Genetic Regulation of Skeletal Muscle Repair

Project Summary Satellite cells are required for the growth and regeneration of skeletal muscle. Molecular genetic analyses, engraftment assays, and single-cell analyses have suggested that satellite cells are a heterogenous population that potentially exhibit very distinct properties. Understanding satellite cell heterogeneity and identifying the long-term self-renewing stem cell within the satellite cell compartment remains one of the most important unresolved issues in muscle biology, and with the advent of technologies that facilitate single-cell analyses, is currently attracting much interest. In preliminary experiments, we have used gene expression analysis to define a gene signature for a subpopulation of satellite cells we term satellite stem cells. This gene signature was then used to identify a novel cluster that contains this subpopulation using single-cell RNA-seq analysis. Transplantation of satellite stem cells after isolation, using cell surface markers that we defined, indicate that satellite stem cells exhibit markedly superior engraftment properties. Therefore, we hypothesize that satellite stem cells represent the bona fide long-term self-renewing muscle stem cell. Here we propose to molecularly characterize this important subpopulation and establish its hierarchal relationship with the larger population of satellite cells and their progenitors. In Aim 1, we propose to characterize the function and potential of satellite stem cells using lineage tracing and following transplantation of prospectively isolated satellite stem cells. We will examine the expression of genes specifically expressed in satellite stem cells during asymmetric cell divisions and lineage progression on cultured myofibers. These experiments will provide important new information concerning the activity, potential, and function of satellite stem cells. In Aim 2, we will define the cellular and molecular composition of satellite stem cell lineage hierarchy at the resolution of single cells. We will use single-cell transcriptomics (RNA-seq) to delineate the differentiation trajectory of satellite stem cells and their derivatives that arise during growth and regeneration using mice containing lineage tracing alleles, and mice lacking genes that perturb regeneration. We will analyze the function of differentially expressed regulatory genes using a novel high-content analysis platform. This data will allow reconstruction of the cellular differentiation trajectories and provide important new information into the molecular regulation of self-renewal and lineage progression. In Aim 3, we will identify and characterize human satellite stem cells by conducting analysis of satellite cells isolated from human muscle biopsies. The notion that satellite cells are heterogenous with a subset representing a long-term self-renewing stem cell remains controversial. Our discovery of a gene- signature specific to satellite stem cells is therefore innovative and paradigm shifting. In particular, the identification of cell surface markers specific to satellite stem cells will without question, facilitate rapid advances in our understanding of the molecular mechanisms that regulate satellite stem cell homeostasis in human muscle wasting diseases and in aging.