RNA-seq of muscle Fibro-Adipogenic Progenitor subpopulations and bulk from different injury contexts

Fibro-Adipogenic Progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, ability to adopt multiple lineages and to perform different biological activities in response to physiological or pathological stimuli. Gene expression analysis at single cell level revealed the intrinsic heterogeneity of FAPs and uncovered discrete subpopulations (subFAPs), which can be prospectively isolated by FACS, based on the expression levels of Tie2 and Vcam1. Tie2-expressing (Tie2+) and Tie2/Vcam1 double negative (DN) subFAPs were present in unperturbed muscles and promptly expanded upon acute injury. By contrast, Vcam1-expressing subFAPs (Vcam1+) were undetectable in unperturbed muscles, but appeared “de novo” upon acute injury and expanded with a shifted kinetic compared to Tie2+ and DN FAPs. While Vcam1+ subFAPs were completely cleared upon resolution of muscle regeneration following acute injury, they persisted in chronically injured muscles from the mdx mouse model of Duchenne Muscular Dystrophy (DMD). “Nested” RNA-seq analysis of subFAPs revealed subpopulation-specific gene expression profiles that were dynamically regulated along the regeneration process, predicted specific biological activities and functional interactions with other cell types, and exhibited DMD-specific signatures. In particular, Vcam1+ subFAPs showed enrichment in fibrotic gene expression and myofibroblast markers, and their altered clearance in acutely injured muscles upon macrophage depletion was associated with fibrosis. These data reveal the individual contribution of distinct subFAPs to the physiological and pathological repair of skeletal muscles, thereby providing new insights into the pathogenesis of muscular dystrophies and targets for selective therapeutic interventions.RNA-seq study of bulk FAPs or subFAPs isolated by Fluorescence Activated Cell Sorting (FACS) from hind limb muscles of young (3 months old) mice.RNA-seq was performed in cells from wild type mice, either unperturbed (WT) or at 3 days post notexin-mediated intramuscular acute injury (WT-inj 3d). We also included dystrophic mice (MDX), the murine model of Duchenne Muscular Dystrophy (DMD) that provides an experimental setting for chronic muscle injury