Study of Skeletal Muscle Differentiation in Human iPS Cells by Knock-in Reporters

 说明（申请人提供）：骨骼肌是人体最大的器官，容易发生多种疾病。肌肉营养不良、由于恶病质或肌肉减少症引起的肌肉消耗和由于损伤引起的肌肉质量损失是最常见的肌肉病症类型。尽管在了解这些疾病的病理基础方面取得了许多进展，但不幸的是，这些病例的治疗选择非常有限或无效。同时，使用基于干细胞的骨骼肌修复疗法已被认为是这些情况下的潜在候选者之一。由于这个原因，多能干细胞是最好的候选者，因为它们具有无与伦比的分化和自我更新潜力。随着人类胚胎干（ES）细胞的成功分离以及随后从体细胞产生诱导多能干细胞（iPS细胞），已经发现了用于疾病建模和设计患者特异性细胞疗法的前所未有的机会。因此，人多能干细胞（即hES/ iPS细胞）向骨骼肌谱系的分化一直是发育研究以及基于干细胞的肌肉疾病再生疗法的关注焦点。事实上，近年来，很少有方法被开发用于从hES/iPS细胞衍生骨骼肌原性前体，例如通过长期培养的肌原性基因过表达或间充质分化。然而，这些方法不能用于临床目的，因为使用病毒的细胞制备不安全，或者因为其他缺点，如低效率或肌原细胞的杂质。这些问题主要是由于缺乏前瞻性的方法来研究hES/iPS细胞向骨骼肌谱系的时序分化，因为大多数这些研究回顾性地评估多能干细胞的肌源性分化。因此，在本研究应用中，设计了实验来克服这些缺点。本申请的第一个目的是在人iPS细胞中产生参与早期骨骼肌发育的重要基因（PAX 7，Myf 5）的敲入报告细胞系。为此目的，利用RNA引导的Cas9介导的同源重组方法来掺入2A-GFP或tdTomato报道基因。这将为研究人ES/iPS细胞体外分化过程中骨骼肌发生的时间模式提供一个独特的机会。本申请的第二个目的是使用化学文库筛选来定义hES/iPS细胞的定向分化。此外，纯化的生肌前体将针对表面标志物和基因表达进行充分表征，以确定源自hES/iPS细胞的生肌前体的特征谱。这将允许将该方法应用于任何其他人iPS细胞系，而不需要在基因组中掺入报道基因。本申请的第三个目的将集中于在两种常见肌肉病变（肌营养不良和肌肉损失）的小鼠模型中评价人iPS衍生的肌原性细胞的体内再生潜力。这将为人类iPS衍生细胞用于肌肉疾病建模或治疗提供宝贵的数据。

 DESCRIPTION (provided by applicant): Skeletal muscle as the largest organ in human body is prone to many disorders. Muscular dystrophies, muscles wasting due to cachexia or sarcopenia and muscle mass loss due to injuries are among most common types of muscle disorders. Despite many advances in understanding the pathologic basis of these disorders, therapeutic options in these cases are unfortunately very limited or ineffective. Meanwhile, using stem cell based therapies for skeletal muscle repair has been considered as one of the potential candidates in these cases. For this reason, pluripotent stem cells are the best candidate due to their unparalleled differentiation and self-renewal potentials. With the successful isolation of human embryonic stem (ES) cells and later on, generation of induced pluripotent stem cells (iPS cells) from the somatic cells, an unprecedented opportunity has been discovered for disease modeling and designing patient specific cell therapies. Therefore, differentiation of human pluripotent stem cells (i.e. hES/ iPS cells) toward skeletal muscle lineage has been the focus of attention for developmental studies as well as stem cell based regenerative therapies for muscle disorders. Indeed, in the recent years, few methods have been developed for derivation of skeletal myogenic precursors from hES/iPS cells such as myogenic gene over-expression or mesenchymal differentiation through long-term cultures. However, these approaches cannot be utilized for clinical purposes due to unsafe cell preps using viruses or due to other shortcomings such as low efficiency or impurity of the myogenic cells. These problems mostly arise from the lack of a prospective approach to study chronological differentiation of hES/iPS cells toward skeletal muscle lineage, as most of these studies evaluate myogenic differentiation of the pluripotent stem cells retrospectively. Therefore, in this research application, experiments have been designed to overcome these shortcomings. In the 1st aim of this application knock-in reporter cell lines in human iPS cells for important genes involved in early skeletal muscle development (PAX7, Myf5) is being generated. For this purpose RNA guided Cas9 mediated homologous recombination approach is utilized to incorporate a 2A- GFP or tdTomato reporter. This will provide a unique opportunity to study the temporal pattern of skeletal myogenesis during in vitro differentiation of the human ES/iPS cells. The 2nd aim of this application is to define directed differentiation of hES/iPS cells using chemical library screen. Moreover, purified myogenic precursors will be fully characterized for surface markers and gene expression in order to determine the signature profile of the myogenic precursors derived from hES/iPS cells. This will allow applying this methodology to any other human iPS cell line without need to incorporate a reporter in the genome. The 3rd aim of this application will focus on evaluation of in vivo regeneration potential of human iPS derived myogenic cells in mice models of two common muscle pathologies (muscular dystrophies and muscle loss). This will provide invaluable data for the application of human iPS derived cells for muscle disease modeling or therapeutics.