Abstract
Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational embryos. Due to the rare and transient nature, the HSC-competent HECs have never been stringently identified and accurately captured, let alone their genuine vascular precursors. Here, we first used high-precision single-cell transcriptomics to unbiasedly examine the relevant EC populations at continuous developmental stages with intervals of 0.5 days from embryonic day (E) 9.5 to E11.0. As a consequence, we transcriptomically identified two molecularly different arterial EC populations and putative HSC-primed HECs, whose number peaked at E10.0 and sharply decreased thereafter, in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by the newly constructed Neurl3-EGFP reporter mouse model, and realized the enrichment further by a combination of surface markers (Procr+Kit+CD44+, PK44). Surprisingly, the endothelial-hematopoietic dual potential was rarely but reliably witnessed in the cultures of single HECs. Noteworthy, primitive vascular ECs from E8.0 experienced two-step fate choices to become HSC-primed HECs, namely an initial arterial fate choice followed by a hemogenic fate conversion. This finding resolves 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.
Highlights
The sampled cells were purified by FACS as CD45−CD31+CD144+, which contained predominantly vascular endothelial cells (ECs) and CD41+ hematopoietic cells
We explored whether endothelial-hematopoietic dual potential could be detected in these hematopoietic stem cells (HSCs)-competent hemogenic endothelial cells (HECs), since a transient intermediate state might be captured in the cell population experiencing fate choice
Enrichment of the HSC-competent HECs by newly established Neurl3-EGFP reporter In an effort to search for single marker to distinguish HSC-primed HECs from non-HECs or those CD45− hematopoietic cells sharing an endothelial immunophenotype, we computationally screened for genes significantly overrepresented in the HEC cluster as compared to each of the other four clusters, including one hematopoietic cluster (HC) and three vascular EC clusters (Supplementary information, Fig. S1f)
Summary
The adult hematopoietic system, consisting mainly of hematopoietic stem cells (HSCs) and their multi-lineage progenies, is believed to be derived from hemogenic endothelial cells (HECs) in mid-gestational embryos.[1,2] It is generally accepted that while still embedded in the endothelial layer and presenting endothelial characteristics, HECs begin to express key hemogenic transcription factor (TF) Runx[1] and have hemogenic potential.[3,4] Different from hematopoietic progenitors, HECs lack the expression of hematopoietic surface markers, such as CD41 and CD45, which mark the population capable of generating hematopoietic progenies when directly tested in colony-forming unit assays.[3,5] Hematopoietic stem and progenitor cells (HSPCs) are visualized to emerge from aortic endothelial cells (ECs) via a transient and dynamic process called endothelial-to-hematopoietic transition to form intra-aortic hematopoietic clusters (IAHCs).[6,7,8,9,10] Being located within IAHCs or to the deeper sub-endothelial layers, pre-HSCs serve as the important cellular intermediates between HECs and HSCs, featured by their inducible repopulating capacity and priming with hematopoietic surface markers.[11,12,13,14,15] The specification of HSCprimed HECs is the initial and one of the most pivotal steps for vascular ECs to choose HSC fate.
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