Skeletal muscle satellite cell-derived mesenchymal stem cells ameliorate acute alcohol-induced liver injury.

Cultured human skeletal-muscle satellite cells have properties of mesenchymal stem cells (skeletal muscle satellite cell-derived mesenchymal stem cells, SkMSCs) and play anti-inflammatory roles by secreting prostaglandin E2 and hepatocyte growth factor (HGF). To evaluate the utility of SkMSCs in treating liver diseases, we determined whether SkMSCs could ameliorate acute liver and gut inflammation induced by binge ethanol administration. Binge drinking of ethanol led to weight loss in the body and spleen, liver inflammation and steatosis, and increased serum ALT and AST levels (markers of liver injury), along with increased IL-1ß, TNF-α, and iNOS expression levels in mice. However, levels of these binge-drinking-induced indicators were reduced by a single intraperitoneal treatment of SkMSCs. Furthermore, levels of bacteria-derived lipopolysaccharide decreased in the livers and sera of ethanol-exposed mice after SkMSC administration. SkMSCs decreased the extent of tissue inflammation and reduced villus and crypt lengths in the small intestine after alcohol binge drinking. SkMSCs also reduced the leakage of blood albumin, an indicator of leaky gut, in the stool of ethanol-exposed mice. Alcohol-induced damage to human colonic Caco-2/tc7 cells was also alleviated by HGF. Therefore, a single treatment with SkMSCs can attenuate alcoholic liver damage by reducing inflammatory responses in the liver and gut, suggesting that SkMSCs could be used in cell therapy to treat alcoholic liver diseases.

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation.

Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

Skeletal muscle regeneration via the chemical induction and expansion of myogenic stem cells in situ or in vitro.

Muscle loss and impairment resulting from traumatic injury can be alleviated by therapies using muscle stem cells. However, collecting sufficient numbers of autologous myogenic stem cells and expanding them efficiently has been challenging. Here we show that myogenic stem cells (predominantly Pax7+ cells)-which were selectively expanded from readily obtainable dermal fibroblasts or skeletal muscle stem cells using a specific cocktail of small molecules and transplanted into muscle injuries in adult, aged or dystrophic mice-led to functional muscle regeneration in the three animal models. We also show that sustained release of the small-molecule cocktail in situ through polymer nanoparticles led to muscle repair by inducing robust activation and expansion of resident satellite cells. Chemically induced stem cell expansion in vitro and in situ may prove to be advantageous for stem cell therapies that aim to regenerate skeletal muscle and other tissues.

FoxO maintains a genuine muscle stem-cell quiescent state until geriatric age.

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34High, with stemness properties (genuine state), and CD34Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.

Inhibition of inflammatory CCR2 signaling promotes aged muscle regeneration and strength recovery after injury.

Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.

Irradiation dependent inflammatory response may enhance satellite cell engraftment.

Skeletal muscle stem (satellite) cells transplanted into host mouse muscles contribute to muscle regeneration. Irradiation of host muscle enhances donor stem cell engraftment by promoting the proliferation of transplanted donor cells. We hypothesised that, similar to other systems, cells damaged by radiation might be effecting this donor cell proliferation. But we found no difference in the percentage of dying (TUNEL+) cells in immunodeficient dystrophic mouse muscles at the times after the irradiation dose that enhances donor cell engraftment. Similarly, irradiation did not significantly increase the number of TUNEL+ cells in non-dystrophic immunodeficient mouse muscles and it only slightly enhanced donor satellite cell engraftment in this mouse strain, suggesting either that the effector cells are present in greater numbers within dystrophic muscle, or that an innate immune response is required for effective donor cell engraftment. Donor cell engraftment within non-irradiated dystrophic host mouse muscles was not enhanced if they were transplanted with either satellite cells, or myofibres, derived from irradiated dystrophic mouse muscle. But a mixture of cells from irradiated muscle transplanted with donor satellite cells promoted donor cell engraftment in a few instances, suggesting that a rare, yet to be identified, cell type within irradiated dystrophic muscle enhances the donor stem cell-mediated regeneration. The mechanism by which cells within irradiated host muscle promote donor cell engraftment remains elusive.

Functionally heterogeneous human satellite cells identified by single cell RNA sequencing.

Although heterogeneity is recognized within the murine satellite cell pool, a comprehensive understanding of distinct subpopulations and their functional relevance in human satellite cells is lacking. We used a combination of single cell RNA sequencing and flow cytometry to identify, distinguish, and physically separate novel subpopulations of human PAX7+ satellite cells (Hu-MuSCs) from normal muscles. We found that, although relatively homogeneous compared to activated satellite cells and committed progenitors, the Hu-MuSC pool contains clusters of transcriptionally distinct cells with consistency across human individuals. New surface marker combinations were enriched in transcriptional subclusters, including a subpopulation of Hu-MuSCs marked by CXCR4/CD29/CD56/CAV1 (CAV1+). In vitro, CAV1+ Hu-MuSCs are morphologically distinct, and characterized by resistance to activation compared to CAV1- Hu-MuSCs. In vivo, CAV1+ Hu-MuSCs demonstrated increased engraftment after transplantation. Our findings provide a comprehensive transcriptional view of normal Hu-MuSCs and describe new heterogeneity, enabling separation of functionally distinct human satellite cell subpopulations.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

