Exploring the Role of Notch Signaling in Purkinje Fiber Development

项目摘要 室性心律失常是一种常见的心脏并发症，可能由遗传突变引起， 在发育过程中传导细胞类型定位不当，或由于 心肌梗死。尽管希氏-浦肯野系统在这些情况下发挥了作用，但人们对此知之甚少。 这些细胞的发育起源以及什么分子线索驱动着它们的特征。这种信息的缺乏 阻碍了基于人类多能干细胞(HPSC)的心室传导模型的产生 系统(VCS)来研究这些细胞如何与周围的心肌结合，以及这是如何形成的 在疾病中调节失调。 在这项提案的目标1中，我们试图定义一种使用hPSCs产生人VCS细胞的方法。vbl.使用 我们通过各种生理测试来了解这些细胞的电生理特性的变化。 细胞在分化过程中走向传导命运，并检查传导细胞类型与 不同培养体系中HPSC来源的心室肌细胞。我们还将描述分子的特征 这些细胞分化的基础是传导命运的变化，并将它们与胎儿 使用RNA测序的对应者。 在目标2中，我们将对引起VCS的祖细胞类型进行单细胞RNA测序 以及小鼠发育过程中的其他血统。使用tSNE和PCA聚类算法，我们试图分析 这些祖细胞类型的异质性并确定在分化过程中存在的亚群 风投公司。使用从脑室发育的不同阶段收集的时间样本，我们将使用谱系 轨迹算法，以澄清VCS规范期间的血统关系。 本文介绍的研究使用互补的模型系统来执行详细的体外研究 HPSC来源的VCS细胞的分子和电生理特性，并将此分析与 在本地信令环境中指定正在开发的VCS时，对其进行单单元分析。这 这项工作将建立一种新的体外模型，用于未来探索细胞类型对心律失常的特殊贡献的研究 并可确定用于功能获得/丧失研究的分子靶点，以确定在 我们的分析在功能上是VCS开发所必需的。

Project Summary Ventricular arrhythmias are a common cardiac complication that may result from inherited mutations, improper positioning of conductive cell types during development, or as a result of fibrotic scarring following a myocardial infarction. Despite the role of the His-Purkinje system in these conditions, still little is known about the developmental origins of these cells and what molecular cues drive their specification. This lack of information has hampered the generation of human pluripotent stem-cell (hPSC) based models of the ventricular conduction system (VCS) to study how these cells couple with the surrounding myocardium, and how this becomes dysregulated in disease. In Aim 1 of this proposal we seek to define a method to generate human VCS cells using hPSCs. Using a variety of physiological assays we aim to understand changes in the electrophysiological properties of these cells during differentiation towards a conductive fate, and examine the interactions of conductive cell types with hPSC-derived ventricular cardiomyocytes in various culture systems. We will also characterize the molecular changes that underlie differentiation of these cells toward a conductive fate and compare them to their fetal counterparts using RNA sequencing. In Aim 2 we will conduct single-cell RNA sequencing of progenitor cell types that give rise to the VCS and other lineages during mouse development. Using tSNE and PCA clustering algorithms we seek to profile the heterogeneity of these progenitor cell types and identify subpopulations present during differentiation of the VCS. Using temporal samples collected from various stages of ventricular development we will employ lineage trajectory algorithms to clarify lineage relationships during VCS specification. The research laid out herein uses complementary model systems to perform detailed in vitro studies of the molecular and electrophysiological properties of hPSC-derived VCS cells and combines this analysis with single-cell profiling of the developing VCS as it become specified within the native signaling environment. This work will establish a new in vitro model for future studies exploring cell-type specific contributions to arrhythmias and may identify molecular targets for gain/loss of functions studies to determine if novel regulators identified in our analysis are functionally required for VCS development.