Abstract
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.
Highlights
Presence of vitamin C (Vc); VcϪ_G2ECs, the GATA2ϩ endothelial cells (G2ECs) generated in the absence of Vc; ATAC-seq, transposase-accessible chromatin with sequencing; CHIP-seq, chromatin immunoprecipitation followed by sequencing; TSS, transcriptional start sites; H3, histone 3; CFU-E, erythroid CFUs; BM, basic medium; MO, morpholinos; bFGF, basic fibroblast growth factor; sgRNA, single guide RNA; qPCR, quantitative PCR; DAPI, 4Ј,6-diamidino-2-phenylindole; BMP, basic metabolic panel
Later at day 10, a certain percentage of CD43ϩ cells became CD45ϩ, a marker indicating mature HPCs [4, 16] (Fig. 1E). Both adult and embryonic globin HBB, HBE, and HBG1 could be detected in the erythroid CFUs (CFU-E) (Fig. 1, H and I), demonstrating both the definitive and primitive hematopoiesis occurred during differentiation
To see if similar mechanisms regulate the generation of hemogenic endothelial cells (HECs), we examined the chromatin accessibility landscapes and epigenetic modifications in VcϪ_G2ECs and Vcϩ_G2ECs by transposase-accessible chromatin with sequencing (ATAC-seq) and ChIP followed by sequencing (CHIP-seq) on active (H3K4me3) and repressive (H3K27me3) histone modifications
Summary
Vitamin C is required for generation of HPCs from hPSCs in a defined condition We sought to develop an efficient approach to differentiate blood cells from hPSCs in a chemically defined, serum-free and monolayer condition. Based on this report and other literatures [4, 14, 15], we developed a stepwise strategy to differentiate hPSCs in a defined, monolayer condition that recapitulates main stages of early hematopoiesis, including the PS, LM, HECs, and HPCs (Fig. 1A). Later at day 10, a certain percentage of CD43ϩ cells became CD45ϩ, a marker indicating mature HPCs [4, 16] (Fig. 1E) Both adult and embryonic globin HBB, HBE, and HBG1 could be detected in the erythroid CFUs (CFU-E) (Fig. 1, H and I), demonstrating both the definitive and primitive hematopoiesis occurred during differentiation. We found that only Vc, but not the other antioxidants could promote the HPC generation (Fig. 1N, Fig. S1H) These data above suggest that Vc is an essential factor for hematopoietic differentiation in hPSCs in a defined condition
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