases muscle strength during recovery, and overcomes MuSC dysfunction and regenerative failure during aging. Levels of NAM and bioactive PN spontaneously decline during aging in model organisms and inter-independently associate with muscle mass and walking speed in a human cohort of 186 aged people. Collectively, our results establish NAM/PN as a new nutritional intervention that stimulates MuSCs, enhances muscle regeneration, and alleviates age-related muscle decline with a direct opportunity for clinical translation.

PTPN1/2 inhibition promotes muscle stem cell differentiation in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal disease caused by mutations in the DMD gene that encodes dystrophin. Dystrophin deficiency also impacts muscle stem cells (MuSCs), resulting in impaired asymmetric stem cell division and myogenic commitment. Using MuSCs from DMD patients and the DMD mouse model mdx, we found that PTPN1 phosphatase expression is up-regulated and STAT3 phosphorylation is concomitantly down-regulated in DMD MuSCs. To restore STAT3-mediated myogenic signaling, we examined the effect of K884, a novel PTPN1/2 inhibitor, on DMD MuSCs. Treatment with K884 enhanced STAT3 phosphorylation and promoted myogenic differentiation of DMD patient-derived MuSCs. In MuSCs from mdx mice, K884 treatment increased the number of asymmetric cell divisions, correlating with enhanced myogenic differentiation. Interestingly, the pro-myogenic effect of K884 is specific to human and murine DMD MuSCs and is absent from control MuSCs. Moreover, PTPN1/2 loss-of-function experiments indicate that the pro-myogenic impact of K884 is mediated mainly through PTPN1. We propose that PTPN1/2 inhibition may serve as a therapeutic strategy to restore the myogenic function of MuSCs in DMD.

Branched actin polymerization drives invasive protrusion formation to promote myoblast fusion during skeletal muscle regeneration.

Skeletal muscle regeneration is a multistep process involving the activation, proliferation, differentiation, and fusion of muscle stem cells, known as satellite cells. The fusion of satellite cell-derived mononucleated muscle cells (SCMs) is indispensable for the generation of multinucleated, contractile myofibers during muscle repair. However, the molecular and cellular mechanisms underlying SCM fusion during muscle regeneration remain poorly understood. In this study, we uncovered an essential role for branched actin polymerization in SCM fusion. Using conditional knockouts of the Arp2/3 complex and its actin nucleation-promoting factors, N-WASP and WAVE, we demonstrated that branched actin polymerization is required for the SCM fusion, but not for satellite cell proliferation, differentiation, and migration. We showed that the N-WASP and WAVE complexes have partially redundant functions in regulating SCM fusion. Furthermore, we showed that branched actin polymerization is essential for generating invasive protrusions at the fusogenic synapses in SCMs. Taken together, our study has identified new components of the myoblast fusion machinery in skeletal muscle regeneration and demonstrated a critical role for branched actin-propelled invasive protrusions in this process.

The proliferation and differentiation of skeletal muscle stem cells are enhanced in a bioreactor.

Skeletal muscle (SKM) is the largest organ in mammalian body and it can repair damages by using the residential myogenic stem cells (MuSC), but this repairing capacity reduces with age and in some genetic muscular dystrophy. Under these circumstances, artificial amplification of autologous MuSC in vitro might be necessary to repair the damaged SKM. The amplification of MuSC is highly dependent on myogenic signals, such as sonic hedgehog (Shh), Wnt3a, and fibroblast growth factors, so formulating an optimum myogenic kit composed of specific myogenic signals might increase the proliferation and differentiation of MuSC efficiently. In this study, various myogenic signals have been tested on C2C12 myoblasts and primary MuSC, and a myogenic kit consists of insulin, lithium chloride, T3, and retinoic acid has been formulated, and we found it significantly increased the fusion index and MHC expression level of both C2C12 and MuSC myotubes. A novel bioreactor providing cyclic stretching (CS) and electrical stimulation (ES) has been fabricated to enhance the myogenic differentiation of both C2C12 and MuSC. We further found that coating the bioreactor substratum with collagen gave the best effect on proliferation and differentiation of MuSC. Furthermore, combining the collagen coating and physical stimuli (CS + ES) in the bioreactor can generate more proliferative primary MuSC cells. Our results have demonstrated that the combination of myogenic kit and bioreactor can provide environment for efficient MuSC proliferation and differentiation. These MuSC and mature myotubes amplified in the bioreactor might be useful for clinical grafting into damaged SKM in the future.

