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Hematopoietic stem cells (HSCs)/progenitor cells (HPCs) are generated from hemogenic endothelial cells (HECs) during the endothelial-to-hematopoietic transition (EHT); however, the underlying mechanism remains poorly understood. Here, using an array of approaches, including CRSPR/Cas9 gene knockouts, RNA-Seq, ChIP-Seq, ATAC-Seq etc., we report that vitamin C (Vc) is essential in HPC generation during human pluripotent stem cell (hPSC) differentiation in defined culture conditions. Mechanistically, we found that the endothelial cells generated in the absence of Vc fail to undergo the EHT because of an apparent failure in opening up genomic loci essential for hematopoiesis. Under Vc deficiency, these loci exhibited abnormal accumulation of histone H3 trimethylation at Lys-27 (H3K27me3), a repressive histone modification that arose because of lower activities of demethylases that target H3K27me3. Consistently, deletion of the two H3K27me3 demethylases, Jumonji domain-containing 3 (JMJD3 or KDM6B) and histone demethylase UTX (UTX or KDM6A), impaired HPC generation even in the presence of Vc. Furthermore, we noted that Vc and jmjd3 are also important for HSC generation during zebrafish development. Together, our findings reveal an essential role for Vc in the EHT for hematopoiesis, and identify KDM6-mediated chromatin demethylation as an important regulatory mechanism in hematopoietic cell differentiation.

Definitive hematopoiesis generates hematopoietic stem/progenitor cells (HSPCs) that give rise to all mature blood and immune cells, but remains poorly defined in human. Here, we resolve human hematopoietic populations at the earliest hematopoiesis stage by single-cell RNA-seq. We characterize the distinct molecular profiling between early primitive and definitive hematopoiesis in both human embryonic stem cell (hESC) differentiation and early embryonic development. We identify CD44 to specifically discriminate definitive hematopoiesis and generate definitive HSPCs from hESCs. The multipotency of hESCs-derived HSPCs for various blood and immune cells is validated by single-cell clonal assay. Strikingly, these hESCs-derived HSPCs give rise to blood and lymphoid lineages in vivo. Lastly, we characterize gene-expression dynamics in definitive and primitive hematopoiesis and reveal an unreported role of ROCK-inhibition in enhancing human definitive hematopoiesis. Our study provides a prospect for understanding human early hematopoiesis and a firm basis for generating blood and immune cells for clinical purposes.

Human pluripotent stem cells (hPSCs) exhibit very limited contribution to interspecies chimeras. One explanation is that the conventional hPSCs are in a primed state and so unable to form chimeras in pre-implantation embryos. Here, we show that the conventional hPSCs undergo rapid apoptosis when injected into mouse pre-implantation embryos. While, forced-expression of BMI1, a polycomb factor in hPSCs overcomes the apoptosis and enables hPSCs to integrate into mouse pre-implantation embryos and subsequently contribute to chimeras with both embryonic and extra-embryonic tissues. In addition, BMI1 also enables hPSCs to integrate into pre-implantation embryos of other species, such as rabbit and pig. Notably, BMI1 high expression and anti-apoptosis are also indicators for naïve hPSCs to form chimera in mouse embryos. Together, our findings reveal that the apoptosis is an initial barrier in interspecies chimerism using hPSCs and provide a rational to improve it., Conventional human pluripotent stem cells (hPSCs) fail to contribute to interspecies chimaeras when injected into mouse blastocysts. Here the authors show that forced expression of BMI1 overcomes apoptosis of hPSCs in blastocysts of mouse, rabbit and pig allowing them to contribute to chimaeras.