Engineering of Human Excitable Tissues from Unexcitable Cells

 描述（由申请人提供）：干细胞注射到心脏中作为心肌梗死和心力衰竭的潜在疗法正在积极寻求。虽然正在进行的成人干细胞试验显示出适度的临床益处，但预计该领域的重大进展将来自于使用诱导多能干细胞衍生的心肌细胞。尽管前景广阔，但多能干细胞衍生的心肌细胞的最终临床应用面临着许多需要解决的挑战，包括细胞成熟不足、表型异质性、胚胎发生、植入后活力低和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新的方法，心脏细胞和基因治疗，不依赖于使用干细胞。相反，我们建议采用体外或原位基因工程的成纤维细胞成电活性细胞与可定制的电表型，可以耦合周围的心肌细胞，并改善其电和收缩功能。具体地说，在目标1中，我们将利用最少的遗传操作来快速有效地将成人成纤维细胞转化为可自发激发和传导动作电位的可兴奋细胞的容易扩展和均匀的来源。在目标2中，将表征具有选择的电生理表型的工程化成纤维细胞在良好控制的体外共培养系统中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化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性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，我们将利用计算机模拟来促进可兴奋细胞的基因工程，并增强对它们与天然心肌细胞在体外和体内的功能相互作用的机制的理解。我们相信，所提出的基因和组织工程方法将为未来工程化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.