GLUD1 determines murine muscle stem cell fate by controlling mitochondrial glutamate levels.

Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal invitro and invivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation.

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.

Effect of gelatin concentration and freezing temperature on porous scaffolds for cultured meat

Cultured meat is an emerging green alternative for livestock and poultry meat, and obtained from cell cultured in laboratories or factories. Three-dimensional (3D) scaffolds are widely used in cultured meat production by simulating extracellular matrix to support cell culture in vitro. Currently, the scaffolds used for the fabrication of cultured meat lack the research on porous structures, which limit cell growth and differentiation and simulation of distinct textural characteristics of traditional meat. Therefore, this study investigated the efficacy of food-grade scaffolds prepared with different gelatin concentrations and freezing temperatures to support cell attachment and growth. The result showed 6% gelatin scaffolds prepared at −80 °C or liquid nitrogen demonstrated suitable porous structures and promoted the proliferation and differentiation of muscle stem cells (MSCs) to improve the expression of genes and proteins (myogenin and myosin) related to myogenic progression. The scaffolds also created 3D-engineered cultured meat tissues that closely mimic the mechanical properties of traditional meat. This study provides the theoretical baseline for developing porous scaffolds for efficient cultured meat production in the meat substitute industry.

Retinoic acid signalling inhibits myogenesis by blocking MYOD translation in pig skeletal muscle cells

Vitamin A is an essential nutrient in animals, playing important roles in animal health. In the pig industry, proper supplementation of vitamin A in the feed can improve pork production performance, while deficiency or excessive intake can lead to growth retardation or disease. However, the specific molecular mechanisms through which vitamin A operates on pig skeletal muscle growth as well as muscle stem cell function remain unexplored. Therefore, in this study, we isolated the pig primary skeletal muscle stem cells (pMuSCs) and treated with retinoic acid (RA), the natural metabolite of vitamin A, and then examined the myogenic capacity of pMuSCs via immunostaining, real-time PCR, CCK8 and western-blot analysis. Unexpectedly, the RA caused a significant decrease in the proliferation and differentiation of pMuSCs. Mechanistically, the RA addition induced the activation of retinoic acid receptor gamma (RARγ), which inhibited the myogenesis through the blockage of protein translation of the master myogenic regulator myogenic differentiation 1 gene (MYOD). Specifically, RARγ inactivate AKT kinase (AKT) signalling and lead to dephosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (eIF4EBP1), which in turn repress the eukaryotic translation initiation factor 4E (eIF4E) complex and block mRNA translation of MYOD. Inhibition of AKT could rescue the myogenic defects of RA-treated pMuSCs. Our findings revealed that retinoid acid signalling inhibits the skeletal muscle stem cell proliferation and differentiation in pigs. Therefore, the vitamin A supplement in the feedstuff should be cautiously optimized to avoid the potential adverse consequences on muscle development associated with the excessive levels of retinoic acid.

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.

The Mediator Med23 controls a transcriptional switch for muscle stem cell proliferation and differentiation in muscle regeneration

Muscle stem cells (MuSCs) contribute to a robust muscle regeneration process after injury, which is highly orchestrated by the sequential expression of multiple key transcription factors. However, it remains unclear how key transcription factors and cofactors such as the Mediator complex cooperate to regulate myogenesis. Here, we show that the Mediator Med23 is critically important for MuSC-mediated muscle regeneration. Med23 is increasingly expressed in activated/proliferating MuSCs on isolated myofibers or in response to muscle injury. Med23 deficiency reduced MuSC proliferation and enhanced its precocious differentiation, ultimately compromising muscle regeneration. Integrative analysis revealed that Med23 oppositely impacts Ternary complex factor (TCF)-targeted MuSC proliferation genes and myocardin-related transcription factor (MRTF)-targeted myogenic differentiation genes. Consistently, Med23 deficiency decreases the ETS-like transcription factor 1 (Elk1)/serum response factor (SRF) binding at proliferation gene promoters but promotes MRTF-A/SRF binding at myogenic gene promoters. Overall, our study reveals the important transcriptional control mechanism of Med23 in balancing MuSC proliferation and differentiation in muscle regeneration.

