In Vitro and In Situ Engineering of Fibroblasts for Cardiac Repair

人胚胎干细胞(HESCs)分化为功能性心肌细胞的研究 几年前，以及ipscs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 医药。尽管如此，将人类ipsc技术转化为心脏病的临床治疗一直是 由于包括未成熟和异种心肌细胞在内的许多挑战而缓慢 表型，低扩张能力，高代谢需求和低生存能力 植入，有可能诱发肿瘤和心律失常，而且成本较高。要解决这些问题 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 不依赖于干细胞的使用。取而代之的是，我们将开发工程方法 将人类成纤维细胞终末分化为具有动作电位传导能力的细胞。 这些单元将以低成本快速生成，具有稳定、同质和可定制的特点 电表型，易于在体外扩展和现成，并能够 与心肌细胞电偶联，显著提高电性和收缩能力 梗塞心脏的功能。具体地说，在目标1中，我们建议利用原核离子 将人成纤维细胞工程成易于扩展的同质来源的通道 自发放电和传导动作电位的电可兴奋细胞。在目标2中，我们将 利用受控良好的体外共培养系统探索工程成纤维细胞如何与 特定的电生理特性会影响人体的电和机械功能。 心肌细胞。在目标3中，我们建议直接比较主动传导的成纤维细胞和 PSC来源的心肌细胞的抗心律失常作用和改善收缩能力 以及梗塞大鼠心脏的血流动力学功能。此外，我们还将利用计算机 模拟促进主动传导成纤维细胞的基因工程并增强 体外培养心肌细胞与其功能相互作用的机制研究 在活体内。我们希望这个项目的成功完成将使未来的应用成为可能 基因工程成纤维细胞在心肌梗死和心律失常的细胞治疗中的应用。

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.