An integrated human stem cell model for elucidating myocardial-endocardial interactions in cardiac development and disease

用于阐明心脏发育和疾病中心肌-心内膜相互作用的综合人类干细胞模型

• 批准号: 10352393
• 负责人: Mingtao Zhao
• 金额: $ 38.5万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10352393
• 关键词: Address; Affect; Animal Model; Animals; Aortic Valve Stenosis; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiac development; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Communication; Congenital Heart Defects; Data; Defect; Development; Disease; Electrophysiology (science); Elements; Embryo; Endocardium; Endothelial Cells; Endothelium; Gene Dosage; Generations; Genetic; Genetic Engineering; Genotype; Growth; Heart; Heart Atrium; Heart Diseases; Human; Hypoplastic Left Heart Syndrome; Interruption; Knowledge; Lead; Left; Left ventricular structure; Link; Mediating; Mitral Valve; Molecular; Morphogenesis; Muscle Fibers; Mutation; Myocardial; Myocardium; NOTCH1 gene; Neonatal Mortality; Newborn Infant; Nodal; Operative Surgical Procedures; Pathogenicity; Patients; Proteomics; Publishing; Running; Side; Signal Transduction; Small RNA; Structural defect; Structure; Study models; System; Variant; Ventricular; aortic valve; aortic valve disorder; ascending aorta; calcification; clinical phenotype; clinically relevant; congenital heart disorder; design; exosome; genome editing; human stem cells; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; intercellular communication; mouse model; notch protein; novel; novel therapeutics; patch clamp; prevent; single-cell RNA sequencing; stem cell model; stem cells; transcriptome sequencing

Hypoplastic left heart syndrome (HLHS) is a severe type of congenital heart defects, which is characterized by the underdevelopment of left side of the heart. The clinical presentation of HLHS includes hypoplasia of the left ventricle and structural defects in mitral valves, aortic valve, and ascending aorta. HLHS newborns usually die within a week without surgical treatment. We and others have linked the pathogenic NOTCH1 mutations to HLHS and calcific aortic valve disease. It appears that abnormal NOTCH signaling interrupts the communication between myocardium and endocardium thus leads to incomplete growth of ventricular chamber. However, the mechanisms by which NOTCH1 mutations results in hypoplasia of the left ventricle are largely unknown due to limited models for studying HLHS. Genetically engineered animals are not capable of reproducing the clinical phenotypes in HLHS patients. Previous studies have focused on the structural and electrophysiological defects in cardiomyocytes from HLHS patient-derived induced pluripotent stem cells (iPSCs), which may not recapitulate the underlying non-cell autonomous scenarios in the hypoplastic ventricles. In this proposal, we hypothesize that NOTCH1-mediated myocardial-endocardial crosstalk is required for normal human ventricular cardiomyocyte differentiation, and NOTCH1 mutations leads to abnormal myocardial-endocardial interactions which cause the hypoplasia of ventricular cardiomyocytes in HLHS. We will employ an integrated stem cell model using HLHS and CRISPR genome-edited iPSCs to investigate how NOTCH1 mutations lead to abnormal myocardial- endocardial interactions in HLHS. We will design a novel co-culture platform using human iPSC-derived cardiomyocytes (iPSC-CMs) and endothelial cells (iPSC-ECs) with distinct NOTCH1 genetic composition to study the intercellular communication between endocardium and myocardium in both healthy and diseased conditions. In Specific Aim 1, we will investigate the cellular and molecular mechanisms by which endothelial NOTCH1 deficiency suppresses human ventricular cardiomyocyte differentiation and proliferation. In Specific Aim 2, we will determine how the crosstalk between myocardium and endocardium affects ventricular cardiomyocyte differentiation and proliferation by co-culture of human iPSC-CMs and iPSC-ECs. In Specific Aim 3, we will decipher the mechanisms by which NOTCH1 mutations results in the dysfunctional myocardial- endocardial interactions and contribute to hypoplasis of the left ventricle using genome-edited HLHS-iPSCs. The completion of this R01 project will have a major impact on the understanding of HLHS through interactions between endocardium and myocardium using clinically relevant and patient-derived cardiomyocytes and endothelial cells.

左心发育不全综合征(HLHS)是一种严重的先天性心脏病，其特点是 由心脏左侧发育不良所致。HLHS的临床表现包括下丘脑发育不良 左心室和二尖瓣、主动脉瓣和升主动脉的结构缺陷。HLHS新生儿通常 在没有手术治疗的情况下在一周内死亡。我们和其他人已经将致病的NOTCH1突变与 HLHS和钙化性主动脉瓣病。似乎异常的槽口信令中断了通信 心肌和心内膜之间的相互作用导致室腔的不完全生长。然而， NOTCH1突变导致左心室发育不全的机制很大程度上是未知的，因为 研究HLHS的有限模型。基因工程动物不能复制临床上的 HLHS患者的表型。以往的研究主要集中在结构和电生理缺陷上。 在来自HLHS患者来源的诱导多能干细胞(IPSCs)的心肌细胞中，可能不能概括 发育不全的脑室中潜在的非细胞自主场景。在这项提案中，我们假设 NOTCH1介导的心肌-心内膜串扰是正常人心肌细胞所必需的 分化和NOTCH1突变导致异常的心肌-心内膜相互作用，从而导致 HLHS中的心肌细胞发育不良。我们将采用使用HLHS的集成干细胞模型 和CRISPR基因组编辑的IPSCs，以研究NOTCH1突变如何导致异常心肌- HLHS中的心内膜相互作用。我们将利用人类IPSC来源的细胞设计一个新的共培养平台 具有不同NOTCH1基因组成的心肌细胞(IPSC-CMS)和内皮细胞(IPSC-ECs) 健康人与疾病人心内膜与心肌细胞间通讯的研究 条件。在特定的目标1中，我们将研究内皮细胞 NOTCH1缺乏抑制人心室肌细胞的分化和增殖。具体而言 目的2，我们将确定心肌和心内膜之间的串扰如何影响心室 人IPSC-CMS与IPSC-ECs共培养条件下心肌细胞的分化与增殖具体而言 目的3，我们将破译NOTCH1突变导致心肌功能障碍的机制。 使用基因组编辑的HLHS-IPSCs，心内膜相互作用并导致左心室发育不良。这个 R01项目的完成将通过互动对HLHS的理解产生重大影响 使用临床相关和患者来源的心肌细胞和 内皮细胞。