Animal-free scaffold from brown algae provides a three-dimensional cell growth and differentiation environment for steak-like cultivated meat

Scaffolds for the production of cultivated meat, a promising sustainable meat alternative, should exhibit physical and chemical properties that enable three-dimensional animal cell culture, along with biological characteristics that support cell attachment, proliferation, and differentiation. Additionally, the scaffold should be crafted from edible materials and offer textural similarities to meat and have minimal influence on flavor and taste. Herein, an edible alginate-based alginate-cellulose hydrogel (ACe-gel) scaffold derived from the brown alga Undaria pinnatifida is developed. In terms of physical characteristics, the scaffold had porosity (119.5 ± 37.2 μm) and moisture-holding capacity (73.03 ± 3.82, 68.66 ± 9.54, and 84.17 ± 9.94 at 25 °C, 37 °C, and 60 °C, respectively) suitable for three-dimensional culture and differentiation of bovine muscle stem cells (bMuSCs). Accordingly, the scaffold was superior to a commercial alginate scaffold in terms of the attachment and proliferation of bMuSCs (5.5-fold over 72 h), and its performance was comparable with that of a lyophilized collagen scaffold (7.8-fold over 72 h, compared with the pure alginate). The bMuSCs cultured on the ACe-gel scaffold were capable of differentiating into muscle fibers, as verified by gene expression profile analysis. Furthermore, the scaffold exhibited minimal heavy metal contents and distinct seaweed odorants, while the stress-strain characteristics of the scaffold cultured with bMuSC (Young's modulus of raw ACe-gel: 285.19 ± 83.37 kPa, cooked ACe-gel meat: 880.60 ± 485.60 kPa) closely resembled that of meat (raw beef: 267.76 ± 156.42 kPa, cooked beef: 1331.94 ± 762.43 kPa). These findings highlight that the seaweed-derived and animal-free ACe-gel scaffold has strong potential for utilization as a food technology for cultured meat production in the future.

First person – Samuele Metti

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Samuele Metti is co-first author on ‘ Native collagen VI delays early muscle stem cell differentiation’, published in JCS. Samuele conducted the research described in this article while a PhD student in Prof. Paolo Bonaldo's lab at University of Padova, Italy. He is now a postdoc in the lab of Prof. Gisou van der Goot at School of Life Science, École Polytechnique Fédérale de Lausanne, Switzerland, investigating how extracellular matrix proteins affect muscle stem cell behaviour.

Augmented mitochondrial apoptotic signaling impairs C2C12 myoblast differentiation following cellular aging through sequential passaging.

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self-renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age-related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high-passage (HP) myoblasts exhibit greater mitochondrial-mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase-9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts.

Division-Independent Differentiation of Muscle Stem Cells During a Growth Stimulus.

Adult muscle stem cells (MuSCs) are known to replicate upon activation before differentiating and fusing to regenerate myofibers. It is unclear whether MuSC differentiation is intrinsically linked to cell division, which has implications for stem cell population maintenance. We use single-cell RNA-sequencing to identify transcriptionally diverse subpopulations of MuSCs after 5 days of a growth stimulus in adult muscle. Trajectory inference in combination with a novel mouse model for tracking MuSC-derived myonuclei and in vivo labeling of DNA replication revealed an MuSC population that exhibited division-independent differentiation and fusion. These findings demonstrate that in response to a growth stimulus in the presence of intact myofibers, MuSC division is not obligatory.

Effect of nonsteroidal anti-inflammatory drugs on pelvic floor muscle regeneration in a preclinical birth injury rat model

