Investigating the direct reprogramming of fibroblasts into skeletal muscle progenitors

研究成纤维细胞直接重编程为骨骼肌祖细胞

• 批准号: 10408751
• 负责人: Konrad Hochedlinger
• 金额: $ 43.17万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10408751
• 关键词: Address; Adult; Affect; Binding; Biological Assay; Biological Models; Cell Differentiation process; Cell Line; Cell Maintenance; Cell Therapy; Cells; ChIP-seq; Characteristics; Chromatin; DNA; Derivation procedure; Disease; Disease model; Dystrophin; Enhancers; Epigenetic Process; Exhibits; Fibroblasts; Future; Gene Expression; Gene Mutation; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Germ Layers; Hepatocyte; Human; In Vitro; Individual; Injury; Knowledge; Maintenance; Measures; Memory; Modeling; Molecular; Mouse Strains; Mus; Muscle; Muscle Development; Muscle Fibers; Muscular Dystrophies; MyoD Protein; Myopathy; Neurons; Pattern; Pharmaceutical Preparations; Phenotype; Population; Process; Property; Protocols documentation; Publishing; Reporting; Role; Signal Transduction; Skeletal Muscle; Source; Specific qualifier value; Specificity; Stem cell transplant; System; Testing; Therapeutic; Transgenes; Transplantation; Undifferentiated; cell dedifferentiation; cell type; cofactor; disease phenotype; experimental study; fetal; gene therapy; in vivo; insight; mdx mouse; muscle regeneration; mutant; myogenesis; novel strategies; progenitor; satellite cell; self renewing cell; self-renewal; small molecule; stem; stem cell self renewal; stem cells; stem-like cell; transcription factor; transcriptome sequencing; transdifferentiation

SUMMARY Transdifferentiation denotes the conversion of one mature cell type into another mature cell upon forced expression of transcription factors or treatment with small molecules. Transdifferentiation systems typically give rise to postmitotic cells, which poses a challenge for mechanistic studies and potential therapeutic applications. To address this shortcoming in the muscle lineage, we recently developed a novel strategy to dedifferentiate fibroblasts directly into “induced myogenic progenitor cells” (iMPCs) by transiently expressing the myogenic transcription factor MyoD in the presence of three small molecules. iMPC cultures are comprised of stem-like cells that give rise to progenitors and mature myofibers exhibiting spontaneous contraction, thus recapitulating key stages of myogenesis in a dish. Moreover, stem-like iMPC subsets can be clonally propagated for at least 20 passages while retaining the ability to produce myotubes, demonstrating long-term self-renewal and differentiation potential in vitro. Accordingly, bulk iMPCs transplanted into mdx dystrophic mice engraft and differentiate into Dystrophin-expressing myotubes in vivo. Thus, our results represent the first successful derivation of stable, expandable and functional muscle stem-like cells directly from fibroblasts and provide the basis for this R01 application using three complementary aims. In SPECIFIC AIM 1, we will compare molecular and functional properties between Pax7+ stem-like cells purified from iMPC cultures and Pax7+ satellite cells purified from skeletal muscle using single-cell expression and chromatin analyses as well as a serial transplantation assay. In addition, we will leverage a tetO-MyoD mouse we recently developed to test whether different cell types are equally amenable to dedifferentiation into iMPCs and whether iMPCs derived from distinct cell types retain a transcriptional memory from their cells of origin. In SPECIFIC AIM 2, we will investigate the molecular mechanisms underlying this dedifferentiation process. First, we will assess whether the establishment and maintenance of iMPCs depend on the same genetic regulators as satellite cells in vivo, with a focus on the transcription factors Pax7, Myf5 and MyoD including MyoD mutants with altered DNA and cofactor binding. We will further explore the specific roles of MyoD and small molecules during iMPC induction by examining enhancer and gene expression dynamics in relation to transdifferentiation (MyoD alone). In SPECIFIC AIM 3, we will test the potential therapeutic utility of iMPCs using mouse and human cells. Briefly, we will assess whether iMPCs from dystrophic mdx mice recapitulate published disease phenotypes in vitro and whether iMPCs are susceptible to gene therapy. Mechanistic insights gained throughout these 3 aims will finally be exploited for efforts to generate human iMPCs. Collectively, our project will provide fundamental insights into the mechanisms by which transcription factors and external signals rewire cell fate using the muscle lineage as a model system and explore how this knowledge could be used in a therapeutic setting. !

摘要 转分化指的是一种成熟细胞类型在强迫下转化为另一种成熟细胞 转录因子的表达或用小分子处理。转分化系统通常会给 升至有丝分裂后细胞，这对机制研究和潜在的治疗应用构成了挑战。 为了解决肌肉谱系的这一缺陷，我们最近开发了一种新的策略来去分化 成纤维细胞通过瞬时表达成肌细胞直接转化为“诱导成肌祖细胞”(IMPC) 转录因子MyoD中存在三个小分子。IMPC培养物由茎状结构组成 产生祖细胞和成熟的肌纤维的细胞，表现出自发收缩，从而重现 培养皿中肌肉发生的关键阶段。此外，茎状iMPC亚集可以克隆繁殖至少 20代，同时保持产生肌管的能力，展示长期自我更新和 体外分化潜能。因此，将大量iMPC移植到MDX营养不良小鼠体内，并 在体内分化为表达肌营养不良蛋白的肌管。因此，我们的结果代表了第一个成功的 直接从成纤维细胞获得稳定的、可扩增的和有功能的肌肉干细胞样细胞，并提供 使用三个相辅相成的目标为此R01应用程序奠定基础。在特定的目标1中，我们将比较分子 从iMPC纯化的Pax7+干细胞与Pax7+卫星细胞的功能特性 使用单细胞表达和染色质分析以及一系列的 移植试验。此外，我们将利用我们最近开发的Teto-MyoD小鼠来测试 不同类型的细胞同样容易去分化为iMPC，以及iMPC是否来源于不同的 细胞类型保留了它们起源的细胞的转录记忆。在具体的AIM 2中，我们将调查 这种去分化过程背后的分子机制。首先，我们会评估编制是否 与体内卫星细胞一样，iMPC的维持依赖于相同的基因调控，重点是 转录因子Pax7、Myf5和MyoD包括DNA和辅因子结合改变的MyoD突变体。我们 将通过检测增强子进一步探讨MyoD和小分子在iMPC诱导过程中的具体作用 以及与转分化相关的基因表达动力学(仅MyoD)。在具体的AIM 3中，我们将测试 使用小鼠和人类细胞的iMPC的潜在治疗效用。简而言之，我们将评估iMPC是否 从营养不良的mdx小鼠在体外总结了已发表的疾病表型以及iMPC是否易感 到基因疗法。通过这三个目标获得的机械性见解最终将被用于努力 产生人类的iMPC。总而言之，我们的项目将提供对这些机制的基本见解 以肌肉谱系为模型，转录因子和外部信号重新连接细胞命运 并探索如何在治疗环境中使用这些知识。 好了！