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.

父母R01的摘要： 与LMNA相关的扩张性心肌病（DCM）是遗传性心脏病最普遍的形式之一， 以严重的收缩功能障碍和心室腔室扩大为特征。主要标志 LMNADCM还涉及非肌细胞功能障碍（包括心肌纤维化和内皮病）的特征。 但是，心脏中细胞间交流的精确机制尚不清楚，部分原因是 迄今为止的人类心脏分泌症的定义很差。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OMICS方法识别和调查 串扰信号通路可能与LMNA-DCM发病机理有关。在AIM 1中，我们将全面 通过采用高通量适体蛋白质组学，概述了每种细胞类型的基线秘密组 方法并执行透孔共培养分析，以系统地评估下游功能 细胞串扰的后果。在AIM 2中，我们将通过进一步研究对这些研究进行补充 不同LMNA-DCM /对照单元的工程心脏组织（EHT）中的细胞间通信机制 类型组成。随后将通过单细胞RNA测序（SCRNA-SEQ）对EHT进行分析 预测细胞细胞串扰方式，并构建跨越独特和共享配体受体对的列表 状况。在AIM 3中，我们将使用使用> 4,000种化合物的大规模高通量筛选 多细胞IPSC衍生的心脏器官（IPSC-CO）与三轮车记者线不同。我们 预计这些研究的成功完成将导致对DCM的新机械洞察力 发病机理并有助于开发新的治疗策略，这些策略可能阻碍和逆转异常串扰 患病心脏中心脏细胞类型之间的信号传导。 拟议的补充： 根据2020年美国的数据，美国有6210万西班牙裔居住 人口普查。在西班牙裔人群中，心脏病已经相对低估了。在这种建议的多样性中 补充说，我们将扩大父母R01的范围，以包括西班牙裔种族的患者进行调查 LMNA相关的扩张性心肌病（LMNA-DCM。具体，在此提案中，我们将包括11个个体 （10个健康，1例患病）来自西班牙裔社区，以扩大父母R01的多样性。这个建议 将首先表征来自诱导多能干细胞和 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。然后在扩展目标2中，我们将生成3D 使用AIM 1中生成的细胞类型的心脏器官，通过 转录组分析和基因表达使用单细胞RNA测序。