BackgroundPelvic floor muscle (PFM) injury is a common consequence of vaginal childbirth. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used postpartum analgesics. Multiple studies report negative effects of these drugs on limb muscle regeneration. The impact of NSAIDs on PFM recovery following birth injury has not been explored. ObjectiveUsing the validated rat model, we assessed the effects of NSAIDs on acute and longer-term PFM recovery following simulated birth injury (SBI). Study DesignThree-month old Sprague-Dawley rats were randomly assigned to (1) controls; (2) SBI; (3) SBI + NSAID; or (4) NSAID group. SBI was induced using a well-established vaginal balloon distention protocol. Ibuprofen was administered in drinking water (0.2 mg/ml) ad lib. Animals were sacrificed at 1,3,5,7,10,28 days post birth injury/ibuprofen administration. The pubocaudalis portion of the rat levator ani, which, like the human pubococcygeus, experiences larger parturition-associated strains, was harvested (N=3-9/timepoint/group). The cross-sectional areas of regenerating (embryonic myosin heavy chain (eMyHC)+) and mature myofibers were assessed at the acute and 28-day timepoints, respectively. The intramuscular collagen content was assessed at the 28-day timepoint. Myogenesis was evaluated using anti-Pax7 and anti-myogenin antibodies to identify activated and differentiated muscle stem cells, respectively. The overall immune infiltrate was assessed using anti-CD45 antibody. Expression of genes coding for pro- and anti-inflammatory cytokines was assessed by qRT-PCR at 3,5,10 days post-injury. ResultsThe pubocaudalis fiber size was significantly smaller in the SBI+NSAID compared to SBI group at 28 days post-injury, P<0.0001. The median size of eMyHC+ fibers was also significantly reduced, with fiber area distribution enriched with smaller fibers in SBI+NSAID compared to SBI group at 3 days post-injury (P<0.0001), suggesting a delay in the onset of regeneration in the presence of NSAIDs. By 10 days post-injury, the median eMyHC+ fiber size in the SBI group decreased from 7 days post-injury (P<0.0001) with a tight cross-sectional area distribution, indicating nearing completion of this state of regeneration. However, in the SBI+NSAID group, the size of eMyHC+ fibers continued to increase (P<0.0001) with expansion of the cross-sectional area distribution, signifying a delay in regeneration in these animals. NSAID decreased the muscle stem cell pool at 7 days post-injury (P<0.0001) and delayed muscle stem cell differentiation, as indicated by persistently elevated number of myogenin+ cells 7 days post-injury (P<0.05). In contrast, the proportion of myogenin+ cells returned to baseline by 5 days post-injury in the SBI group. The analysis of expression of genes coding for pro- and anti-inflammatory cytokines demonstrated only transient elevation of Tgfb1 in SBI+NSAID group at 5 but not at 10 days post-injury. Consistent with previous studies, SBI+NSAID resulted in increased deposition of intramuscular collagen compared to uninjured animals. There were no significant differences in any outcomes of interest between the NSAID group and the unperturbed controls. ConclusionsNSAIDs negatively impact PFM regeneration in the preclinical SBI model. The above appears to be driven by the negative impact of NSAIDs on pelvic muscle stem cell function, resultant in delayed temporal progression of PFM regeneration following birth injury. These novel findings provide impetus to investigate the impact of postpartum NSAID administration on muscle regeneration in women at high risk for PFM injury.

Effect of standard and physiological cell culture temperatures on in vitro proliferation and differentiation of primary broiler chicken pectoralis major muscle satellite cells.

Culture temperatures for broiler chicken cells are largely based on those optimized for mammalian species, although normal broiler body temperature is typically more than 3°C higher. The objective was to evaluate the effects of simulating broiler peripheral muscle temperature, 41°C, compared with standard temperature, 38°C, on the in vitro proliferation and differentiation of primary muscle-specific stem cells (satellite cells; SC) from the pectoralis major (PM) of broiler chickens. Primary SC cultures were isolated from the PM of 18-day-old Ross 708 × Yield Plus male broilers. SC were plated in triplicate, 1.8-cm2, gelatin-coated wells at 40,000 cells per well. Parallel plates were cultured at either 38°C or 41°C in separate incubators. At 48, 72, and 96h post-plating, the culture wells were fixed and immunofluorescence-stained to determine the expression of the myogenic regulatory factors Pax7 and MyoD as well as evaluated for apoptosis using a TUNEL assay. After 168h in culture, plates were immunofluorescence-stained to visualize myosin heavy chain and Pax7 expression and determine myotube characteristics and SC fusion. Population doubling times were not impacted by temperature (p ≥ 0.1148), but culturing broiler SC at 41°C for 96h promoted a more rapid progression through myogenesis, while 38°C maintained primitive populations (p ≤ 0.0029). The proportion of apoptotic cells increased in primary SC cultured at 41°C (p ≤ 0.0273). Culturing at 41°C appeared to negatively impact fusion percentage (p < 0.0001) and tended to result in the formation of thinner myotubes (p = 0.061) without impacting the density of differentiated cells (p = 0.7551). These results indicate that culture temperature alters primary broiler PM SC myogenic kinetics and has important implications for future in vitro work as well as improving our understanding of how thermal manipulation can alter myogenesis patterns during broiler embryonic and post-hatch muscle growth.

