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

ABSTRACT: 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.

SUBMITTER: Marg A

PROVIDER: S-EPMC6920394 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Skeletal muscle stem cells, called satellite cells, 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. The transcription factor Pax7 defines satellite cells across species 1-4 . We previously established human PAX7-positive cell colonies with high regenerative potential 5 . We now identified PAX7-negative human muscle-derived cell colonies (PAX7neg) also positive for the myogenic markers desmin and MYF5. These included cells from a unique myopathic patient with rigid spine and respiratory insufficiency due to a homozygous PAX7 c.86-1G>A mutation (PAX7null). Single cell and bulk transcriptome analysis showed high intra- and inter-donor heterogeneity and revealed the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7neg cell populations, including PAX7null, formed myofibers after transplantation into mice, and regenerated muscle after reinjury. Transplanted PAX7neg cells repopulated the satellite cell niche where they re-expressed PAX7. Strikingly, PAX7null cells expressing CLEC14A were also identified below the basal lamina. In summary, transplanted human cells do not depend on PAX7 for muscle regeneration. Thus, Pax7 is not a suitable marker for selection of optimal cells for muscle regenerative therapies.

2019-11-11 | GSE117382 | GEO

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

Project description:Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7

| PRJNA481958 | ENA

Derivation and expansion of PAX7-positive muscle progenitors from human and mouse embryonic stem cells.

Project description:Cell therapies treating pathological muscle atrophy or damage requires an adequate quantity of muscle progenitor cells (MPCs) not currently attainable from adult donors. Here, we generate cultures of approximately 90% skeletal myogenic cells by treating human embryonic stem cells (ESCs) with the GSK3 inhibitor CHIR99021 followed by FGF2 and N2 supplements. Gene expression analysis identified progressive expression of mesoderm, somite, dermomyotome, and myotome markers, following patterns of embryonic myogenesis. CHIR99021 enhanced transcript levels of the pan-mesoderm gene T and paraxial-mesoderm genes MSGN1 and TBX6; immunofluorescence confirmed that 91% ± 6% of cells expressed T immediately following treatment. By 7 weeks, 47% ± 3% of cells were MYH(+ve) myocytes/myotubes surrounded by a 43% ± 4% population of PAX7(+ve) MPCs, indicating 90% of cells had achieved myogenic identity without any cell sorting. Treatment of mouse ESCs with these factors resulted in similar enhancements of myogenesis. These studies establish a foundation for serum-free and chemically defined monolayer skeletal myogenesis of ESCs.

| S-EPMC4266001 | biostudies-literature

Metformin alleviates muscle wasting post-thermal injury by increasing Pax7-positive muscle progenitor cells.

Project description:BACKGROUND:Profound skeletal muscle wasting and weakness is common after severe burn and persists for years after injury contributing to morbidity and mortality of burn patients. Currently, no ideal treatment exists to inhibit muscle catabolism. Metformin is an anti-diabetic agent that manages hyperglycemia but has also been shown to have a beneficial effect on stem cells after injury. We hypothesize that metformin administration will increase protein synthesis in the skeletal muscle by increasing the proliferation of muscle progenitor cells, thus mitigating muscle atrophy post-burn injury. METHODS:To determine whether metformin can attenuate muscle catabolism following burn injury, we utilized a 30% total burn surface area (TBSA) full-thickness scald burn in mice and compared burn injuries with and without metformin treatment. We examined the gastrocnemius muscle at 7 and 14?days post-burn injury. RESULTS:At 7 days, burn injury significantly reduced myofiber cross-sectional area (CSA) compared to sham, p?<?0.05. Metformin treatment significantly attenuated muscle catabolism and preserved muscle CSA at the sham size. To investigate metformin's effect on satellite cells (muscle progenitors), we examined changes in Pax7, a transcription factor regulating the proliferation of muscle progenitors. Burned animals treated with metformin had a significant increase in Pax7 protein level and the number of Pax7-positive cells at 7?days post-burn, p?<?0.05. Moreover, through BrdU proliferation assay, we show that metformin treatment increased the proliferation of satellite cells at 7?days post-burn injury, p?<?0.05. CONCLUSION:In summary, metformin's various metabolic effects and its modulation of stem cells make it an attractive alternative to mitigate burn-induced muscle wasting while also managing hyperglycemia.

| S-EPMC6950874 | biostudies-literature

Mesodermal iPSC-derived progenitor cells functionally regenerate cardiac and skeletal muscle.

