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. !

概括转变表示在强迫时将一种成熟的细胞类型转化为另一个成熟的细胞成熟转录因子的表达或用小分子处理。升至蒙单后细胞，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通过透射表达肌源性在小分子存在下转录因子myod myod。引起祖细胞和成熟的肌纤维的细胞，表现出自发收缩，因此概括了盘子中的肌发生的关键阶段。 20段落的同时保留了生产肌管的能力，展示了长期自我更新和相应地，分化潜力。在体内分为表达肌营养不良的肌管。直接从成纤维细胞中推导稳定，可扩展和功能性肌肉干细胞，并提供使用三个比较分子的R01应用的基础 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从骨骼肌中纯化移植测定。不同的细胞类型同样适合于对IMPC的脱发以及IMPC是否来自不同的IMPC 细胞类型在特定AIM 2中保留其原始单元的转录记忆。这种去分化过程的分子机制。 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和辅助因子的Myod突变体将进一步探讨IMPC诱导过程中Myod和小分子的特定作用与转变的基因表达动力学（单独的MYOD）。使用小鼠和人类细胞的IMPC的潜在治疗效用。从营养不良的MDX小鼠中，在体外概括了已发表的疾病表型，以及IMPC是否可察觉基因疗法。共同产生人类的IMPC，我们的项目将提供基本的见解通过哪些转录因子和外部用肌肉谱系作为模型的重新布线细胞命运系统并探索在治疗环境中该知识如何。呢