Generation of mature human embryonic stem cell-derived left ventricular cardiomyocytes for transplantation in a large animal model

摘要 人胚胎干细胞 (hESC) 可以成为心血管细胞生成的无限来源 他们的发展的不同阶段。心脏干细胞治疗的最终目标是提供治疗 受损心脏的相关细胞可以重新填充损伤区域，与宿主机电耦合 心肌，并提供功能益处。然而，基于 hESC 的细胞疗法的临床应用还很有限。 受到许多技术挑战的限制，包括无法提供纯心肌细胞群 可以功能性地整合到宿主心肌中，而没有心律失常的风险。此外，缺乏 具有临床相关损伤原型的适当大型动物模型限制了我们对命运的理解 移植后的 hESC 衍生细胞。 迄今为止，hESC 来源的心肌细胞的递送尝试依赖于分化方案，该方案导致 在培养皿中敲打细胞，这些细胞被认为是用于移植的心肌细胞。然而，它们的杂质， 可能包括心肌细胞亚型，例如起搏细胞，可能导致严重的安全问题，例如 心律失常。因此，分离出与左心室（LV）心肌细胞非常相似的纯群体 内源性心肌细胞是细胞治疗的一个重要目标。最近，我们隔离并 表征了第一和第二心脏心肌细胞，以及分化的起搏细胞 人类胚胎干细胞。此外，我们与计算生物学家合作者报告了心血管祖细胞如何 在发展过程中做出命运决定。最后，我们小组在加州大学洛杉矶分校建立了第一个大型动物设施 我们在猪梗塞心脏中进行了 hESC 衍生移植。我们相信专业知识和 我们小组的经验以及可用资源将有助于拟议项目的成功。 我们假设hESC衍生的左心室心肌细胞可以移植到宿主心肌中并提供 功能性效益。在具体目标 1 中，我们计划通过以下方式从分化的 hESC 中分离出 LV 心肌细胞： 修改 Activin/Nodal 和视黄酸信号通路。我们将根据这些细胞的特征来全面表征它们 基因和蛋白质表达，以及单细胞水平的电生理学。在具体目标2中，通过分析 hESC来源的心肌细胞和内源性心肌细胞在不同阶段的单细胞基因表达谱 开发过程中，我们计划探索介导体外分化和成熟的分子特征。 最后，在具体目标 3 中，我们建议将 hESC 衍生的 LV 心肌细胞移植到临床相关的细胞中。 猪心肌梗塞模型。我们将进行侵入性和非侵入性测试以确定结构 以及移植细胞与宿主心肌的功能整合。我们相信本次活动的成功 项目将增强我们对 hESC 分化和成熟的理解 心肌细胞，深入了解移植后的命运，并为未来的基于细胞的奠定平台 治疗心脏病的疗法。

ABSTRACT Human embryonic stem cells (hESCs) can be an unlimited source for generation of cardiovascular cells at different stages of their development. The ultimate goal of cardiac stem cell therapy is to deliver therapeutically relevant cells to damaged hearts that can repopulate the injury area, electromechanically couple with the host myocardium, and provide functional benefit. However, the clinical application of hESC-based cell therapy is limited by many technical challenges including the inability to deliver a pure population of cardiomyocytes that can functionally integrate into the host myocardium without the risk of arrhythmias. Additionally, lack of appropriate large animal models with clinically relevant injury prototype has limited our understanding of the fate of hESC-derived cells after transplantation. Attempts to date for delivery of hESC-derived cardiac cells has relied on differentiation protocols that result in beating cells in a dish, which are considered cardiomyocytes for transplantation. However, their impurity, which may include cardiomyocyte subtypes such as pacemaker cells, may lead to serious safety issues, such as arrhythmias. Therefore, isolation of a pure population left ventricular (LV) cardiomyocytes that closely resemble the endogenous cardiomyocytes is an important goal of cell-based therapy. Recently, we isolated and characterized first- and second-heart filed cardiomyocytes, as well as pacemaker cells from differentiating hESCs. Additionally, with our computational biologist collaborator, we reported how cardiovascular progenitors make fate decisions during development. And finally, our group established the first large animal facility at UCLA where we have performed hESC-derived transplantation in porcine infarct hearts. We believe the expertise and experience of our group along with the available resources will contribute to the success of the proposed project. We hypothesize that hESC-derived LV cardiomyocytes can engraft into the host myocardium and provide functional benefit. In specific aim 1, we plan to isolate LV cardiomyocytes from differentiating hESCs by modifying Activin/Nodal and retinoic acid signaling pathway. We will fully characterize these cells based on their gene and protein expression, as well as electrophysiology at a single cell level. In specific aim 2, by analyzing single cell gene expression profile of hESC-derived and endogenous cardiomyocytes at different stages of development, we plan to explore the molecular signatures that mediate differentiation and maturation in vitro. And finally, in specific aim 3, we propose to transplant hESC-derived LV cardiomyocytes in a clinically relevant model of a porcine myocardial infarction. We will perform invasive and non-invasive tests to determine structural and functional integration of the transplanted cells into the host myocardium. We believe that the success of this project will enhance our understanding of hESC differentiation and maturation to chamber-specific cardiomyocytes, provide insight into their fate after transplantation, and set the platform for future cell-based therapies to treat heart disease.