Tanshinone IIA promotes the proliferation and differentiation ability of primary muscle stem cells via MAPK and Akt signaling

Salvia miltiorrhiza Bunge is a widely-used traditional Chinese medicine to treat a variety of diseases including muscle disorders. The underlying pharmacological mechanisms of which active component and how it functions are still unknown. Tanshinone IIA (Tan IIA) is the main active lipophilic compound in Salvia miltiorrhiza Bunge. Muscle stem cells (MuSCs) play a crucial role in maintaining healthy physiological function of skeletal muscle. For the purpose of this study, we investigated the effects of Tan IIA on primary MuSCs as well as mechanism. The EdU staining, cell counts assay and RT-qPCR results of proliferative genes revealed increased proliferation ability of MuSCs after Tan IIA treatment. Immunofluorescent staining of MyHC and RT-qPCR results of myogenic genes found Tan IIA contributed to promoting differentiation of MuSCs. In addition, enrichment analysis of RNA-seq data and Western blot assay results demonstrated activated MAPK and Akt signaling after treatment of Tan IIA during proliferation and differentiation. The above proliferative and differentiative phonotypes could be suppressed by the combination of MAPK inhibitor U0126 and Akt inhibitor Akti 1/2, respectively. Furthermore, HE staining found significantly improved myofiber regeneration of injured muscle after Tan IIA treatment, which also contributed to muscle force and running performance recovery. Thus, Tan IIA could promote proliferation and differentiation ability of MuSCs through activating MAPK and Akt signaling, respectively. These beneficial effects also significantly contributed to muscle regeneration and muscle function recovery after muscle injury.

Histone methyltransferases and regenerative myogenesis: A focus on 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.

Migration and differentiation of muscle stem cells are coupled by RhoA signalling during regeneration.

Skeletal muscle is highly regenerative and is mediated by a population of migratory adult muscle stem cells (muSCs). Effective muscle regeneration requires a spatio-temporally regulated response of the muSC population to generate sufficient muscle progenitor cells that then differentiate at the appropriate time. The relationship between muSC migration and cell fate is poorly understood and it is not clear how forces experienced by migrating cells affect cell behaviour. We have used zebrafish to understand the relationship between muSC cell adhesion, behaviour and fate in vivo. Imaging of pax7-expressing muSCs as they respond to focal injuries in trunk muscle reveals that they migrate by protrusive-based means. By carefully characterizing their behaviour in response to injury we find that they employ an adhesion-dependent mode of migration that is regulated by the RhoA kinase ROCK. Impaired ROCK activity results in reduced expression of cell cycle genes and increased differentiation in regenerating muscle. This correlates with changes to focal adhesion dynamics and migration, revealing that ROCK inhibition alters the interaction of muSCs to their local environment. We propose that muSC migration and differentiation are coupled processes that respond to changes in force from the environment mediated by RhoA signalling.

Amniotic Membrane-Derived Stromal Cells Release Extracellular Vesicles That Favor Regeneration of Dystrophic Skeletal Muscles.

Duchenne muscular dystrophy (DMD) is a muscle disease caused by mutations in the dystrophin gene characterized by myofiber fragility and progressive muscle degeneration. The genetic defect results in a reduced number of self-renewing muscle stem cells (MuSCs) and an impairment of their activation and differentiation, which lead to the exhaustion of skeletal muscle regeneration potential and muscle replacement by fibrotic and fatty tissue. In this study, we focused on an unexplored strategy to improve MuSC function and to preserve their niche based on the regenerative properties of mesenchymal stromal cells from the amniotic membrane (hAMSCs), that are multipotent cells recognized to have a role in tissue repair in different disease models. We demonstrate that the hAMSC secretome (CM hAMSC) and extracellular vesicles (EVs) isolated thereof directly stimulate the in vitro proliferation and differentiation of human myoblasts and mouse MuSC from dystrophic muscles. Furthermore, we demonstrate that hAMSC secreted factors modulate the muscle stem cell niche in dystrophic-mdx-mice. Interestingly, local injection of EV hAMSC in mdx muscles correlated with an increase in the number of activated Pax7+/Ki67+ MuSCs and in new fiber formation. EV hAMSCs also significantly reduced muscle collagen deposition, thus counteracting fibrosis and MuSCs exhaustion, two hallmarks of DMD. Herein for the first time we demonstrate that CM hAMSC and EVs derived thereof promote muscle regeneration by supporting proliferation and differentiation of resident muscle stem cells. These results pave the way for the development of a novel treatment to counteract DMD progression by reducing fibrosis and enhancing myogenesis in dystrophic muscles.