Regulation of endothelial cell specification

内皮细胞规格的调节

• 批准号: 10569601
• 负责人: Juan M Melero-Martin
• 金额: $ 60.36万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569601
• 关键词: Biological Assay; Blood Vessels; Cell Communication; Cell Differentiation process; Cell Line; Cell Reprogramming; Cells; ChIP-seq; Chromatin; Co-Immunoprecipitations; Competence; Data; Development; Endothelial Cells; Endothelium; Epigenetic Process; Exhibits; Family; Foundations; Genes; Genetic Transcription; Goals; Homeostasis; Human; Impairment; In Vitro; Knowledge; Molecular; Mus; Mutagenesis; Myeloid Cells; Natural regeneration; Nucleic Acid Regulatory Sequences; Pattern; Perfusion; Phenotype; Process; Property; Proteins; Proteomics; Regulation; Regulatory Element; Solid; Specific qualifier value; Supporting Cell; System; Technology; Time; Vascular Diseases; Vascular Graft; Vascular System; Vascular regeneration; candidate validation; cell type; directed differentiation; in vitro Assay; in vivo; induced pluripotent stem cell; insight; organ regeneration; organ repair; protein protein interaction; repaired; single-cell RNA sequencing; stem cells; transcription factor

SUMMARY Approaches to vascular regeneration and repair require specification of endothelial cells that are competent to form functioning blood vessels. However, the process by which endothelial cells are specified from mesoder- mal precursors remains poorly understood. A key transcriptional regulator of endothelial cell (EC) specification is the ETS-family transcription factor ETV2. Our preliminary data shows that mesodermal progenitor cells (MPCs), differentiated from human induced pluripotent stem cells (iPSCs), are rapidly and efficiently repro- grammed by ETV2 into endothelial cell-like cells (iEC-Ms). These iEC-Ms exhibit properties of endothelial cells in vitro, and assemble into perfused vascular networks in the in vivo microvascular graft assay. In contrast, ETV2 expression directly in iPSCs yielded cells that expressed endothelial cell markers (iEC-Ps) and exhibited a subset of endothelial cell properties in vitro, but did not form perfused vascular networks in microvascular grafts. The overarching goal of this proposal is to use this experimental paradigm to define the mechanisms by which ETV2 drives reprogramming to iECs, and to dissect the mechanisms by which the starting cell type (MPC vs iPSCs) influences the functional properties of the resulting iECs. We propose 3 Specific Aims to achieve these goals: (1) To dissect the transcriptional regulatory landscape of endothelial differentiation. (2) To determine the molecular mechanisms that limit functionality of iECs differ- entiated directly from iPSCs. (3) To characterize the protein-protein interactions required for ETV2 to drive iEC specification. To achieve these aims, we will use cutting edge technologies including single cell RNA-seq, ChIP-seq, and proximity proteomics. Together, these studies will define the molecular mechanisms that underlie the earliest stages of endothe- lial cell specification and that establish endothelial cell competence for interaction with support cells and forma- tion of functional vessels. This fundamental knowledge will form the foundation for strategies to promote vessel development in organ repair and regeneration.

总结 血管再生和修复的方法需要内皮细胞的特化， 形成功能性血管。然而，内皮细胞从中胚层分化为中胚层的过程是不确定的。 人们对疾病前体的了解仍然很少。内皮细胞（EC）特化的关键转录调节因子 是ETS家族转录因子ETV 2。我们的初步数据显示中胚层祖细胞 从人诱导多能干细胞（iPSC）分化而来的多能干细胞（MPCs）能够快速有效地再生， 通过ETV 2编程为内皮细胞样细胞（iEC-MS）。这些iEC-Ms表现出内皮细胞的特性 在体外，并在体内微血管移植物测定中组装成灌注的血管网络。与此相反， 直接在iPSC中表达ETV 2产生表达内皮细胞标志物（iEC-Ps）的细胞，并表现出 一个子集的内皮细胞特性在体外，但没有形成灌注的血管网络，在微血管 移植物这个建议的首要目标是使用这个实验范式来定义机制 ETV 2驱动重新编程为iEC的机制，并剖析起始细胞 类型（MPC vs iPSC）影响所得iEC的功能性质。 我们提出了3个具体的目标来实现这些目标：（1）解剖转录调控景观 内皮细胞分化。(2)为了确定限制iEC功能的分子机制， 直接从iPSC中获得。(3)表征ETV 2驱动iEC所需的蛋白质-蛋白质相互作用 规范.为了实现这些目标，我们将使用尖端技术，包括单细胞RNA-seq， ChIP-seq和邻近蛋白质组学。 总之，这些研究将定义内皮细胞最早阶段的分子机制， lial细胞特化和建立内皮细胞与支持细胞相互作用能力， 功能性血管。这些基本知识将成为促进船舶 器官修复和再生的发展。