Examining the Therapeutic Potential of iPS cells in Duchenne Muscular Dystrophy

描述（由申请人提供）：最近通过引入Oct3/4、Sox2、Klf4和c-Myc，对成年小鼠和人成纤维细胞进行直接重编程以达到多能状态的突破，为产生患者和疾病特异性干细胞带来了新的希望，而没有与人类胚胎干细胞（ES）衍生相关的伦理问题。然而，在诱导多能干细胞用于治疗之前，有必要评估这些细胞向所需细胞类型分化的能力。例如，在胚胎干细胞向胚状体（EBs）的体外分化过程中，骨骼肌祖细胞的生成效率非常低。这是由于EBs中近轴中胚层的缺乏。我们最近证明，Pax3能够使分化的ES细胞产生肌源性祖细胞，这些细胞在植入mdx小鼠后被赋予恢复肌肉功能的能力。因此，在这里，我们计划通过将iPS细胞的生成与我们通过条件表达Pax7获得肌源性祖细胞的方法相结合，来检验iPS细胞可能在未来用于治疗DMD的原理证明。此外，如果想将iPS细胞转化为临床，必须证明从人类胚胎干细胞中可以成功获得功能性肌源性祖细胞。本文将通过将Pax7的条件表达应用于人类胚胎干细胞来评估这一点，目的是将这一知识应用于从杜氏肌营养不良症患者获得的人类iPS细胞的未来研究。

DESCRIPTION (provided by applicant): The recent breakthrough of direct reprogramming adult mouse and human fibroblasts toward a pluripotent state by introducing Oct3/4, Sox2, Klf4, and c-Myc, has brought new hope for the generation of patient- and disease- specific stem cells without the ethical issues associated with the derivation of human embryonic stem (ES) cells. However before induced pluripotent stem (iPS) cells can be applied for therapeutic applications, it is necessary to assess the ability of these cells to differentiate towards the desired cell type. For instance, skeletal myogenic progenitors are generated very inefficiently during in vitro differentiation of ES cells into embryoid bodies (EBs). This is due to the scarcity of paraxial mesoderm within EBs. We have recently demonstrated that Pax3 enables the generation of myogenic progenitors from differentiating ES cells that are endowed with the capacity to restore muscle function following their engraftment in mdx mice. Thus here we plan to examine the proof of principle that iPS cells may be used in the future for the treatment of DMD by combining the generation of iPS cells with our approach to derive myogenic progenitors by conditional expression of Pax7. Moreover, if one envisions translating iPS cells to the clinic, one has to demonstrate that functional myogenic progenitors can be successfully obtained from human ES cells. This will be assessed here by applying conditional expression of Pax7 to human ES cells with the goal to apply this knowledge to future studies involving human iPS cells obtained from patients with Duchene muscular dystrophy. PUBLIC HEALTH RELEVANCE: Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells hold great promise for the treatment of degenerative diseases, however to date studies on their potential use in the treatment of muscular dystrophies have been hampered by the difficulty of differentiating ES cells into skeletal muscle progenitors. This application builds on a novel method we have developed to generate muscle progenitors from mouse ES cells. We have shown that such progenitors can be transplanted into normal injured, and dystrophic mice, where they contribute to muscle fiber regeneration, and improve muscle function after injury. In these studies, we will apply this approach to wild-type mouse iPS cells (Aim 1) as well as ex vivo genetically corrected dystrophic mouse iPS cells (Aim 2), thus assessing proof-of-principle to whether these cells are endowed with in vivo regenerative potential. In Aim 3, we will investigate the mechanisms controlling muscle differentiation in human ES cells with the goal to apply this knowledge to future studies involving human iPS cells obtained from patients with Duchenne muscular dystrophy.