| Experiment Id | GSE139389 | Name | Embryonic Endothelial Evolution towards First Hematopoietic Stem Cells Revealed by Single-Cell Transcriptomic and Functional Analyses |
| Experiment Type | RNA-Seq | Study Type | Baseline |
| Source | GEO | Curation Date | 2024-03-19 |
| description | 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. |
