Engineering of Human Excitable Tissues from Unexcitable Cells

 描述(申请人提供)：干细胞注入心脏正在被积极寻求作为心肌梗死和心力衰竭的潜在治疗方法。虽然正在进行的成人来源干细胞的试验显示出适度的临床益处，但该领域的重大进展有望通过使用诱导多能干细胞来源的心肌细胞来实现。尽管前景看好，但多能干细胞来源的心肌细胞的最终临床应用仍面临许多需要解决的挑战，包括细胞成熟不足、表型异质性、心律失常、植入后存活率低以及规模扩大等关键问题。因此，在这个项目中，我们的目标是建立一种不依赖于干细胞的心脏细胞和基因治疗的新方法。相反，我们建议在体外或原位将成纤维细胞的基因工程转化为具有可定制的电表型的电活性细胞，这种细胞可以与周围的心肌细胞偶联并改善其电和收缩功能。具体地说，在目标1中，我们将利用最少的基因操作来快速有效地将成人成纤维细胞转化为易于扩展和同质的可兴奋细胞来源，这些细胞可以自主地激发和传导动作电位。在目标2中，具有选择的电生理表型的工程化成纤维细胞将在控制良好的体外共培养系统中表征其与新生大鼠心肌细胞的功能相互作用。在目标3中，我们将确定是否可以通过植入工程的可兴奋成纤维细胞或将内源性成纤维细胞逆转录病毒转化为电活动细胞来改善梗死大鼠心脏的收缩功能。除了上述实验研究外，我们还将利用计算机模拟来促进可兴奋细胞的基因工程，并加强对其在体外和体内与天然心肌细胞功能相互作用的机制的了解。我们相信，所提出的基因和组织工程方法将为未来工程成纤维细胞在心肌梗死和心律失常的细胞和基因治疗中的实验和临床应用提供坚实的基础。

 DESCRIPTION (provided by applicant): Stem cell injections into the heart are actively being pursued as a potential therapy for myocardial infarction and heart failure. While the ongoing trials with adult-derived stem cells show moderate clinical benefits, significant progress in the field is expected to arise from the use of cardiomyocytes derived from induced pluripotent stem cells. Despite great promise, eventual clinical use of pluripotent stem cell-derived cardiomyocytes faces a number of challenges that need to be resolved including key issues with inadequate cell maturation, phenotypic heterogeneity, arrhythmogenesis, low viability after implantation, and scale-up. Therefore, in this project we aim to establish a novel approach for cardiac cell and gene therapy that does not rely on the use of stem cells. Instead we propose to employ in vitro or in situ genetic engineering of fibroblasts into electrically active cells with customizable electrical phenotype that can couple with surrounding cardiomyocytes and improve their electrical and contractile function. Specifically, in Aim 1 we will utilize minimum st of genetic manipulations to rapidly and efficiently convert adult human fibroblasts into a readily expandable and homogeneous source of excitable cells that autonomously fire and conduct action potentials. In Aim 2, engineered fibroblasts with select electrophysiological phenotypes will be characterized for their functional interactions with neonatal rat cardiomyocytes in well-controlled in vitro co- culture systems. In Aim 3, we will establish if contractile function of infarcted rat hearts can be improved by implantation of engineered excitable fibroblasts or retroviral conversion of endogenous fibroblasts into electrically active cells. In addition to abov experimental studies, we will utilize computer simulations to facilitate genetic engineering of excitable cells and enhance mechanistic understanding of their functional interactions with native cardiomyocytes in vitro and in vivo. We believe that the proposed genetic and tissue engineering approach will provide strong foundation for the future experimental and clinical use of engineered fibroblasts in cell- and gene-based therapies for cardiac infarction and arrhythmias.