Examining the Therapeutic Potential of iPS cells in Duchenne Muscular Dystrophy

描述(申请人提供)：最近通过引入Oct3/4、Sox2、Klf4和c-Myc，直接将成年小鼠和人类成纤维细胞重新编程为多能状态的突破，为产生患者和疾病特异性干细胞带来了新的希望，而不存在与人类胚胎干细胞(ES)派生相关的伦理问题。然而，在诱导多能干细胞(IPS)可以应用于治疗应用之前，有必要评估这些细胞向所需细胞类型分化的能力。例如，在ES细胞体外分化为类胚体(EBS)的过程中，骨骼肌生肌祖细胞的生成效率非常低。这是由于EBS内缺乏轴旁中胚层所致。我们最近已经证明，Pax3能够从分化的ES细胞中产生肌源性祖细胞，ES细胞被赋予了在MDX小鼠体内植入后恢复肌肉功能的能力。因此，在这里，我们计划通过结合iPS细胞的产生和我们的方法来检验iPS细胞未来可能用于治疗DMD的原理证据，我们的方法是通过条件表达Pax7来获得肌源性祖细胞。此外，如果人们设想将iPS细胞转化到临床，人们必须证明可以成功地从人类ES细胞中获得有功能的肌源性祖细胞。这里将通过将Pax7的条件表达应用于人类ES细胞来评估这一点，目的是将这一知识应用于未来涉及从Duchene肌营养不良患者获得的人iPS细胞的研究。 公共卫生相关性：胚胎干细胞(ES)和诱导多能干细胞(IPS)在治疗退行性疾病方面前景看好，然而到目前为止，由于难以将ES细胞分化为骨骼肌祖细胞，其在肌肉营养不良治疗中的潜在应用研究一直受到阻碍。这一应用建立在我们开发的一种新方法之上，该方法可以从小鼠的ES细胞中产生肌肉祖细胞。我们已经证明，这些祖细胞可以移植到正常受损和营养不良的小鼠体内，在那里它们有助于肌肉纤维的再生，并改善损伤后的肌肉功能。在这些研究中，我们将把这种方法应用于野生型小鼠iPS细胞(Aim 1)以及体外基因纠正的营养不良小鼠iPS细胞(Aim 2)，从而评估这些细胞是否具有体内再生潜力的原则证明。在目标3中，我们将研究控制人类ES细胞肌肉分化的机制，目标是将这一知识应用于未来涉及从Duchenne肌营养不良患者获得的人iPS细胞的研究。

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.