Human iPSCs for Elucidating Intercellular Crosstalk Signaling in DCM - Diversity Supplement

父R01摘要： LMNA相关扩张型心肌病(DCM)是最常见的遗传性心脏病之一， 以严重的收缩功能障碍和心室室扩大为特征。的主要特征 LMNADCM还涉及非心肌细胞功能障碍，包括心肌纤维化和内皮病变。 然而，心脏细胞间通讯的确切机制仍不清楚，部分原因是 到目前为止，人类心脏分泌组的定义并不明确。为了克服这一挑战，我们建议利用 人类ipscs、基因组编辑技术和最先进的组学方法来识别和研究 串扰信号通路可能参与LMNA-DCM的发病机制。在目标1中，我们将全面 利用基于高通量适配子的蛋白质组学分析每种细胞类型的基线分泌组 方法并进行跨井共培养试验，系统评价下游功能 蜂窝串扰的后果。在目标2中，我们将通过进一步调查来补充这些研究 不同LMNA-DCM/对照细胞在工程心脏组织中的细胞间通讯机制 文字成分。EHTS随后将通过单细胞RNA测序(scRNA-seq)进行分析，以 预测细胞-细胞串扰模式，并构建一个唯一和共享的配体受体对列表 条件。在目标3中，我们将对&gt；4,000种化合物进行大规模高通量筛选，使用 多细胞IPSC衍生的心脏器质(IPSC-CO)是从三系报告系分化而来的。我们 预计这些研究的成功完成将带来对DCM的新的机械论见解 发病机制，并帮助开发新的治疗策略，可以阻止和逆转异常串扰 病变心脏中不同类型的心肌细胞之间的信号传递。 拟议补编： 根据2020年美国的数据，有6210万拉美裔美国人生活在美国 人口普查。在拉美裔人群中，心脏病的研究相对较少。在这一提议的多样性中 作为补充，我们将扩大父母R01的范围，以包括西班牙裔患者进行调查 LMNA相关性扩张型心肌病具体地说，在这项提案中，我们将包括11名个人 (10名健康，1名患病)来自西班牙裔社区，以扩大亲本R01的多样性。这项建议 将首先鉴定来自诱导多能干细胞的健康和患病的西班牙裔心脏细胞，并 将使用CRISPR基因组编辑技术的LMNA-DCM变体引入9个健康的西班牙裔细胞系 验证健康和患病细胞之间的功能变化。然后在扩展的AIM 2中，我们将生成3D 使用AIM 1中产生的细胞类型的心脏有机体通过以下方式捕获细胞-细胞信号通路 利用单细胞RNA测序进行转录分析和基因表达。

Summary of Parent R01: LMNA-related dilated cardiomyopathy (DCM) is among the most prevalent forms of inherited heart disease, characterized by severe systolic dysfunction and ventricular chamber enlargement. Major hallmarks of LMNADCM also involve features of non-myocyte dysfunction including myocardial fibrosis and endotheliopathy. However, precise mechanisms of intercellular communication in the heart remain unclear, in part because the human cardiac secretome to date has been poorly defined. To overcome this challenge, we propose to leverage human iPSCs, genome-editing technology, and state-of-the-art omics methods to identify and investigate crosstalk signaling pathways potentially involved in LMNA-DCM pathogenesis. In Aim 1, we will comprehensively profile the baseline secretomes of each cell type by employing high-throughput aptamer-based proteomics methods and perform trans-well co-culture assays to systematically evaluate the downstream functional consequences of cellular crosstalk. In Aim 2, we will complement these studies with further investigation into intercellular communication mechanisms in engineered heart tissues (EHTs) of varying LMNA-DCM / control cell type compositions. The EHTs will be subsequently analyzed by single-cell RNA sequencing (scRNA-seq) to predict cell-cell crosstalk modalities and construct a list of unique and shared ligand receptor pairs across conditions. In Aim 3, we will perform large-scale high-throughput screening of >4,000 compounds using multicellular iPSC-derived cardiac organoid (iPSC-CO) differentiated from tri-lineage reporter lines. We anticipate that the successful completion of these studies will lead to new mechanistic insight into DCM pathogenesis and help develop novel therapeutic strategies that can impede and reverse aberrant crosstalk signaling between cardiac cell types in the diseased heart. Proposed Supplement: There are 62.1 million Hispanics living in the United States, according to data from the 2020 United States Census. In the Hispanic population, heart diseases have been relatively understudied. In this proposed diversity supplement, we will extend the scope of the parent R01 to include patients from Hispanic ethnicity to investigate LMNA-related dilated cardiomyopathy (LMNA-DCM. Specifically, in this proposal, we will include 11 individuals (10 healthy, 1 diseased) from the Hispanic community to expand the diversity of the parent R01. This proposal will first characterize healthy and diseased Hispanic heart cells derived from induced pluripotent stem cells and introduce LMNA-DCM variants using CRISPR genome-editing technology to 9 healthy Hispanic cell lines to validate functional changes between healthy and diseased cells. Then in extended Aim 2, we will generate 3D Cardiac Organoids using the cell types generated in Aim 1 to capture cell-cell signaling pathways by way of transcriptomic analysis and gene expression using single-cell RNA sequencing.