Angiomyogenesis With Myoblast Derived iPS Cells

描述（由申请人提供）：从成纤维细胞中生成诱导多能干细胞（iPS）的创新方法为生成用于个体化细胞治疗的自体、患者特异性多能干细胞系打开了令人惊叹的新大门。然而，在非病毒诱导多能干细胞用于临床应用之前，还有许多重要的研究工作要做，以优化其生成、安全性和有效性。我们的研究将使用人类骨骼肌来源的成肌细胞而不是最终分化的成纤维细胞来产生iPS并将其分化为心脏祖细胞。我们的主要假设是，诱导多能性所需的特定因子组合是由体细胞的细胞类型和分化状态决定的。因此，我们提出骨骼肌母细胞（SMs）是诱导多能状态的优越候选者，单独或与小分子治疗联合使用较少的因素。主要假设将在以下不同的目标中进行检验。特异性Aim-1将致力于通过采用非病毒策略，以更少的因子重编程人类SMs的多能性。拟议的研究将使用人类SMs作为候选细胞，它与成纤维细胞不同，具有多能性，并且与最终分化的成纤维细胞相比更容易重编程。其次，在没有基因组整合的情况下，使用miRs对SMs进行重编程将是一种高度创新的策略。特异性Aim-2将专注于从SM来源的iPS细胞（SM-iPS）中分离心脏和血管源性细胞系，并研究其体外分化行为。特异性Aim-3将比较SM-iPS细胞及其预编程衍生物的体内行为、移植细胞在不同时间点的存活以及急性心肌梗死小动物模型中的心肌修复能力。一旦确定了它们的致心潜能，最佳选择的细胞类型将在Specific Aim-4的大型临床前动物模型中进行评估，以确保SM-iPS细胞的翻译数据。体内研究的终点将是移植细胞的肌血管生成分化，梗死面积的衰减以及在改善整体心脏功能方面的功能益处。这些研究将涉及多学科方法，将采用最先进的分子生物学，组织化学和免疫组织化学技术，以及完善的综合生理学，包括建立实验动物模型，压力-容量环路和动物心功能经胸超声检查。我们的结果有望增强对SM-iPS细胞作为心肌修复的潜在供体细胞来源的潜在理解，而不存在致心律失常和免疫原性问题。

DESCRIPTION (provided by applicant): The innovative approach of generating induced pluripotent stem (iPS) cells from fibroblasts opens amazing new doors for generating autologous, patient-specific pluripotent stem cell lines for individualized cell therapy. There is however much important research to be done on non-viral generation of iPS cells to optimize their generation, safety and efficacy before these are feasible for clinical application. Our research will use human skeletal muscle derived myoblasts rather than terminally differentiated fibroblasts for generation of iPS and their differentiation into cardiac progenitor cells. Our main hypothesis is that the specific combination of factors necessary for induction of the pluripotency is determined by the cell type and differentiation status of the somatic cells. We therefore propose that skeletal myoblasts (SMs) are superior candidates for induction to pluripotent state with fewer factors either alone or in combination with treatment with small molecules. The main hypothesis will be tested in the following distinct Aims. Specific Aim-1 will be devoted to reprogram human SMs for pluripotency with fewer factors by employing non-viral strategy. The proposed studies will use human SMs as the candidate cells which unlike fibroblasts are multipotent and are easier to be reprogrammed as compared to the terminally differentiated fibroblasts. Secondly, the use of miRs for reprogramming of SMs without genomic integration would be highly innovative strategy. Specific Aim-2 will focus on isolation of cardiac and vasculogenic lineages from SM derived iPS (SM-iPS) cells and study their in vitro differentiation behavior. Specific Aim-3 will compare SM-iPS cells and their pre-programmed derivatives for their in vivo behavior, survival of the cell graft at various time-points and myocardial reparability in small animal model of acute myocardial infarction. Once their cardiogenic potential will be established, the best chosen cell types will be assessed in a large preclinical animal model in Specific Aim-4 to secure translational data for SM-iPS cells. The end points of the in vivo studies will be myoangiogenic differentiation of the engrafted cells, attenuation of infarct size and the functional benefits in terms of improved global heart function. These studies will involve multidisciplinary approach which will employ state of the art molecular biology, histochemical and immunohistochemical techniques and well integrative physiology involving well-established experimental animal model, pressure-volume loop and transthoracic ultrasonography for animal heart function. Our results are expected to enhance understanding of the potential of SM-iPS cells as a potential source of donor cells for myocardial repair without the problem of arrhythmogenicity and immunogenicity.