Spatiotemporal And Functional Heterogeneity Of Hematopoietic Stem Cell-competent Hemogenic Endothelial Cells In Mouse Embryos

As the cornerstone of the adult hematopoietic system,self-renewal and multi-lineage differentiation are the dominant feature of hematopoietic stem cells(HSCs).HSCs have been widely proposed that they are derived from a special subgroup of endothelial cells,called hemogenic endothelial cells(HECs),in the aorta-gonad-mesonephros(AGM)region around embryonic day(E)10,through the endothelial-to-hematopoietic transition(EHT)process.It has been widely proposed that HECs express CD47,CD61 and Dll4 in addition to pan-endothelial markers like Flk1and VE-Cadherin,but lack the expression of hematopoietic markers CD41,CD43 and CD45.The expression of Runx1,a transcription factor critically required for the process of EHT,represents a hallmark of HECs.Based on this,Runx1+23GFP(GFP transgene on the Runx1+23 enhancer locus)transgenic reporter mouse model has been established to enrich HECs for expressional and functional analysis.Similarly,Gfi1-Tomato transgenic mice are used to delineate the process of EHT,but the enrichment of these surface markers or reporters mentioned above is insufficient and the HSC competence of the HECs has not been directly confirmed.Recently,the HSC-primed HECs have been efficiently captured by the marker combination CD41~-CD43~-CD45~-CD31~+CD201~+Kit~+CD44~+(PK44)in the AGM region of mouse embryos.Additionally,functional heterogeneity of PK44 population was revealed in vitro at the single-cell level,involving three kinds of differentiation potentials,namely,only endothelial potential(23%),endothelial-hematopoietic dual potential(2.7%)and only hematopoietic potential(40%).In the present study,we investigated the spatiotemporal and functional heterogeneity of PK44 cells around the time of emergence of HSCs.First,PK44 cells in E10 AGM region could be further divided into three molecularly different populations showing endothelial-or hematopoietic-biased characteristics.Specifically,with the combination of Kit,the expression of CD93 or CD146 could divide PK44 cells into endothelial-and hematopoietic-feature biased populations,which was further functionally validated at single cell level through flow cytometry index sorting function.Next,PK44 population could also be detected in the yolk sac,showing a developmental dynamics and functional diversification similar to those in the AGM region.Unexpectedly,the three differentiation potentials like that in AGM region were found within yolk sac PK44 cells.Importantly,PK44 cells in the yolk sac demonstrated an unambiguous long-term multi-lineage reconstitution capacity after in vitro incubation.Regardless of the functional similarity,PK44 cells in the yolk sac displayed transcriptional features different to those in the AGM region.Taken together,our work delineated the spatiotemporal characteristics of HECs represented by PK44,and revealed a previously unknown HSC competence of HECs in the yolk sac.These findings provided a fundamental basis for in-depth studying the different origins and molecular programs of HSC generation in the future.