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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. !

总结