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

基本信息

批准号：
10032776
负责人：
Konrad Hochedlinger
金额：
$ 45.1万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
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/10032776
关键词：
Address Adult Affect Binding Biological Assay Biological Models Cell Culture Techniques 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 cells stem-like cell transcription factor transcriptome sequencing transdifferentiation

项目摘要

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. !

概括转分化表示一种成熟的细胞类型在强迫时转化为另一个成熟的细胞转录因子的表达或用小分子处理。转变系统通常给出升至蒙霉菌细胞，这对机械研究和潜在的治疗应用构成了挑战。为了解决肌肉谱系中的这种缺点，我们最近制定了一种新型策略来推测成纤维细胞直接通过瞬时表达肌源性，直接进入“诱导肌源性祖细胞”（IMPC）在存在三个小分子的情况下，转录因子myod。 IMPC文化已完成类似茎的培养物产生祖细胞和成熟的肌纤维的细胞，表现出赞助，从而概括菜肴中肌发生的关键阶段。此外，至少可以将类似茎状的IMPC子集传播 20次通过，同时保留生产肌管的能力，展示长期自我更新和分化潜力在体外。彼此之间，移植到MDX营养不良的小鼠植入和在体内区分表达肌营养不良蛋白的肌管。那，我们的结果代表了第一个成功直接从成纤维细胞中推导稳定，可扩展和功能性肌肉样细胞，并提供使用三个完整目标的R01应用程序的基础。在特定的目标1中，我们将比较分子 PAX7+茎样细胞之间从IMPC培养物纯净的PAX7+卫星细胞之间的功能特性使用单细胞表达和染色质分析和串行从骨骼肌纯化移植测定法。此外，我们将利用我们最近开发的一种teto-myod鼠标来测试是否不同的细胞类型同样适合于将IMPC推导到IMPC中，以及IMPC是否来自不同的IMPC 细胞类型从其原始细胞中保留转录记忆。在特定目标2中，我们将调查该去分化过程的基础分子机制。首先，我们将评估建立是否 IMPC的维持取决于体内与卫星细胞相同的遗传调节剂，重点是转录因子PAX7，MYF5和MYOD，包括具有改变DNA和辅因子结合的MYOD突变体。我们通过检查增强剂，将进一步探索IMPC诱导过程中MYOD和小分子的特定作用和基因表达动力学与转分化的关系（单独使用Myod）。在特定目标3中，我们将测试使用小鼠和人类细胞的IMPC的潜在理论实用性。简而言之，我们将评估IMPC是否从营养不良的MDX小鼠概括了体外已发表的疾病表型，以及IMPC是否易感进行基因治疗。在这三个目标中获得的机械洞察力最终将被探索以进行努力产生人类的IMPC。总的来说，我们的项目将提供有关机制的基本见解通过哪些转录因子和外部信号使用肌肉谱系作为模型恢复细胞命运系统并探索如何在治疗环境中使用这些知识。呢