In Vitro and In Situ Engineering of Fibroblasts for Cardiac Repair

从人胚胎干细胞（hESC）衍生功能性心肌细胞十五 几年前，以及iPSC的发现，为人类工程学打开了大门。 用于药物发现、疾病建模和再生的心脏组织替代物 药尽管如此，将人类iPSC技术转化为心脏病的临床治疗仍然是一个挑战。 由于许多挑战，包括不成熟和异质性心肌细胞， 表型，它们的低扩张能力，高代谢需求和低生存能力后， 植入、诱发肿瘤和心律失常的可能性以及高成本。解决这些 局限性，我们建议探索一种基于细胞和基因的心脏修复的新策略， 并不依赖于干细胞的使用。相反，我们将开发工程方法， 终末分化成能够传导动作电位的细胞。 这些细胞将以低成本快速生成，具有稳定，均匀和可定制的特性。 电表型，容易在体外扩展和现成可用，并且能够 与心肌细胞电偶联，并显著改善电和收缩 梗塞心脏的功能。具体而言，在目标1中，我们提出利用原核离子 将人成纤维细胞工程化为一种易于扩展和均匀的 自发放电和传导动作电位的电兴奋细胞。在目标2中，我们 利用良好控制的体外共培养系统，探索工程化成纤维细胞如何与 特定电生理学特性影响天然神经元的电和机械功能 心肌细胞在目标3中，我们提出直接比较主动传导的成纤维细胞和 PSC衍生的心肌细胞的抗心律失常作用和改善心肌收缩的能力 和血流动力学功能。此外，我们将利用计算机 模拟，以促进基因工程的积极进行成纤维细胞和增强 体外与天然心肌细胞功能相互作用的机制研究 和体内。我们预计，该项目的成功完成将使未来的应用 在心肌梗死和心律失常的细胞治疗中的工程化成纤维细胞。

The derivation of functional cardiomyocytes from human embryonic stem cells (hESCs) fifteen years ago, as well as the discovery of iPSCs, has opened doors to the engineering of human cardiac tissue surrogates for use in drug discovery, disease modeling, and regenerative medicine. Still, translating human iPSC technology to clinical therapy for heart disease has been slow due to a number of challenges including immature and heterogeneous cardiomyocyte phenotype, their low expansion capacity, high metabolic demand and low viability after implantation, potential for tumor and arrhythmia induction, and high costs. To address these limitations, we propose to explore a novel strategy for cell- and gene-based cardiac repair that does not rely on the use of stem cells. Instead, we will develop methods for engineering of terminally differentiated human fibroblasts into cells capable of action potential conduction. These cells will be generated rapidly, at low cost, have stable, homogeneous, and customizable electrical phenotype, be readily expandable in vitro and available off-the-shelf, and be able to electrically couple with cardiomyocytes and significantly improve electrical and contractile function of the infarcted heart. Specifically, in Aim 1 we propose to utilize prokaryotic ion channels to engineer human fibroblasts into a readily expandable and homogeneous source of electrically excitable cells that autonomously fire and conduct action potentials. In Aim 2, we will utilize well-controlled in vitro co-culture systems to explore how engineered fibroblasts with specific electrophysiological properties affect electrical and mechanical function of native cardiomyocytes. In Aim 3, we propose to directly compare actively conducting fibroblasts and PSC-derived cardiomyocytes for their antiarrhythmic action and ability to improve contractile and hemodynamic function of infarcted rat hearts. In addition, we will utilize computer simulations to facilitate genetic engineering of actively conducting fibroblasts and enhance mechanistic understanding of their functional interactions with native cardiomyocytes in vitro and in vivo. We expect that successful completion of this project will enable future applications of engineered fibroblasts in cell-based therapies for myocardial infarction and arrhythmias.