Project description:Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles.

| S-EPMC4665797 | biostudies-other

Myf5-positive satellite cells contribute to Pax7-dependent long-term maintenance of adult muscle stem cells.

Project description:Skeletal muscle contains Pax7-expressing muscle stem or satellite cells, enabling muscle regeneration throughout most of adult life. Here, we demonstrate that induced inactivation of Pax7 in Pax7-expressing cells of adult mice leads to loss of muscle stem cells and reduced heterochromatin condensation in rare surviving satellite cells. Inactivation of Pax7 in Myf5-expressing cells revealed that the majority of adult muscle stem cells originate from myogenic lineages, which express the myogenic regulators Myf5 or MyoD. Likewise, the majority of muscle stem cells are replenished from Myf5-expressing myogenic cells during adult life, and inactivation of Pax7 in Myf5-expressing cells after muscle damage leads to a complete arrest of muscle regeneration. Finally, we demonstrate that a relatively small number of muscle stem cells are sufficient for efficient repair of skeletal muscles. We conclude that Pax7 acts at different levels in a nonhierarchical regulatory network controlling muscle-satellite-cell-mediated muscle regeneration.

| S-EPMC4082715 | biostudies-literature

An absolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration.

Project description:Skeletal muscle tissue provides mechanical force for locomotion of all vertebrate animals. It is prone to damage from acute physical trauma and physiological stress. To cope with this, it possesses a tremendous capacity for rapid and effective repair that is widely held to be accomplished by the satellite cells lying between the muscle fiber plasmalemma and the basement membrane. Cell transplantation and lineage-tracing studies have demonstrated that Pax7-expressing (Pax7(+)) satellite cells can repair damaged muscle tissue repeatedly after several bouts of acute injury. These findings provided evidence that Pax7(+) cells are muscle stem cells. However, stem cells from a variety of other origins are also reported to contribute to myofibers upon engraftment into muscles, questioning whether satellite cells are the only stem cell source for muscle regeneration. Here, we have engineered genetic ablation of Pax7(+) cells to test whether there is any significant contribution to muscle regeneration after acute injury from cells other than this source. We find that such elimination of Pax7(+) cells completely blocks regenerative myogenesis either following injury to the tibialis anterior (TA) muscle or after transplantation of extensor digitorum longus (EDL) muscles into nude mice. As Pax7 is specifically expressed in satellite cells, we conclude that they are essential for acute injury-induced muscle regeneration. It remains to be established whether there is any significant role for stem cells of other origins. The implications of our results for muscle stem cell-based therapy are discussed.

| S-EPMC3152922 | biostudies-literature

Regenerating human skeletal muscle forms an emerging niche in vivo to support PAX7 cells.

Project description:Skeletal muscle stem and progenitor cells including those derived from human pluripotent stem cells (hPSCs) offer an avenue towards personalized therapies and readily fuse to form human-mouse myofibres in vivo. However, skeletal muscle progenitor cells (SMPCs) inefficiently colonize chimeric stem cell niches and instead associate with human myofibres resembling foetal niches. We hypothesized competition with mouse satellite cells (SCs) prevented SMPC engraftment into the SC niche and thus generated an SC ablation mouse compatible with human engraftment. Single-nucleus RNA sequencing of SC-ablated mice identified the absence of a transient myofibre subtype during regeneration expressing Actc1. Similarly, ACTC1+ human myofibres supporting PAX7+ SMPCs increased in SC-ablated mice, and after re-injury we found SMPCs could now repopulate into chimeric niches. To demonstrate ACTC1+ myofibres are essential to supporting PAX7 SMPCs, we generated caspase-inducible ACTC1 depletion human pluripotent stem cells, and upon SMPC engraftment we found a 90% reduction in ACTC1+ myofibres and a 100-fold decrease in PAX7 cell numbers compared with non-induced controls. We used spatial RNA sequencing to identify key factors driving emerging human niche formation between ACTC1+ myofibres and PAX7+ SMPCs in vivo. This revealed that transient regenerating human myofibres are essential for emerging niche formation in vivo to support PAX7 SMPCs.

| S-EPMC10709143 | biostudies-literature

Prepubertal skeletal muscle growth requires Pax7-expressing satellite cell-derived myonuclear contribution.

Project description:The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+ ; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+ ; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth.

| S-EPMC6215399 | biostudies-literature

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Project description:Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

| S-EPMC4154594 | biostudies-literature

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