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

人类 iPSC 用于阐明 DCM 中的细胞间串扰信号传导 - 多样性补充

• 批准号: 10730997
• 负责人: Lei Stanley Qi
• 金额: $ 3.39万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730997
• 关键词: 3-Dimensional; Arrhythmia; Biological Assay; Cardiac; Cardiomyopathies; Cell Line; Cells; Censuses; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complement; Data; Dilated Cardiomyopathy; Disease; Disease model; Ethnic Origin; Functional disorder; Gene Expression; Genetic; Heart; Heart Diseases; Hispanic; Hispanic Populations; Human; Individual; Inherited; Investigation; Ligands; Methods; Modality; Organoids; Parents; Pathogenesis; Patients; Proteomics; Reporter; Signal Pathway; Signal Transduction; System; Technology; United States; Variant; Ventricular; aptamer; cardiac tissue engineering; cell type; coronary fibrosis; genome editing; heart cell; high throughput screening; hispanic community; induced pluripotent stem cell; insight; intercellular communication; novel therapeutic intervention; receptor; single-cell RNA sequencing; sudden cardiac death; transcriptomics

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.

母版 R01 总结： LMNA 相关的扩张型心肌病 (DCM) 是最常见的遗传性心脏病之一， 其特点是严重的收缩功能障碍和心室扩大。主要特点 LMNADCM 还涉及非肌细胞功能障碍的特征，包括心肌纤维化和内皮病。 然而，心脏细胞间通讯的精确机制仍不清楚，部分原因是 迄今为止，人类心脏分泌蛋白组的定义还很不明确。为了克服这一挑战，我们建议利用 人类 iPSC、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 发病机制。在目标1中，我们将全面 通过采用基于适体的高通量蛋白质组学来分析每种细胞类型的基线分泌组 方法并进行跨孔共培养测定，以系统地评估下游功能 细胞串扰的后果。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/对照细胞的工程心脏组织 (EHT) 中的细胞间通讯机制 类型组合物。 EHT 随后将通过单细胞 RNA 测序 (scRNA-seq) 进行分析，以 预测细胞间的串扰模式并构建一系列独特且共享的配体受体对 状况。在目标 3 中，我们将使用以下方法对 >4,000 种化合物进行大规模高通量筛选 多细胞 iPSC 衍生的心脏类器官 (iPSC-CO) 从三系报告系分化而来。我们 预计这些研究的成功完成将为 DCM 带来新的机制见解 发病机制并帮助开发可以阻止和逆转异常串扰的新治疗策略 患病心脏中心肌细胞类型之间的信号传导。 建议补充： 根据2020年美国数据，有6210万拉美裔居住在美国 人口普查。在西班牙裔人群中，心脏病的研究相对较少。在这个提议的多样性中 补充，我们将扩大父 R01 的范围以包括来自西班牙裔的患者进行调查 LMNA 相关扩张型心肌病 (LMNA-DCM)。具体而言，在本提案中，我们将包括 11 名个体 （10 名健康者，1 名患病者）来自西班牙裔社区，以扩大亲本 R01 的多样性。这个提议 将首先表征源自诱导多能干细胞的健康和患病的西班牙心脏细胞， 使用 CRISPR 基因组编辑技术将 LMNA-DCM 变体引入 9 个健康的西班牙细胞系，以 验证健康细胞和患病细胞之间的功能变化。然后在扩展目标2中，我们将生成3D 心脏类器官使用目标 1 中生成的细胞类型通过以下方式捕获细胞间信号传导途径： 使用单细胞 RNA 测序进行转录组分析和基因表达。