Single cell RNA-sequencing of murine dermal cells and hindlimb skeletal muscle stromal cells.
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ABSTRACT: Traumatic injury often results in muscle loss and impairment, which is worsened under aged and diseased conditions. Activation of resident stem cells or transplantation of myogenic stem cells can promote muscle regeneration. However, major challenges remain in harvesting sufficient autologous myogenic stem cells and expanding such cells efficiently for muscle regeneration therapies. Here, we identified a chemical cocktail that selectively induced a robust expansion of myogenic stem cells from readily-obtainable dermal cells and from muscle stromal cells. By differential plating and lineage tracing, we showed that Pax7+ cells were the major source for chemical-induced myogenic stem cells (CiMCs). We further performed single-cell RNA sequencing (scRNA-seq) analysis to characterize the transcriptomic profile of CiMCs and demonstrate a specific expansion of myogenic cells from heterogeneous dermal cell population. Upon transplantation into the injured muscle, CiMCs were efficiently engrafted and improved functional muscle regeneration in both adult and aged mice. In addition, CiMC transplantation rescued muscle function in mice with Duchenne muscular dystrophy (DMD). Furthermore, an in situ therapeutic modality using this cocktail was developed by loading the chemical cocktail into injectable nanoparticles, which enabled a sustained release of the cocktail in injured muscle and a local expansion of resident satellite cells for muscle regeneration in adult and aged mice. These findings will lead to the development of novel in vitro and in situ stem cell therapies for effective skeletal muscle repair.
Project description:Traumatic injury often results in muscle loss and impairment, which is worsened under aged and diseased conditions. Activation of resident stem cells or transplantation of myogenic stem cells can promote muscle regeneration. However, major challenges remain in harvesting sufficient autologous myogenic stem cells and expanding such cells efficiently for muscle regeneration therapies. Here, we identified a chemical cocktail that selectively induced a robust expansion of myogenic stem cells from readily-obtainable dermal cells and from muscle stromal cells. By differential plating and lineage tracing, we showed that Pax7+ cells were the major source for chemical-induced myogenic stem cells (CiMCs). We further performed single-cell RNA sequencing (scRNA-seq) analysis to characterize the transcriptomic profile of CiMCs and demonstrate a specific expansion of myogenic cells from heterogeneous dermal cell population. Upon transplantation into the injured muscle, CiMCs were efficiently engrafted and improved functional muscle regeneration in both adult and aged mice. In addition, CiMC transplantation rescued muscle function in mice with Duchenne muscular dystrophy (DMD). Furthermore, an in situ therapeutic modality using this cocktail was developed by loading the chemical cocktail into injectable nanoparticles, which enabled a sustained release of the cocktail in injured muscle and a local expansion of resident satellite cells for muscle regeneration in adult and aged mice. These findings will lead to the development of novel in vitro and in situ stem cell therapies for effective skeletal muscle repair.
Project description:Skeletal muscle function and regenerative capacity decline during aging, yet factors driving these changes are incompletely understood. Muscle regeneration requires temporally coordinated transcriptional programs to drive myogenic stem cells to activate, proliferate, fuse to form myofibers, and to mature myonuclei, restoring muscle function after injury. We assessed global changes in myogenic transcription programs distinguishing muscle regeneration in aged mice from young mice by comparing pseudotime trajectories from single-nucleus RNA sequencing of myogenic nuclei. Aging-specific differences in coordinating myogenic transcription programs necessary for restoring muscle function occur following muscle injury, likely contributing to compromised regeneration in aged mice. Differences in pseudotime alignment of myogenic nuclei when comparing aged to young mice via Dynamic Time-Warping revealed pseudotemporal differences becoming progressively more severe as regeneration proceeds. Disruptions in timing of myogenic gene expression programs may contribute to incomplete skeletal muscle regeneration and declines in muscle function as organisms age.
