Single Cell Resolution of Human Hematoendothelial Cells Defines Transcriptional Signatures of Hemogenic Endothelium.
ABSTRACT: Endothelial-to-hematopoietic transition (EHT) is an important stage in definitive hematopoietic development. However, the genetic mechanisms underlying human EHT remain poorly characterized. We performed single cell RNA-seq using 55 hemogenic endothelial cells (HECs: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c+ ), 47 vascular endothelial cells without hematopoietic potential (non-HE: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c- ), and 35 hematopoietic progenitor cells (HPCs: CD34+ CD43+ RUNX1c+ ) derived from human embryonic stem cells (hESCs). HE and HP were enriched in genes implicated in hemogenic endothelial transcriptional networks, such as ERG, GATA2, and FLI. We found transcriptional overlap between individual HECs and HPCs; however, these populations were distinct from non-HE. Further analysis revealed novel biomarkers for human HEC/HPCs, including TIMP3, ESAM, RHOJ, and DLL4. Collectively, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE are more heterogeneous and transcriptionally distinct. Our findings provide a novel strategy to test new genetic targets and optimize the production of definitive hematopoietic cells from human pluripotent stem cells. Stem Cells 2018;36:206-217.
Project description:The transition from hemogenic endothelial cells (HECs) to hematopoietic stem/progenitor cells (HS/PCs), or endothelial to hematopoietic transition (EHT), is a critical step during hematopoiesis. However, little is known about the molecular determinants of HECs due to the challenge in defining HECs. We report here the generation of GATA2w/eGFP reporter in human embryonic stem cells (hESCs) to mark cells expressing GATA2, a critical gene for EHT. We show that during differentiation, functional HECs are almost exclusively GATA2/eGFP+. We then constructed a regulatory network for HEC determination and also identified a panel of positive or negative surface markers for discriminating HECs from non-hemogenic ECs. Among them, ITGB3 (CD61) precisely labeled HECs both in hESC differentiation and embryonic day 10 mouse embryos. These results not only identify a reliable marker for defining HECs, but also establish a robust platform for dissecting hematopoiesis in vitro, which might lead to the generation of HSCs in vitro.
Project description: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.
Project description:Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the "heptad" complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs.
Project description:Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34(+) CD31(+) CD144(+) population derived from hPSCs contain hemato-endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial-to-hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34(+) CD31(+) CD144(+) HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation.
Project description:Embryonic hematopoiesis is a complex process. Elucidating the mechanism regulating hematopoietic differentiation from pluripotent stem cells would allow us to establish a strategy to efficiently generate hematopoietic cells. However, the mechanism governing the generation of hematopoietic progenitors from human embryonic stem cells (hESCs) remains unknown. Here, on the basis of the emergence of CD43(+) hematopoietic cells from hemogenic endothelial (HE) cells, we demonstrated that VEGF was essential and sufficient, and that bFGF was synergistic with VEGF to specify the HE cells and the subsequent transition into CD43(+) hematopoietic cells. Significantly, we identified TGF? as a novel signal to regulate hematopoietic development, as the TGF? inhibitor SB 431542 significantly promoted the transition from HE cells into CD43(+) hematopoietic progenitor cells (HPCs) during hESC differentiation. By defining these critical signaling factors during hematopoietic differentiation, we can efficiently generate HPCs from hESCs. Our strategy could offer an in vitro model to study early human hematopoietic development.
Project description:Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational mouse embryos. Due to rare and transient nature, the HSC-competent ECs have never been stringently identified and accurately captured, let alone their genuine vasculature precursors. Here, we firstly used high-precision single-cell transcriptomics to unbiasedly examine relevant EC populations at continuous developmental stages and transcriptomically identified putative HSC-primed HECs. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by newly constructed Neurl3-EGFP reporter mouse model, and realized enrichment further by surface marker combination. Surprisingly, endothelial-hematopoietic bi-potential was rarely but reliably witnessed in culture of single HECs. Noteworthy, primitive vascular ECs experienced two-step fate choices to become HSC-primed HECs, resolving 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. Overall design: We initially sequenced 662 single cells from E9.5-E11.0 body and DA of totally 29 embryos. Additionally, we also sequenced 96 single cells with a PK44 immunophenotype (CD41-CD43-CD45-CD31+CD201+Kit+CD44+) from E10.0 AGM regions of totally 9 embryos, 47 T1 pre-HSCs (CD31+CD45-CD41lowKit+CD201high) from E11.0 AGM regions of totally 18 embryos, 48 single cells with an immunophenotype of CD41-CD43-CD45-CD31+CD44+Neurl3-EGFP+ from Neurl3-EGFP reporter mouse embryos and 579 single cells from E8.0-E9.0 body of 24 embryos. In total, 1,432 single cells were sequenced. For each embryo, the embryo proper was isolated and the head, limb buds, heart, visceral bud, and vitelline and umbilical vessels outside the embryo proper were excluded. To specifically capture aortic luminal ECs of AGM region, we performed microinjection of fluorescent dye Oregon green into the dorsal aortas of E10.0-E11.0 embryos as reported. The sampled cells were purified by FACS as CD45-CD31+CD144+, which contained predominantly vascular ECs and CD41+ hematopoietic cells. Meanwhile, CD45-CD31-CD144- non-EC cells in the body were used as negative controls.
