Regulation of endothelial cell specification

摘要 血管再生和修复的方法需要指定有能力的内皮细胞 形成有功能的血管。然而，内皮细胞从中层到中层的过程- MAL的前体仍然知之甚少。内皮细胞(EC)的关键转录调控因子 是ETS家族转录因子ETV2。我们的初步数据显示，中胚层祖细胞 从人诱导的多能干细胞(IPSCs)分化而来的基质细胞(MPC)能够快速、高效地增殖、分化、分化为多能干细胞。 经ETV2扩增为内皮细胞样细胞(IEC-ms)。这些IEC-ms显示出内皮细胞的特性 在体外，并在体内微血管移植实验中组装成灌流的血管网络。相比之下， 直接在IPSCs中表达ETV2可产生表达内皮细胞标志(IEC-Ps)的细胞，并显示 体外培养的内皮细胞特性的一个亚群，但在微血管中没有形成灌流的血管网络 嫁接。这一提议的首要目标是使用这一实验范式来定义机制 ETV2通过它驱动重新编程到IECS，并剖析起始细胞 类型(MPC与IPSCS)会影响生成的IECS的功能属性。 为了实现这些目标，我们提出了3个具体目标：(1)剖析转录调控格局 血管内皮细胞分化。(2)为了确定限制IECS功能的分子机制不同- 直接从iPSCs中获得。(3)描述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， 芯片序列和邻近蛋白质组学。 总而言之，这些研究将确定内毒素最早阶段的分子机制。 建立内皮细胞与支持细胞相互作用能力，并形成 功能血管的破裂。这些基础知识将构成推广船舶的战略的基础 器官修复和再生的研究进展。

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.