Angiomyogenesis With Myoblast Derived iPS Cells

描述（由申请人提供）：从成纤维细胞产生诱导多能干（iPS）细胞的创新方法为产生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iPS细胞的非病毒生成方面还有许多重要的研究要做，以优化它们的生成，安全性和有效性，然后才能用于临床应用。我们的研究将使用人骨骼肌来源的成肌细胞而不是终末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，诱导多能性所必需的因子的特定组合由体细胞的细胞类型和分化状态决定。因此，我们建议骨骼肌成肌细胞（SM）是上级候选人诱导多能状态与较少的因素，无论是单独或与小分子治疗的组合。主要假设将在以下不同的目标中进行检验。Specific Aim-1将致力于通过采用非病毒策略以较少的因子重编程人类SM以获得多能性。拟议的研究将使用人SM作为候选细胞，与成纤维细胞不同，SM是多能的，并且与终末分化的成纤维细胞相比更容易重编程。其次，使用miR重编程SM而不进行基因组整合将是高度创新的策略。Specific Aim-2将专注于从SM衍生的iPS（SM-iPS）细胞中分离心脏和血管生成谱系，并研究其体外分化行为。具体目标-3将比较SM-iPS细胞及其预编程衍生物在急性心肌梗死小动物模型中的体内行为、细胞移植物在不同时间点的存活率和心肌可修复性。一旦确定了它们的心源性潜力，将在Specific Aim-4中的大型临床前动物模型中评估最佳选择的细胞类型，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、梗死面积的减小和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综合生理学，包括完善的实验动物模型、压力-容积环和经胸超声检查动物心脏功能。我们的研究结果有望增强对SM-iPS细胞作为心肌修复供体细胞的潜力的理解，而不会产生致炎性和免疫原性的问题。 公共卫生相关性：本研究旨在通过我们的非病毒/microRNA重编程方法研究人骨骼肌成肌细胞来源的iPS细胞的血管肌生成行为。我们预期在用这些重编程的干细胞治疗后，梗死心脏的血管肌生成得到改善，而没有肿瘤发生和细胞排斥的问题。使用大型动物模型评估移植的iPS细胞的体内效应预期将产生具有转化意义的临床前数据。

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. PUBLIC HEALTH RELEVANCE: This proposal is designed to investigate angiomyogenic behavior of iPS cells derived from human skeletal myoblasts by using our novel non-viral/microRNA reprogramming approach. We anticipate improved angiomyogenesis in the infarcted heart after treatment with these reprogrammed stem cells without the problem of tumorigenesis and cell rejection. The use of large animal model for the assessment of in vivo effects of the transplanted iPS cells is expected to generate preclinical data with translational significance.