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: 10633236
• 负责人: Konrad Hochedlinger
• 金额: $ 43.6万
• 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/10633236
• 关键词: Address; Adult; Affect; Binding; Biological Assay; Biological Models; Cell Differentiation process; Cell Line; Cell Maintenance; Cell Separation; Cell Therapy; Cells; ChIP-seq; Characteristics; Chromatin; DNA; Derivation procedure; Disease; Disease model; Dystrophin; Engraftment; 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; Predisposition; Process; Proliferating; 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; postmitotic; progenitor; satellite cell; self renewing cell; self-renewal; small molecule; stem; stem cell self renewal; stem cells; stem-like cell; synergism; 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. !

总结 转分化是指一种成熟细胞类型在受迫分化时转化为另一种成熟细胞。 转录因子的表达或用小分子处理。转分化系统通常给出 上升到有丝分裂后的细胞，这对机制研究和潜在的治疗应用提出了挑战。 为了解决肌肉谱系中的这一缺陷，我们最近开发了一种新的去分化策略， 通过瞬时表达肌源性成纤维细胞直接转化为“诱导的肌源性祖细胞”（iMPC）， 转录因子MyoD在三个小分子的存在下。iMPC培养物由干样 产生祖细胞和成熟肌纤维的细胞，表现出自发收缩，从而重演 培养皿中肌细胞生成的关键阶段。此外，茎样iMPC子集可以克隆繁殖至少 20代，同时保留产生肌管的能力，证明了长期的自我更新， 体外分化潜能。因此，移植到mdx营养不良小鼠中的批量iMPC植入并 在体内分化成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 直接从成纤维细胞衍生稳定的、可扩增的和功能性的肌肉干细胞样细胞，并提供 本R 01应用程序的基础上使用三个互补的目标。在具体目标1中，我们将比较分子 从iMPC培养物纯化的Pax 7+干细胞样细胞和Pax 7+卫星细胞之间的功能特性 使用单细胞表达和染色质分析从骨骼肌中纯化， 移植试验此外，我们将利用我们最近开发的tetO-MyoD小鼠来测试是否 不同的细胞类型同样易于去分化成iMPC，并且iMPC是否来源于不同的 细胞类型保留来自其起源细胞的转录记忆。在具体目标2中，我们将研究 这种去分化过程的分子机制。首先，我们会评估是否有 和维持iMPCs依赖于相同的遗传调控作为卫星细胞在体内，重点是 转录因子Pax 7、Myf 5和MyoD，包括DNA和辅因子结合改变的MyoD突变体。我们 将通过检查增强子，进一步探索MyoD和小分子在iMPC诱导过程中的特定作用。 和与转分化相关的基因表达动力学（单独MyoD）。在具体目标3中，我们将测试 使用小鼠和人细胞的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mdx小鼠中重现体外已发表的疾病表型，以及iMPC是否易感 基因治疗在这三个目标中获得的机制见解最终将被用于努力， 产生人iMPC。总的来说，我们的项目将提供基本的见解， 转录因子和外部信号以肌肉谱系为模型， 系统，并探索如何在治疗环境中使用这些知识。 !