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.

SummaryInvestigation of human muscle regeneration requires robust methods to purify and transplant muscle stem and progenitor cells that collectively constitute the human satellite cell (HuSC) pool. Existing approaches have yet to make HuSCs widely accessible for researchers, and as a result human muscle stem cell research has advanced slowly. Here, we describe a robust and predictable HuSC purification process that is effective for each human skeletal muscle tested and the development of storage protocols and transplantation models in dystrophin-deficient and wild-type recipients. Enzymatic digestion, magnetic column depletion, and 6-marker flow-cytometric purification enable separation of 104 highly enriched HuSCs per gram of muscle. Cryostorage of HuSCs preserves viability, phenotype, and transplantation potential. Development of enhanced and species-specific transplantation protocols enabled serial HuSC xenotransplantation and recovery. These protocols and models provide an accessible system for basic and translational investigation and clinical development of HuSCs.

Background: Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white and beige adipose tissue (BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in numerous injury models. Purpose: To characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their promyogenic effect is mediated via exosomes, and analyze human FAPs for an analogous promyogenic exosome-rich subpopulation. Study Design: Controlled laboratory study. Methods: FAPs from UCP1 reporter mice were isolated via fluorescence-activated cell sorting and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1–), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNA sequencing was performed on UCP1–, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ FAP-BAT exosomes (Exo-FB) as well as UCP1– non–FAP-BAT exosomes (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and mouse embryonic fibroblasts in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat wild type C57B/L6J mice that had undergone a massive rotator cuff tear. At 6 weeks mice were sacrificed, and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing was then performed on FAPs isolated from human muscle that were treated with the beta-agonist formoterol or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans. Results: Flow cytometry analysis of sorted UCP1 reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with 3 distinct transcriptomic profiles characterized with bulk RNA sequencing. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers; UCP1– cells were marked by fibrogenic markers; and UCP1+ cells were characterized differential enrichment of white adipose tissue markers. Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, while treatment of mouse embryonic fibroblasts resulted in differentiation into myotubes. Treatment of cells with Exo-nFB resulted in poor myotube formation. Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and fatty infiltration as compared with treatment with Exo-nFB or phosphate-buffered saline. Single-cell RNA sequencing of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with one subpopulation demonstrating the presence of UCP1+ beige adipocytes with a distinct profile of BAT, mitochondrial, and extracellular vesicle–associated markers. Conclusion: FAP-BAT cells form a subpopulation of FAPs with upregulated beige gene expression and exosome production that mediate promyogenic effects in vitro and in vivo, and they are present as a transcriptomically similar subpopulation of FAPs in humans. Clinical Relevance: FAP-BAT cells and their exosomes represent a potential therapeutic avenue for treating rotator cuff muscle degeneration.

Rotator cuff (RC) tears are frequently complicated by muscle atrophy. Muscle stem cells (MuSCs) repair damaged myofibers following injury, but their role in the prevention or pathogenesis of atrophy following RC tears remains undefined. We hypothesized that the RC MuSC population would be affected by supraspinatus (SS) and infraspinatus (IS) tendon transection (TT) compared to uninjured muscle in a mouse model of RC tear. C57BL6/J mice underwent unilateral SS and IS TT and contralateral sham surgery. At 3, 8, or 14 weeks after injury, mice were euthanized, and SS and IS were harvested for FACS sorting of CD31-/CD45-/Sca1-/ITGa7+/VCAM+ MuSCs or histological analysis. Ki-67+ MuSCs from injured muscle increased 3.4-fold at 3 weeks (p = 0.03) and 8.1-fold at 8 weeks (p = 0.04) following TT injury, but returned to baseline by 14 weeks (p = 0.91). Myod1 remained upregulated 3.3-fold at 3 weeks (p = 0.03) and 2.0-fold at 14 weeks (p = 0.0003), respectively. Myofiber cross-sectional area was decreased at both 3 and 14 weeks after injury, but the number of MuSCs per fiber remained relatively constant at 3 (p = 0.3) and 14 (p = 0.6) weeks after TT. In this study, we characterized the longitudinal effect of RC tendon injury on the MuSC population in supraspinatus and infraspinatus muscles. MuSCs are transiently activated, and are not depleted, in spite of persistent muscle atrophy. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1370-1376, 2018.