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

 说明(申请人提供)：骨骼肌作为人体最大的器官，易患多种疾病。肌肉营养不良、恶病质或肉质减少引起的肌肉萎缩以及因受伤导致的肌肉质量下降是最常见的肌肉疾病类型。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。与此同时，使用干细胞为基础的治疗骨骼肌修复一直被认为是这些情况下的潜在候选之一。因此，多能干细胞因其无与伦比的分化和自我更新潜力而成为最佳候选细胞。随着人类胚胎干细胞的成功分离，以及后来体细胞诱导多能干细胞的产生，为疾病建模和设计患者特异性细胞疗法提供了前所未有的机会。因此，人类多能干细胞(即HES/iPS细胞)向骨骼肌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治疗的关注焦点。事实上，近年来，很少有方法可以从HES/iPS细胞中获得骨骼肌源性前体细胞，如肌源性基因过度表达或通过长期培养向间充质分化。然而，这些方法不能用于临床，因为使用病毒的细胞制备不安全，或者由于其他缺点，如肌源性细胞的低效率或不纯。这些问题主要是由于缺乏一种前瞻性的方法来研究HES/iPS细胞向骨骼肌谱系的时序分化，因为这些研究大多回顾地评估了多能干细胞的肌源性分化。因此，在本文的研究应用中，设计了实验来克服这些不足。在这项应用的第一个目的是建立人iPS细胞中与早期骨骼肌发育有关的重要基因(PAX7，Myf5)的敲入报告细胞系。为此，利用RNA引导的Cas9介导的同源重组方法来掺入2A-GFP或tdTomato报告基因。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利用化学文库筛选确定HES/iPS细胞的定向分化。此外，纯化的成肌前体将完全表征表面标志和基因表达，以确定来自HES/iPS细胞的成肌前体的特征图谱。这将允许将这种方法应用于任何其他人类iPS细胞系，而不需要在基因组中加入报告。这项应用的第三个目标将集中在评估人iPS来源的肌源性细胞在两种常见肌肉病变(肌肉营养不良和肌肉丢失)小鼠模型中的体内再生潜力。这将为人类iPS来源的细胞在肌肉疾病建模或治疗中的应用提供宝贵的数据。

 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.