Regulation of endothelial cell specification

内皮细胞规格的调节

• 批准号: 10343756
• 负责人: Juan M Melero-Martin
• 金额: $ 65.2万
• 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/10343756
• 关键词: Biological Assay; Blood Vessels; Cell Communication; Cell Differentiation process; Cell Line; 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; Organ; Pattern; Phenotype; Process; Property; Proteins; Proteomics; Regulation; Regulatory Element; Solid; Specific qualifier value; Supporting Cell; System; Technology; Time; Vascular Diseases; Vascular System; Vascular regeneration; cell type; 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.

概括 血管再生和修复的方法需要有能力的内皮细胞的规格 形成有功能的血管。然而，内皮细胞从中胚层分化的过程 对mal前体的了解仍知之甚少。内皮细胞 (EC) 规范的关键转录调节因子 是 ETS 家族转录因子 ETV2。我们的初步数据表明，中胚层祖细胞 (MPC) 是从人类诱导多能干细胞 (iPSC) 分化而来，能够快速有效地再生 通过 ETV2 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 体外，并在体内微血管移植测定中组装成灌注血管网络。相比之下， ETV2 直接在 iPSC 中表达，产生表达内皮细胞标记物 (iEC-Ps) 的细胞，并表现出 体外内皮细胞特性的一个子集，但未在微血管中形成灌注血管网络 移植物。该提案的总体目标是使用这种实验范式来定义机制 ETV2 通过 ETV2 驱动 iEC 重编程，并剖析起始细胞的机制 类型（MPC 与 iPSC）影响所得 iEC 的功能特性。 我们提出了 3 个具体目标来实现这些目标：(1) 剖析转录调控格局 内皮分化。 (2) 确定限制 iEC 功能差异的分子机制 直接从 iPSC 获得。 (3) 表征ETV2驱动iEC所需的蛋白质-蛋白质相互作用 规格。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。 细胞规范并建立内皮细胞与支持细胞和形成相互作用的能力 功能血管的灰化。这些基础知识将为促进船舶发展的战略奠定基础。 器官修复和再生的发展。