Embryonic Endothelial Evolution towards First Hematopoietic Stem Cells Revealed by Single-Cell Transcriptomic and Functional Analyses

Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational mouse embryos. Due to rare and transient nature, the HSC-competent ECs have never been stringently identified and accurately captured, let alone their genuine vasculature precursors. Here, we firstly used high-precision single-cell transcriptomics to unbiasedly examine relevant EC populations at continuous developmental stages and transcriptomically identified putative HSC-primed HECs. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by newly constructed Neurl3-EGFP reporter mouse model, and realized enrichment further by surface marker combination. Surprisingly, endothelial-hematopoietic bi-potential was rarely but reliably witnessed in culture of single HECs. Noteworthy, primitive vascular ECs experienced two-step fate choices to become HSC-primed HECs, resolving several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro. We initially sequenced 662 single cells from E9.5-E11.0 body and DA of totally 29 embryos. Additionally, we also sequenced 96 single cells with a PK44 immunophenotype (CD41-CD43-CD45-CD31+CD201+Kit+CD44+) from E10.0 AGM regions of totally 9 embryos, 47 T1 pre-HSCs (CD31+CD45-CD41lowKit+CD201high) from E11.0 AGM regions of totally 18 embryos, 48 single cells with an immunophenotype of CD41-CD43-CD45-CD31+CD44+Neurl3-EGFP+ from Neurl3-EGFP reporter mouse embryos and 579 single cells from E8.0-E9.0 body of 24 embryos. In total, 1,432 single cells were sequenced. For each embryo, the embryo proper was isolated and the head, limb buds, heart, visceral bud, and vitelline and umbilical vessels outside the embryo proper were excluded. To specifically capture aortic luminal ECs of AGM region, we performed microinjection of fluorescent dye Oregon green into the dorsal aortas of E10.0-E11.0 embryos as reported. The sampled cells were purified by FACS as CD45-CD31+CD144+, which contained predominantly vascular ECs and CD41+ hematopoietic cells. Meanwhile, CD45-CD31-CD144- non-EC cells in the body were used as negative controls.成年造血干细胞（Hematopoietic stem cells, HSCs）被认为起源于小鼠胚胎妊娠中期的生血内皮细胞（hemogenic endothelial cells, HECs）。由于具备造血干细胞潜能的内皮细胞（HSC-competent ECs）具有稀有性和瞬时性，此前从未被严格鉴定并精准捕获，更遑论其真正的血管前体。本研究首次利用高精度单细胞转录组学技术，无偏倚地分析了连续发育阶段的相关内皮细胞群体，并从转录组层面鉴定出潜在的HSC定向生血内皮细胞。结合计算预测与体内功能验证，我们通过新建的Neurl3-EGFP报告基因小鼠模型精准捕获了具备HSC潜能的HECs，并通过表面标志物组合进一步实现了富集。令人意外的是，在单个HECs的培养中，我们罕见但可靠地观察到了内皮-造血双潜能特性。值得注意的是，原始血管内皮细胞需经历两步命运抉择才能转化为HSC定向生血内皮细胞，这解决了此前多项观测到的矛盾结果。综上，对体内HSC定向生血内皮细胞特化过程中的内皮演化及分子程序的全面解析，将为未来体外诱导造血干细胞生成的研究提供重要支撑。我们最初对来自29个E9.5-E11.0胚胎的体部及背主动脉的662个单细胞进行了测序。此外，我们还对来自9个E10.0胚胎主动脉-性腺-中肾（AGM）区的、具有PK44免疫表型（CD41⁻CD43⁻CD45⁻CD31⁺CD201⁺Kit⁺CD44⁺）的96个单细胞，来自18个E11.0胚胎AGM区的47个T1型前造血干细胞（CD31⁺CD45⁻CD41lowKit⁺CD201high），来自Neurl3-EGFP报告基因小鼠胚胎的、具有CD41⁻CD43⁻CD45⁻CD31⁺CD44⁺Neurl3-EGFP⁺免疫表型的48个单细胞，以及来自24个E8.0-E9.0胚胎体部的579个单细胞进行了测序。总计共测序1432个单细胞。对于每枚胚胎，我们均分离了胚体本身，并排除了胚体以外的头部、肢芽、心脏、内脏芽以及卵黄囊和脐血管。为特异性捕获AGM区的主动脉腔面内皮细胞，我们参照已发表的方法，向E10.0-E11.0胚胎的背主动脉中显微注射了奥里根绿（Oregon green）荧光染料。采样细胞通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）以CD45⁻CD31⁺CD144⁺表型进行纯化，该群体主要包含血管内皮细胞与CD41⁺造血细胞。同时，我们将体内CD45⁻CD31⁻CD144⁺的非内皮细胞用作阴性对照。