Project description:Healthy skeletal muscle can regenerate after ischaemic, mechanical, or toxin-induced injury, but ageing impairs that regeneration potential. This has been largely attributed to dysfunctional satellite cells and reduced myogenic capacity. Understanding which signalling pathways are associated with reduced myogenesis and impaired muscle regeneration can provide valuable information about the mechanisms driving muscle ageing and prompt the development of new therapies. To investigate this, we developed a high-throughput in vitro model to assess muscle regeneration in chemically injured C2C12 and human myotube-derived young and aged myoblast cultures. We observed a reduced regeneration capacity of aged cells, as indicated by an attenuated recovery towards preinjury myotube size and myogenic fusion index at the end of the regeneration period, in comparison with younger muscle cells that were fully recovered. RNA-sequencing data showed significant enrichment of KEGG signalling pathways, PI3K-Akt, and downregulation of GO processes associated with muscle development, differentiation, and contraction in aged but not in young muscle cells. Data presented here suggests that repair in response to in vitro injury is impaired in aged vs. young muscle cells. Our study establishes a framework that enables further understanding of the factors underlying impaired muscle regeneration in older age.
Project description:Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength and cross-sectional area compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.
Project description:Loss of regenerative capacity during ex vivo expansion of muscle stem cells hampers the development of cell-based therapies for skeletal muscle disorders. Here, we developed a method to isolate regenerative reserve cell fractions from expanded murine primary cultures using differential adhesion properties and the induction of subsequent cycles of proliferation and differentiation. Fast-adhering reserve cells (FRCs) were enriched for cells expressing myogenin and contributed efficiently to muscle regeneration following transplantation in immunodeficient hosts. Slow adhering reserve cells (SRCs) generated higher number of PAX7+ cells under differentiating conditions and predominantly regenerated muscle after a secondary injury, suggesting engraftment as muscle stem cells. Transcriptomic analysis revealed that FRCs were enriched for markers of myogenic differentiation, while SRCs were characterized by a distinctive set of cell-adhesion genes. Overall, reserve cells provide a valuable source for the study and identification of molecular determinants of regeneration using ex vivo-expanded muscle cells.
Project description:Skeletal muscle regeneration is driven by the interaction of myogenic and non-myogenic cells. In aging, regeneration is impaired due to dysfunctions of myogenic and non-myogenic cells, but this is not understood comprehensively. We collected an integrated atlas of 273,923 single-cell transcriptomes from muscles of young, old, and geriatric mice (~5, 20, 26 months-old) at six time-points following myotoxin injury. We identified eight cell types, including T and NK cells and macrophage subtypes, that displayed accelerated or delayed response dynamics between ages. Through pseudotime analysis, we observed myogenic cell states and trajectories specific to old and geriatric ages. To explain these age differences, we assessed cellular senescence by scoring experimentally derived and curated gene-lists. This pointed to an elevation of senescent-like subsets specifically within muscle stem cells in aged muscles in both single-cell and spatial transcriptomics datasets. This resource provides a holistic portrait of the altered cellular states underlying skeletal muscle regenerative decline across mouse lifespan.
Project description:Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but the application of this approach has been limited due to the deleterious effects of cell expansion in vitro and poor engraftment efficiency. In an effort to overcome this hurdle, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small molecule screening platform employing zebrafish and mouse, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to identify those that could increase myogenic engraftment in zebrafish and mice. These efforts identified two lipids, lysophosphatidic acid (LPA) and niflumic acid (NFA), both linked to activation of intracellular calcium ion flux, which showed conserved, dose-dependent and synergistic effects in promoting muscle engraftment across these vertebrate species.
Project description:Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to Dystrophin-expressing myofibers upon transplantation, a subset of which maintain Pax7 expression in vivo and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that iMPCs can be established from muscle tissue following small molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple differentiated cell types.
Project description:Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to Dystrophin-expressing myofibers upon transplantation, a subset of which maintain Pax7 expression in vivo and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that iMPCs can be established from muscle tissue following small molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple differentiated cell types.
Project description: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, branching off from 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 MuSCs activation and expansion of myogenic progenitors through production of the metabolite a-ketoglutarate (a-KG) and the a-KG-generated amino acid glutamine. Genetic ablation of Psat1 in MuSCs resulted in defective expansion of MuSCs and impaired regeneration. Psat1, a-KG, and glutamine were reduced in MuSCs of old mice and myoblasts of old individuals. a-KG or glutamine re-established appropriate muscle regeneration of adult Psat1-/- mice, old mice, and improved proliferation of old human myoblasts. These findings contribute insights into the metabolic role of Psat1 during muscle regeneration and suggest a-KG and glutamine as potential therapeutic interventions to ameliorate muscle regeneration during aging.