Department of Cardiology, Boston Children's Hospital, Boston, MA (Jian Chen, X.Z., M.A.T., S.S., F.X., Jiehui Chen, P.Z., W.T.P.).
Department of Genetics, Harvard Medical School, Boston, MA (D.M.D., C.E.S., J.G.S.).
Circ Res. 2024 Mar;134(5):529-546. doi: 10.1161/CIRCRESAHA.123.323956. Epub 2024 Feb 13.
Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized.
We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs).
From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (, , , and ), venous ( and ), and capillary () marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages.
Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.
成熟的内皮细胞（ECs）是异质的，其亚型由组织起源和在血管床内的位置定义（即动脉、毛细血管、静脉和淋巴管）。在血管系统发育过程中，特别是内皮细胞的动静脉特化过程中，这种异质性是如何建立的，仍不完全清楚。
我们使用基于液滴的单细胞 RNA 测序和多重纠错荧光原位杂交技术，从 E9.5、E12.5 和 E15.5 小鼠胚胎中定义 EC 和 EC 祖细胞亚型。我们使用轨迹推断分析 EC 祖细胞中动脉 EC（aEC）和静脉 EC（vEC）的特化。网络分析确定了动静脉分化的候选转录调节因子，我们通过 CRISPR（成簇规律间隔短回文重复）在人类诱导多能干细胞中的功能丧失来测试这些因子，这些细胞正在进行定向分化为 aEC 或 vEC（人类诱导多能干细胞-aEC 或人类诱导多能干细胞-vEC）。
从 7682 个 E9.5 至 E15.5 的 EC 的单细胞转录组中，我们鉴定出 19 种 EC 亚型，包括 EC 祖细胞。15448 个 EC 的空间转录组分析为这些 EC 亚型提供了正交验证，并确定了它们的空间分布。大多数胚胎 EC 根据其血管床类型分组，而来自大脑、心脏、肝脏和肺的 EC 根据其组织起源分组。鉴定出了动脉（、、、和）、静脉（和）和毛细血管（）标记基因。与 aEC 相比，胚胎 vEC 和毛细血管 EC 与其成人对应物共享的标记物较少。具有静脉特征的早期毛细血管 EC 作为 aEC 和 vEC 谱系分化的分支点发挥作用。
我们的研究结果以单细胞分辨率提供了胚胎 EC 异质性的时空图谱，并表明胚胎中 EC 的多样性既来自组织起源，也来自血管床位置。发育中的 aEC 和 vEC 共享共同的具有静脉特征的毛细血管前体，并受到不同的转录调控网络的调节。
