Exploring the Role of Notch Signaling in Purkinje Fiber Development

探索Notch信号在浦肯野纤维发育中的作用

• 批准号: 10407637
• 负责人: David Matthew Gonzalez
• 金额: $ 2.54万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10407637
• 关键词: Action Potentials; Adopted; Algorithms; Arrhythmia; Biological Assay; Biological Models; Cardiac; Cardiac Myocytes; Cardiac conduction system; Cardiovascular system; Cell Differentiation process; Cell Line; Cell Therapy; Cells; Characteristics; Cicatrix; Complication; Coupled; Coupling; Cues; Data; Development; Disease; Electrolytes; Electrophysiology (science); Endocardium; Environment; Epicardium; Epigenetic Process; Frequencies; Future; Generations; Genes; Hereditary Disease; Heterogeneity; Human; In Vitro; Inherited; Ion Channel; Light; Maintenance; Measures; Methods; Molecular; Molecular Target; Morphology; Mus; Mutation; Myocardial; Myocardial Infarction; Myocardium; Nodal; Pathway interactions; Physiological; Physiology; Play; Pluripotent Stem Cells; Positioning Attribute; Potassium Channel; Property; Regulation; Reporting; Research; Role; Sampling; Signal Pathway; Signal Transduction; Specific qualifier value; Structure of purkinje fibers; System; Systems Development; Therapeutic Agents; Time; Tissues; Transgenic Organisms; Translating; Ventricular; Ventricular Arrhythmia; Work; cell type; drug discovery; fetal; fiber cell; human RNA sequencing; human pluripotent stem cell; in vitro Model; in vivo; loss of function; mouse development; multi-electrode arrays; notch protein; novel; novel therapeutics; progenitor; promoter; segregation; single cell sequencing; single-cell RNA sequencing; spatiotemporal; stem cell model; stem cells; transcriptome; transcriptome sequencing; transcriptomics

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.

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，传导细胞类型的定位不当，或由于 心肌梗死尽管希氏-浦肯野系统在这些疾病中的作用， 这些细胞的发育起源以及什么样的分子线索驱动它们的特化。缺少资料 阻碍了基于人多能干细胞（hPSC）的心室传导模型的产生 研究这些细胞如何与周围心肌耦合，以及这如何成为 在疾病中失调。 在本提案的目的1中，我们试图定义一种使用hPSC产生人PBMC的方法。使用 我们的目的是了解这些细胞的电生理特性的变化， 细胞在分化过程中向导电命运，并检查导电细胞类型的相互作用， 在各种培养系统中的hPSC衍生的心室心肌细胞。我们还将描述 这些细胞向传导性命运分化的基础变化，并将它们与胎儿细胞进行比较， 使用RNA测序的对应物。 在Aim 2中，我们将对引起肿瘤的祖细胞类型进行单细胞RNA测序。 和其他谱系在小鼠发育过程中。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， - 是的使用从心室发育的各个阶段收集的时间样本，我们将使用谱系 轨迹算法，以澄清血统关系，在规范化过程中。 本文所述的研究使用互补的模型系统来进行详细的体外研究， hPSC衍生的细胞的分子和电生理特性，并将该分析与 当发育中的细胞在天然信号环境中变得特定时，其单细胞谱分析。这 这项工作将建立一个新的体外模型，用于未来研究探索细胞类型特异性对心律失常的作用 并且可以鉴定用于功能获得/丧失研究的分子靶点，以确定是否在细胞中鉴定出新的调节剂。 我们的分析在功能上是可扩展性开发所必需的。