Project description:Hematopoietic stem cells (HSCs) are derived from hemogenic endothelial cells (HECs) during embryogenesis. The HSC-primed HECs are peak at embryonic day (E) 10 and have been efficiently captured by the marker combination CD41-CD43-CD45-CD31+CD201+Kit+CD44+ (PK44) in the aorta-gonad-mesonephros (AGM) region of mouse embryos most recently. 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. 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. Importantly, PK44 cells in the yolk sac demonstrated an unambiguous 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. Overall design: PK44 (CD41-CD43-CD45-CD31+CD201+Kit+CD44+) population have been identified as HSC-competent HECs in our precious study.With the aim of further exploring whether yolk sac PK44 cells can undergo EHT and have endothelial-hematopoietic dual potential like that in AGM region, we sorted and performed scRNA-seq.
Project description:The transcriptional factor GATA2 is required for blood and hematopoietic stem cell formation during the hemogenic endothelium (HE) stage of development in the embryo. However, it is unclear if GATA2 controls HE lineage specification or if it solely regulates endothelial-to-hematopoietic transition (EHT). To address this problem, we innovated a unique system, which involved generating GATA2 knockout human embryonic stem cell (hESC) lines with conditional GATA2 expression (iG2-/- hESCs). We demonstrated that GATA2 activity is not required for VE-cadherin+CD43-CD73+ non-HE or VE-cadherin+CD43-CD73- HE generation and subsequent HE diversification into DLL4+ arterial and DLL4- non-arterial lineages. However, GATA2 is primarily needed for HE to undergo EHT. Forced expression of GATA2 in non-HE failed to induce blood formation. The lack of GATA2 requirement for generation of HE and non-HE indicates the critical role of GATA2-independent pathways in specification of these two distinct endothelial lineages.
Project description:BACKGROUND:Hematopoiesis is a progressive process collectively controlled by an elaborate network of transcription factors (TFs). Among these TFs, GATA2 has been implicated to be critical for regulating multiple steps of hematopoiesis in mouse models. However, whether similar function of GATA2 is conserved in human hematopoiesis, especially during early embryonic development stage, is largely unknown. RESULTS:To examine the role of GATA2 in human background, we generated homozygous GATA2 knockout human embryonic stem cells (GATA2 (-/-) hESCs) and analyzed their blood differentiation potential. Our results demonstrated that GATA2 (-/-) hESCs displayed attenuated generation of CD34(+)CD43(+) hematopoietic progenitor cells (HPCs), due to the impairment of endothelial to hematopoietic transition (EHT). Interestingly, GATA2 (-/-) hESCs retained the potential to generate erythroblasts and macrophages, but never granulocytes. We further identified that SPI1 downregulation was partially responsible for the defects of GATA2 (-/-) hESCs in generation of CD34(+)CD43(+) HPCs and granulocytes. Furthermore, we found that GATA2 (-/-) hESCs restored the granulocyte potential in the presence of Notch signaling. CONCLUSION:Our findings revealed the essential roles of GATA2 in EHT and granulocyte development through regulating SPI1, and uncovered a role of Notch signaling in granulocyte generation during hematopoiesis modeled by human ESCs.
Project description:We generated homozygous GATA2 knockout human embryonic stem cells (GATA2-/- hESCs) and analyzed their blood differentiation potential. Paritcularly at the hemogenic endothelium (HE) stage and hematopoietic progenitor cell (HPC) stage. Our result revealed that GATA2-/- hESCs displayed attenuated generation of CD34+CD43+ HPCs, due to the impairment of endothelial to hematopoietic transition (EHT). However, GATA2-/- hESCs retained the potential to generate erythroblasts, macrophages, but never granulocytes. Through RNA-Seq and further rescue experiment, we further identified that SPI1 was responsible for the defect of GATA2-/- hESCs in generation of CD34+CD43+ HPCs and granulocytes. Overall design: We compared the transcriptome of GATA2-/- hESCs, HE, HPC and their wildtype counterparts by RNA-Seq.