Disease Modeling of Skeletaland Cardiac Muscle in DMD/BMD using Patient-Specific iPS Cells

使用患者特异性 iPS 细胞对 DMD/BMD 中的骨骼肌和心肌进行疾病建模

• 批准号: 10586035
• 负责人: Rita C. R. Perlingeiro
• 金额: $ 17.05万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-07 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10586035
• 关键词: 3-Dimensional; Action Potentials; Affect; Amyotrophic Lateral Sclerosis; Axon; Becker Muscular Dystrophy; Biological; Biological Assay; Biological Process; Biology; Calcium; Cardiac; Cardiac Myocytes; Cell Line; Cells; Chemicals; Communities; Creatine Kinase; Defect; Detection; Development; Disease; Disease model; Drug Screening; Duchenne muscular dystrophy; Dystrophin; Electrophysiology (science); Functional disorder; Generations; Goals; Heart Atrium; In Vitro; Investigation; Laboratories; Link; Literature; Long QT Syndrome; Measurement; Membrane; Methods; Microelectrodes; Modeling; Molecular; Molecular Profiling; Mus; Muscle Fibers; Muscle function; Muscle satellite cell; Mutation; Myocardium; Myopathy; Nodal; Paper; Pathogenesis; Patients; Phenotype; Pluripotent Stem Cells; Population; Proteins; Publishing; Regenerative Medicine; Research; Resources; Role; Sampling; Signal Pathway; Skeletal Muscle; Somatic Cell; Spinal Muscular Atrophy; System; Technology; Therapeutic; Tissues; Validation; Ventricular; Work; cell type; cohort; disease phenotype; drug discovery; dystrophinopathy; gene correction; human pluripotent stem cell; in vivo; induced pluripotent stem cell; induced pluripotent stem cell technology; insight; instrument; mutant; osteoprogenitor cell; patch clamp; programs; screening; skeletal; small molecule; stem cells; transcriptome; transcriptomic profiling

Summary This project aims to establish a reliable platform to use patient-specific induced pluripotent stem (iPS) cells to model Duchenne Muscular Dystrophy (DMD) and Becker MD (BMD) in the Petri dish, allowing not only for better insight on the pathogenesis of dystrophin-associated disorders, but specially for the development of a relevant system for drug screening. Reprogramming technology provides an unprecedented opportunity to generate large numbers of patient-specific cell types for in vitro disease modeling and drug discovery. Accordingly, iPS cells have been used extensively for these purposes for several disorders, including amyotrophic lateral sclerosis, long-QT syndrome, and spinal muscular atrophy, among others. However to date, there has been scarce literature on the use of this approach to model skeletal muscle disorders, and none studying both skeletal and cardiac muscle in the context of DMD and BMD. Our research group has pioneered methods to derive large quantities of skeletal myogenic progenitor cells from mouse and human pluripotent stem cells, and validated these in vitro and in vivo. Specifically relevant for this proposal, we have recently published a paper focusing on the in vitro maturation of iPS cells towards the skeletal muscle lineage (eLIFE, 2019), which is critical for proper disease modeling. For this project, we have samples from ten DMD/BMD patients, encoding various distinct mutations and displaying mild to severe phenotypes. iPS cells from these samples will be used to produce large numbers of DMD/BMD skeletal myotubes and cardiomyocytes. The hypothesis behind this project is that we will be able to recapitulate disease phenotypes in vitro, including membrane fragility, calcium handling, contraction using three-dimensional myobundles, and electrophysiology measurements. We foresee that molecular/biological signatures resulting from this work will not only enhance our understanding of the pathophysiology behind DMD/BMD, but may also serve as in vitro screening for potential treatments. To validate this hypothesis, we will have control cohort samples, in which we genetically introduce selected DMD mutations in unaffected iPS cells and/or correct selected DMD mutations. The work we propose here, of not only generating skeletal and cardiac muscle from iPS cells but also studying the underlying biology, holds tremendous potential for exploring phenotypes of different mutations, in vitro disease modeling in general, and drug discovery.

概括 该项目旨在建立一个可靠的平台，利用患者特异性诱导多能干（iPS）细胞 培养皿中的杜氏肌营养不良症 (DMD) 和贝克尔肌营养不良症 (BMD) 模型，不仅可以更好地 对肌营养不良蛋白相关疾病的发病机制的深入了解，但特别是为了开发相关的 药物筛选系统。重编程技术提供了前所未有的机会来产生大量 用于体外疾病建模和药物发现的患者特异性细胞类型的数量。因此，iPS细胞 已被广泛用于治疗多种疾病，包括肌萎缩侧索硬化症， 长QT综合征和脊髓性肌萎缩症等。但迄今为止，还很少有 关于使用这种方法来模拟骨骼肌疾病的文献，并且没有研究骨骼肌和骨骼肌疾病的文献 DMD 和 BMD 背景下的心肌。 我们的研究小组开创了从骨骼中提取大量骨骼肌源性祖细胞的方法 小鼠和人类多能干细胞，并在体外和体内验证了这些细胞。与此具体相关 根据提案，我们最近发表了一篇论文，重点关注 iPS 细胞向骨骼的体外成熟 肌肉谱系（eLIFE，2019），这对于正确的疾病建模至关重要。对于这个项目，我们有样品 来自 10 名 DMD/BMD 患者，编码各种不同的突变并表现出轻度至重度表型。 iPS 这些样本中的细胞将用于生产大量 DMD/BMD 骨骼肌管和 心肌细胞。该项目背后的假设是，我们将能够重现疾病表型 体外，包括膜脆性、钙处理、使用三维肌束的收缩，以及 电生理学测量。我们预计这项工作产生的分子/生物特征将 不仅增强了我们对 DMD/BMD 背后病理生理学的理解，而且还可以作为体外 筛选潜在的治疗方法。为了验证这个假设，我们将有对照队列样本，其中我们 在未受影响的 iPS 细胞中通过基因引入选定的 DMD 突变和/或纠正选定的 DMD 突变。 我们在此提出的工作不仅是从 iPS 细胞生成骨骼肌和心肌，而且还研究 基础生物学，具有在体外探索不同突变表型的巨大潜力 一般疾病建模和药物发现。