Characterization of mesoangioblast cell fate and improved promyogenic potential of a satellite cell-like subpopulation upon transplantation in dystrophic murine muscles.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease caused by the lack of dystrophin in muscle fibers that is currently without curative treatment. Mesoangioblasts (MABs) are multipotent progenitor cells that can differentiate to a myogenic lineage and that can be used to express Dystrophin upon transplantation into muscles, in autologous gene therapy approaches. However, their fate in the muscle environment remains poorly characterized. Here, we investigated the differentiation fate of MABs following their transplantation in DMD murine muscles using a mass cytometry strategy. This allowed the identification and isolation of a fraction of MAB-derived cells presenting common properties with satellite muscle stem cells. This analysis also indicated that most cells did not undergo a myogenic differentiation path once in the muscle environment, limiting their capacity to restore dystrophin expression in transplanted muscles. We therefore assessed whether MAB treatment with cytokines and growth factors prior to engraftment may improve their myogenic fate. We identified a combination of such signals that ameliorates MABs capacity to undergo myogenic differentiation in vivo and to restore dystrophin expression upon engraftment in myopathic murine muscles.

The effect of type 2 diabetes mellitus and obesity on muscle progenitor cell function.

In addition to its primary function to provide movement and maintain posture, the skeletal muscle plays important roles in energy and glucose metabolism. In healthy humans, skeletal muscle is the major site for postprandial glucose uptake and impairment of this process contributes to the pathogenesis of type 2 diabetes mellitus (T2DM). A key component to the maintenance of skeletal muscle integrity and plasticity is the presence of muscle progenitor cells, including satellite cells, fibroadipogenic progenitors, and some interstitial progenitor cells associated with vessels (myo-endothelial cells, pericytes, and mesoangioblasts). In this review, we aim to discuss the emerging concepts related to these progenitor cells, focusing on the identification and characterization of distinct progenitor cell populations, and the impact of obesity and T2DM on these cells. The recent advances in stem cell therapies by targeting diabetic and obese muscle are also discussed.

Exogenous skeletal muscle satellite cells promote the repair of levator palpebrae superioris mechanical damage in rat.

AIM OF THE STUDY: Blepharoptosis is a drooping of the upper eyelid, usually due to dysfunction of the levator palpebrae superioris (LPS). Recently, skeletal muscle satellite cells (SSCs) have been reported to promote the repair of damaged skeletal muscle. This study aims to investigate the potential contribution of exogenous SSCs to the regeneration of mechanically damaged LPS. MATERIALS AND METHODS: Thirty-two rats were randomly divided into four groups, including control group, SSCs-treated group, SSCs-treated injury group and non-treated injury group. After rats in injury groups were artificially lacerated on both the left and right LPS, HBBS (Hank's Balanced Salt Solution) containing SSCs was injected into upper eyelid tissue. After 7 days, the LPS muscle tissues were excised. In addition, skeletal muscle cells (SMCs) and SSCs were cocultured for use as an in vitro model, and the protective effects of SSCs on cultured SMCs were also investigated. RESULTS: Histological staining revealed that exogenous SSCs repaired the damaged muscle fibers and attenuated the fibrosis of LPS, possibly due to the increased level of IGF-1. In contrast, the level of IL-1ß, IL-6, TGF-ß1 and Smad2/3 (phospho-T8) were significantly reduced in the SSCs-treated group. The in vitro model using coculture of skeletal muscle cells (SMCs) and SSCs also revealed an increased level of IGF-1 and reduced level of inflammatory factors, resulting in a better cell survival rate. CONCLUSIONS: This study found that exogenous SSCs can promote the repair of LPS mechanical damage and provides new insight into the development of novel therapeutic approaches for blepharoptosis.

Synthetic matrix enhances transplanted satellite cell engraftment in dystrophic and aged skeletal muscle with comorbid trauma.

Muscle satellite cells (MuSCs) play a central role in muscle regeneration, but their quantity and function decline with comorbidity of trauma, aging, and muscle diseases. Although transplantation of MuSCs in traumatically injured muscle in the comorbid context of aging or pathology is a strategy to boost muscle regeneration, an effective cell delivery strategy in these contexts has not been developed. We engineered a synthetic hydrogel-based matrix with optimal mechanical, cell-adhesive, and protease-degradable properties that promotes MuSC survival, proliferation, and differentiation. Furthermore, we establish a biomaterial-mediated cell delivery strategy for treating muscle trauma, where intramuscular injections may not be applicable. Delivery of MuSCs in the engineered matrix significantly improved in vivo cell survival, proliferation, and engraftment in nonirradiated and immunocompetent muscles of aged and dystrophic mice compared to collagen gels and cell-only controls. This platform may be suitable for treating craniofacial and limb muscle trauma, as well as postoperative wounds of elderly and dystrophic patients.

PITX2 Enhances the Regenerative Potential of Dystrophic Skeletal Muscle Stem Cells.

