Dissecting The Ontology Of Human Embryonic Hematopoietic Stem Cell And Megakaryocyte By Single-cell RNA Sequencing

Mammalian hematopoietic system is derived from mesoderm,with multisite of emergency and multilineage of hierarchy during the early embryogenesis.Dissecting the origin and progeny of hematopoietic cells,as well as their microenvironment,offers great promise in the field of regenerative medicine.Hematopoietic stem cells(HSCs)are self-renewing,multipotent cells that give rise to all mature blood and immune cell populations and maintain lifelong blood production.HSCs originate from a specialized endothelial population with hemogenic potential,namely HEC(Hemogenic endothelial cell),in the ventral aspect of the dorsal aorta within the AGM(Aorta-gonad-mesonephros)region.Such process of HSC production is termed as EHT(Endothelial to hematopoietic transition).So far,most studies of hematopoiesis rely on model organisms and,in particular,the mouse and zebrafish.The limited access to obtain embryos and rare cells of interest,preclude the comprehensive understanding of human HSC generation.Therefore,the characterization and enrichment strategy of human HECs need to be resolved by advanced and efficient technical means.In this study,we firstly performed single-cell RNA sequencing(sc RNA-seq)on cells of AGM region at Carnegie stage(CS)13 based on 10x Genomics platform,and identified a subpopulation of ECs highly expressed RUNX1 and enriched RNA metabolism.By screening the cell surface marker from the differentially expressed genes(DEGs),we speculated that CD44 could be used as a cell surface marker to enrich HECs from AGM ECs.To confirm this finding,we performed Single cell tagged reverse transcription sequencing(STRT-Seq)on CD44~+and CD44~-cells FAC-isolated from AGM ECs at CS 12-14.The results showed that CD44~+cells exhibited arterial features while CD44~-cells exhibited venous features.Notably,CD44~+cells comprise the HECs highly expressed RUNX1,MYB and ANGPT1 and enriched terms of ribosome biogenesis and metabolic process.Similarly,we performed STRT-Seq on CD34~+CD45~+HSPCs from dorsal aorta at CS 15,and subdivided the cells into 5 subpopulations including lymphoid and myeloid progenitor cell clusters,as well as 3 HSPC clusters of arterial GJA5~+HSPCs,proliferating Cycling HSPC and mature GFI1B~+HSPC respectively.By combining CS12-14 AGM-ECs with HSPCs,we revealed the transcriptomic EHT trajectory and upregulated genes such as EMCN,PROCR and RUNX1T1 upon the hemogenic fate choice of arterial ECs.Through integrated sc RNA-seq analysis of embryonic body part at CS 10 and caudal half at CS 11,we uncovered a HEC population featured by endothelial and hemogenic genes,but lack of arterial characteristics and Notch signaling enrichment,was emerged at CS 10.We hypothesized that these early HECs undergo the direct differentiation to hematopoietic cells without being arterialized as what the AGM HECs experienced.Finally,we dissected the potential cellular interactions between mesenchymal,epithelial,endothelial cells and HECs respectively,by computing the significant ligand-receptor pairs.The results suggested that Notch,BMP4,TGF-beta,etc.signaling may serve as the initiation of cell fate specification towards HSCs.Likewise,we resolved the cellular heterogeneity in yolk sac(YS)at CS 10/11 and fetal liver at CS 20/23 by unbiased sc RNA-seq analysis,and transcriptomically characterized the megakaryocytes(MKs).Comparative analysis of MKs from YS and FL suggested that YS MKs were featured with significant glycolytic metabolism while FL MKs were featured with higher proliferative activity.By pooling together MKs,we recognized the platelet-producing MKs,and identified two subpopulations of MKs including one was involved in niche-forming with highly expressing extracellular matrix genes,and the other exhibited high expression of genes associated with immune responses.Developmental trajectories analysis underpinned the two distinct program of MK maturation,which was highly regulated by a coordinated group of transcription factors.Taken together,using sc RNA-seq analysis,we have successfully deciphered the generation of first HSCs and cellular heterogeneity of MKs during human early embryogenesis.The transcriptomically defined HECs were captured in AGM region,showing an unambiguous feature of arterial ECs with the up-regulating hemogenic properties.We then identified the CD44 as a cell surface marker to sufficiently enrich HECs from AGM ECs.The cellular heterogeneity of HSPCs in the dorsa aorta was analyzed,followed by mapping the developmental path from arterial ECs via HSC-primed HECs to hematopoietic stem progenitor cells(HSPCs),and revealed a distinct expression pattern of genes that were transiently over-represented during the EHT process.We also uncovered another intra-embryonic HEC population,which was detected much earlier and lacked the arterial feature.Moreover,we revealed the cellular components of the putative aortic niche and potential cellular interactions acting on the HSC-primed HECs.Finally,we revealed the cellular heterogeneity and developmental trajectories of early megakaryopoiesis.These findings have filled the current gaps in the understanding of human hematopoietic development,and provide the important insights and valuable resources for seeking the therapeutic applications of blood disorders,especially generating functional HSCs and platelets from the dish.