NOTCH signaling on the underdeveloped cardiac vascularization of hypoplastic left heart syndrome in the hiPSC-derived vascularized cardiac organoids

在 hiPSC 衍生的血管化心脏类器官中，NOTCH 信号传导对左心发育不全综合征的不发达心脏血管化的影响

• 批准号: 10439137
• 负责人: Huaxiao Yang
• 金额: $ 43.82万
• 依托单位: UNIVERSITY OF NORTH TEXAS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-08 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10439137
• 关键词: BMP10 gene; Binding; Blood Vessels; Blood capillaries; CRISPR/Cas technology; Calcium; Cardiac; Cardiac Myocytes; Cardiovascular system; Cell Line; Cell model; Cell physiology; Cells; Complex; Data; Defect; Development; EGF gene; ERBB2 gene; Endothelial Cells; Endothelium; Fetal Heart; Functional disorder; Gene Expression; Goals; Growth Factor; Heart; Heart Diseases; Heart Valves; Human; Hypoplastic Left Heart Syndrome; Image; Knock-out; Left ventricular structure; Ligands; Link; Measures; Mediating; Mesoderm; Modeling; Molecular; Mutation; Myocardial; Myocardium; NOTCH1 gene; NRG1 gene; Newborn Infant; Nitric Oxide; Organoids; Palliative Surgery; Pathogenesis; Pathway interactions; Patients; Postpartum Period; Process; Proteome; Proteomics; Protocols documentation; Regulation; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Structure; Study models; Tissues; Transgenic Mice; Up-Regulation; Validation; Vascularization; Ventricular; angiogenesis; ascending aorta; cardiac vasculature; cardiogenesis; congenital heart disorder; density; endothelial dysfunction; endothelial stem cell; extracellular; gamma secretase; genetic variant; genome editing; induced pluripotent stem cell; inhibitor; malformation; progenitor; receptor; receptor binding; single-cell RNA sequencing; small molecule; three dimensional cell culture

PROJECT SUMMARY Hypoplastic left heart syndrome (HLHS) is one of the most fatal congenital heart diseases (CHD) that occurs in 1 of every 4,344 newborns. The HLHS patients’ hearts show the severe underdevelopment of the left ventricle, heart valves, and ascending aorta, and specifically lower capillary density in the myocardium. Moreover, the genetic variant of NOTCH1 is implicated in HLHS patients showing the relevance to endocardial defects, endothelial dysfunction, and NOTCH1 signaling pathways. However, how the NOTCH1 signaling determines the underdevelopment of cardiac vasculature in the HLHS patients is still inconclusive. There is still a missing link between the pathogenesis in the cardiovascular underdevelopment and dysfunction found in the HLHS hearts and the NOTCH1 mutation. In this proposal, the genome-edited NOTCH1+/- hiPSCs will be co- differentiated in an already optimized protocol into the vascularized cardiac organoid (VCO) with cardiomyocytes, endothelial cells, smooth muscle cells, and other cardiac cells to recapitulate the underdevelopment and dysfunction of the cardiovascular system in the early development of HLHS heart with NOTCH1 mutation and to further delineate the pathogenesis of HLHS in the dynamic multicellular crosstalk of cardiovascular cells via NOTCH-DLL-JAG receptor-ligand interactions. In the preliminary results, we observed a reduced vascular formation with a smaller myocardial-like ring in VCO when NOTCH signaling was inhibited by g-secretase inhibitor, and smaller organoid size was noticed in the NOTCH1+/- VCO compared to the isogenic wildtype VCO. We will further explore the NOTCH1 signaling on vascular and cardiac formation in VCOs by measuring the cardiovascular structure, proliferation, function, and corresponding gene expressions (Aim 1). Moreover, our preliminary study also shows upregulation of NOTCH1 signaling via the NOTCH-DLL- JAG ligand-receptor interactions in the VCO compared to the non-vascularized cardiac organoid by the single- cell RNA-seq and ligand-receptor analysis. We will further investigate the ligand-receptor interactions and pseudotime trajectories of NOTCH1 signaling in the process of NOTCH1+/- VCO formation. The proteome profile over the HLHS VCO development will be uncovered by the proteomic analysis in correlation and validation with the single-cell RNA-seq data (Aim 2). The completion of the proposed project will be for better determining the NOTCH1 signaling-mediated multicellular crosstalk and downstream pathways of underdeveloped cardiac vascularization in HLHS.

项目摘要 左心脏综合征（HLHS）是发生在 每4,344名新生儿中有1个。 HLHS患者的心脏显示出左心室的严重欠发达， 心脏瓣膜和升主动脉，特别是心肌中毛细血管密度较低。而且， 在HLHS患者中暗示了Notch1的遗传变异，表现出与心内部缺陷相关的性质， 内皮功能障碍和Notch1信号通路。但是，Notch1信号如何确定 HLHS患者心脏脉管系统的不发达仍然尚无定论。仍然缺少 在HLHS中发现的心血管欠发育和功能障碍的发病机理之间的联系 心脏和Notch1突变。在此提案中，基因组编辑的Notch1 +/- HIPSC将是共同的 在已经优化的方案中分化为血管化心脏器官（VCO） 心肌细胞，内皮细胞，平滑肌细胞和其他心脏细胞，以概括 HLHS心脏发展中心血管系统的发育不足和功能障碍 Notch1突变，并进一步描述HLHS在动态多细胞串扰中的发病机理 通过Notch-DLL-JAG受体配体相互作用的心血管细胞。在初步结果中，我们观察到 抑制Notch信号时，在VCO中具有较小的心肌样环的血管形成还原 与G-分泌酶抑制剂相比，Notch1 +/- VCO中注意到了较小的器官大小。 等源性野生型VCO。我们将进一步探讨在血管和心脏形成的Notch1信号传导中 VCO通过测量心血管结构，增殖，功能和相应基因表达 （目标1）。此外，我们的初步研究还显示了Notch1信号的上调 与非血管化心脏器官相比，VCO中的JAG配体 - 受体相互作用 细胞RNA-seq和配体受体分析。我们将进一步研究配体受体相互作用，并 Notch1 +/- VCO形成过程中Notch1信号传导的伪段轨迹。蛋白质组 有关HLHS VCO开发的概况将通过相关性和 使用单细胞RNA-seq数据验证（AIM 2）。拟议项目的完成将是更好的 确定Notch1信号介导的多细胞串扰和下游途径 HLHS中发育不足的心脏血管化。