Duchenne muscular dystrophy (DMD), one of the most lethal genetic disorders, involves progressive muscle degeneration resulting from the absence of DYSTROPHIN. Lack of DYSTROPHIN expression in DMD has critical consequences in muscle satellite stem cells including a reduced capacity to generate myogenic precursors. Here, we demonstrate that the c-isoform of PITX2 transcription factor modifies the myogenic potential of dystrophic-deficient satellite cells. We further show that PITX2c enhances the regenerative capability of mouse DYSTROPHIN-deficient satellite cells by increasing cell proliferation and the number of myogenic committed cells, but importantly also increasing dystrophin-positive (revertant) myofibers by regulating miR-31. These PITX2-mediated effects finally lead to improved muscle function in dystrophic (DMD/mdx) mice. Our studies reveal a critical role for PITX2 in skeletal muscle repair and may help to develop therapeutic strategies for muscular disorders.

Recapitulation of Extracellular LAMININ Environment Maintains Stemness of Satellite Cells In Vitro.

Satellite cells function as precursor cells in mature skeletal muscle homeostasis and regeneration. In healthy tissue, these cells are maintained in a state of quiescence by a microenvironment formed by myofibers and basement membrane in which LAMININs (LMs) form a major component. In the present study, we evaluated the satellite cell microenvironment in vivo and found that these cells are encapsulated by LMα2-5. We sought to recapitulate this satellite cell niche in vitro by culturing satellite cells in the presence of recombinant LM-E8 fragments. We show that treatment with LM-E8 promotes proliferation of satellite cells in an undifferentiated state, through reduced phosphorylation of JNK and p38. On transplantation into injured muscle tissue, satellite cells cultured with LM-E8 promoted the regeneration of skeletal muscle. These findings represent an efficient method of culturing satellite cells for use in transplantation through the recapitulation of the satellite cell niche using recombinant LM-E8 fragments.

Myogenic Satellite Cells: Biological Milieu and Possible Clinical Applications.

Adult skeletal muscle is a post-mitotic terminally differentiated tissue that possesses an immense potential for regeneration after injury. This regeneration can be achieved by adult stem cells named satellite cells that inhabit the muscular tissue. These cells were first identified in 1961 and were described as being wedged between the plasma membrane of the muscle fiber and the surrounding basement membrane. Since their discovery, many researchers investigated their embryological origin and the exact role they play in muscle regeneration and repair. Under normal conditions, satellite cells are retained in a quiescent state and when required, these cells are activated to proliferate and differentiate to repair pre-existing muscle fibers or to a lesser extent fuse with each other to form new myofibers. During skeletal muscle regeneration, satellite cell actions are regulated through a cascade of complex signaling pathways that are influenced by multiple extrinsic factors within the satellite cell micro-environment. Here, the basic concepts were studied about satellite cells, their development, function, distribution and the different cellular and molecular mechanisms that regulate these cells. The recent findings about some of their clinical applications and potential therapeutic use were also discussed.

Human Satellite Cell Isolation and Xenotransplantation.

Satellite cells are mononucleated cells of the skeletal muscle lineage that exist beneath the basal lamina juxtaposed to the sarcolemma of skeletal muscle fibers. It is widely accepted that satellite cells mediate skeletal muscle regeneration. Within the satellite cell pool of adult muscle are skeletal muscle stem cells (MuSCs), also called satellite stem cells, which fulfill criteria of tissue stem cells: They proliferate and their progeny either occupies the adult MuSC niche during self-renewal or differentiates to regenerate mature muscle fibers. Here, we describe robust methods for the isolation of enriched populations of human satellite cells containing MuSCs from fresh human muscle, utilizing mechanical and enzymatic dissociation and purification by fluorescence-activated cell sorting. We also describe a process for xenotransplantation of human satellite cells into mouse muscle by injection into irradiated, immunodeficient, mouse leg muscle with concurrent notexin or bupivacaine muscle injury to increase engraftment efficiency. The engraftment of human MuSCs and the formation of human muscle can then be analyzed by histological and immunofluorescence staining, or subjected to in vivo experimentation.

Autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of Fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type 2I.

α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP -ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a "glycan remodelling" process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase.

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation.

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive ß-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.

Engineered matrices for skeletal muscle satellite cell engraftment and function.

Regeneration of traumatically injured skeletal muscles is severely limited. Moreover, the regenerative capacity of skeletal muscle declines with aging, further exacerbating the problem. Recent evidence supports that delivery of muscle satellite cells to the injured muscles enhances muscle regeneration and reverses features of aging, including reduction in muscle mass and regenerative capacity. However, direct delivery of satellite cells presents a challenge at a translational level due to inflammation and donor cell death, motivating the need to develop engineered matrices for muscle satellite cell delivery. This review will highlight important aspects of satellite cell and their niche biology in the context of muscle regeneration, and examine recent progresses in the development of engineered cell delivery matrices designed for skeletal muscle regeneration. Understanding the interactions of muscle satellite cells and their niche in both native and engineered systems is crucial to developing muscle pathology-specific cell- and biomaterial-based therapies.