Single-cell analysis of the muscle stem cell hierarchy identifies heterotypic communication signals involved in skeletal muscle regeneration

Abstract

Muscle stem cells (MuSCs) are an essential adult stem cell population with the capacity to self-renew and regenerate muscle tissue. Functionally heterogeneous subpopulations of MuSCs have been identified based on their expression of myogenic regulatory factors and surface markers. However, a unified organization of muscle stem and progenitor cells and their subpopulations remains unresolved.Here, we performed temporal analysis of skeletal muscle regeneration using single-cell RNA-sequencing (scRNA-seq) of myotoxin-injured adult mouse hindlimb muscles. We generated over 34,000 single-cell transcriptomes spanning four muscle regeneration time-points and identified 15 distinct cell types, including a heterogeneous population of MuSCs and progenitor cells. Our analysis provides a hierarchical map of myogenic cell populations and identifies stage-specific regulatory programs that govern their contributions to muscle regeneration. In this transcriptomic atlas, we observed cell type-specific regenerative dynamics, exemplified by waves of transient amplification and diversification of multiple immune cell types and, subsequently, myogenic cells. Unbiased trajectory inference organized the myogenic cell populations within the atlas into a continuum, consisting of a hierarchy of quiescent MuSCs, cycling progenitors, committed myoblasts, and terminally differentiated myocytes. This myogenic trajectory matched prior understanding and also revealed that MuSC stages are defined by synchronous changes in regulatory factors, cell cycle-associated, and surface receptor gene expression. Lastly, we analyzed the transcriptomic atlas to identify over 100 candidate heterotypic communication signals between myogenic and non-myogenic cell populations, including many involving the fibroblast growth factor (FGF), Notch, and Syndecan receptor families and their associated ligands. Syndecan receptors were implicated in a large fraction of these cell communication interactions and were observed to exhibit transcriptional heterogeneity within the myogenic continuum. Using multiparameter mass cytometry (CyTOF), we confirmed that cycling MuSCs exhibit diversified Syndecan-1/2 expression, which suggests that dynamic alterations in Syndecan signaling interactions may coordinate stage-specific myogenic cell fate regulation. This scRNA-seq reference atlas provides a resolved hierarchical organization of myogenic subpopulations as a resource to investigate cell-cell interactions that regulate myogenic stem and progenitor cell fates in muscle regeneration.suggest that myogenic stem/progenitor cell lineage can be interpreted as a continuum of hierarchical cell states. However, it remains an unresolved challenge how global profiles in cell cycle mediators, regulatory factors and surface markers define this myogenic continuum.Recent advances in single-cell analyses and algorithms provide potent new strategies to infer cell differentiation trajectories (Hwang et al., 2018;Wagner et al., 2016). Here, we generated a single-cell transcriptomic atlas of mous...

Aging induces aberrant state transition kinetics in murine muscle stem cells

“…S3A, B). Our data are consistent with previous reports of decreased Pax7 with activation at the ensemble level (Cornelison and Wold, 1997;De Micheli et al, 2019;DellOrso et al, 2019) when we consider only the mean expression of Pax7 at the quiescent and activated time points (Fig. S3C).…”

Spatial Heterogeneity of Delta-like 4 Within a Multinucleated Niche Cell Maintains Muscle Stem Cell Diversity

“…The copyright holder for this preprint this version posted October 20, 2020. ; https://doi.org/10.1101/2020.10.20.347484 doi: bioRxiv preprint a continuum of molecular states at tissue homeostasis. This supports models inferred from scRNAseq analysis on SCs (De Micheli et al, 2020;Dell'Orso et al, 2019;Kimmel et al, 2020).…”

“…The copyright holder for this preprint this version posted October 20, 2020. ; https://doi.org/10.1101/2020.10.20.347484 doi: bioRxiv preprint state for the majority of adult life. Through single cell RNA-sequencing (scRNA-seq) and transgenic reporter mice it is appreciated that the QSC pool is molecularly and phenotypically heterogeneous (Chakkalakal et al, 2014;De Micheli et al, 2020;Dell'Orso et al, 2019;Kimmel et al, 2020;Kuang et al, 2007;Rocheteau et al, 2012). These different cell states enable SCs to exhibit functional heterogeneity in response to activation and injury cues.…”

Single-cell Transcriptomics Identify Extensive Heterogeneity in the Cellular Composition of Mouse Achilles Tendons

“…Single cell isolation. The Achilles tendons were digested to obtain a single cell suspension, as modified from a previous study (13). Two Achilles tendons from each animal were processed together as a single sample, and the tendons of N=4 mice were used.…”

“…Tendons were finely minced using a scalpel, and then digested for 1 h at 37ºC in a vigorously shaking Single cell RNA sequencing and analysis. Single cell RNA sequencing and analysis was performed, as modified from a previous study (13). Libraries were prepared using a Chromium Single Cell 3' Reagent Kit (version 3, 10X Genomics, Pleasanton, CA, USA) following the directions of the manufacturer.…”