Genetic Regulation of Skeletal Muscle Repair

骨骼肌修复的基因调控

• 批准号: 10477403
• 负责人: Michael A Rudnicki
• 金额: $ 27.27万
• 依托单位: OTTAWA HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10477403
• 关键词: Aging; Alleles; Biological Assay; Biology; Biopsy; Brown Fat; CD36 gene; CXCL12 gene; Cell Compartmentation; Cell Lineage; Cell division; Cell surface; Cells; Data; Deductibles; Development; Disease; Duchenne muscular dystrophy; Engraftment; Exhibits; Expression Profiling; Gene Expression; Gene Expression Profiling; Genes; Genetic; Growth; Health; Heterogeneity; Human; Immune; Informal Social Control; Kinetics; Knowledge; Label; Methodology; Molecular; Molecular Analysis; Molecular Genetics; Mus; Muscle; Muscle satellite cell; Muscular Atrophy; Natural regeneration; Neuromuscular Diseases; Population; Property; Regulation; Regulator Genes; Resolution; Skeletal Muscle; Skeletal Muscle Satellite Cells; Technology; Testing; Therapeutic Intervention; Translating; Transplantation; Wasting Syndrome; differential expression; experimental study; genetic analysis; genetic signature; innovation; insight; interest; loss of function mutation; mdx mouse; mouse model; muscle regeneration; novel; progenitor; prospective; reconstruction; repaired; satellite cell; self-renewal; single cell analysis; single-cell RNA sequencing; stem cell biology; stem cell function; stem cell homeostasis; stem cell population; stem cells; transcriptome sequencing; transcriptomics

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.

项目摘要 卫星细胞是骨骼肌生长和再生所必需的。分子遗传分析， 植入分析和单细胞分析表明，卫星细胞是异质种群 这可能表现出非常不同的特性。了解卫星细胞异质性并确定 卫星细胞室内的长期自我更新干细胞仍然是最重要的 肌肉生物学中未解决的问题，并且随着促进单细胞分析的技术的出现， 目前引起了很多兴趣。在初步实验中，我们使用了基因表达分析到 定义用于卫星细胞亚群的基因特征，我们称为卫星干细胞。这个基因签名 然后被用于识别一个新的簇，该簇使用单细胞RNA-seq分析，该群集包含该亚群。 分离后的卫星干细胞移植，使用我们定义的细胞表面标记，表明 卫星干细胞表现出明显的上植入特性。因此，我们假设该卫星 干细胞代表真正的长期自我更新肌肉干细胞。在这里，我们提出分子 表征这一重要的亚种群，并与较大的人群建立了层次关系 卫星细胞及其祖细胞。在AIM 1中，我们建议表征卫星的功能和潜力 干细胞使用谱系追踪和前瞻性分离的卫星干细胞移植后。我们 将检查在不对称细胞中特异性表达的基因的表达 培养的肌纤维的分裂和谱系进展。这些实验将提供重要的新 有关卫星干细胞活动，潜力和功能的信息。在AIM 2中，我们将定义 卫星干细胞谱系层次结构的细胞和分子组成在单个细胞的分辨率下。我们 将使用单细胞转录组学（RNA-SEQ）来描述卫星干细胞的分化轨迹 及其在生长和再生过程中使用的衍生物，使用含有谱系追踪等位基因的小鼠， 和缺乏扰动再生基因的小鼠。我们将分析差异表达的功能 使用新型的高含量分析平台进行调节基因。这些数据将允许重建细胞 分化轨迹并为自我更新的分子调节提供重要的新信息 和谱系进展。在AIM 3中，我们将通过进行识别和表征人类卫星干细胞 从人类肌肉活检中分离出的卫星细胞的分析。卫星细胞是异质的观念 代表长期自我更新干细胞的子集仍然存在争议。我们发现了一个基因 因此，特定于卫星干细胞的签名是创新的和范式转移的。特别是 识别特定于卫星干细胞的细胞表面标记将毫无疑问，促进快速 我们对调节卫星干细胞体内平衡的分子机制的理解的进步 人类的肌肉浪费疾病和衰老。