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Characterization and Generation of Human Definitive Multipotent Zhu et al. Cell Discovery (2020) 6:89 Cell Discovery https://doi.org/10.1038/s41421-020-00213-6 www.nature.com/celldisc ARTICLE Open Access Characterization and generation of human definitive multipotent hematopoietic stem/ progenitor cells Yanling Zhu1,2,TianyuWang 1,2,JiamingGu 1,2,3,KeHuang1,2,TianZhang1,2, Zhishuai Zhang1,2,3,HeLiu1,2, Jun Tang1,2,YuchanMai1,2, Yanqi Zhang1,2,3,YuhangLi1,2,3,YashuFeng4, Baoqiang Kang1,2,JinbingLi1,2,3, Yongli Shan1,2, Qianyu Chen1,2, Jian Zhang1,2,BingLong4,JunweiWang1,2,MinghuiGao1,2,DiZhang1,2,3, Min Zhou1,2, Xiaofen Zhong1,2,JiekaiChen1,2,DuanqingPei 1,2,JinfuNie1,2, Bing Liu 5 and Guangjin Pan 1,2,6,7 Abstract 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 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; human early hematopoiesis and a firm basis for generating blood and immune cells for clinical purposes. – Introduction lymphoid progenitors (LPs)1 7. However, many studies In embryogenesis, definitive hematopoiesis generates show that the uni-potent progenitors derive directly from hematopoietic stem cells and multipotent progenitors HSPCs without going through oligo-potent progenitors, (HSC/MPPs) that further give rise to all mature blood and and thus the classical model of human hematopoiesis is – immune cells. Classically, the multi-potent HSPCs revised1,8 11. Nonetheless, the multipotent HSC/MPPs undergo a stepwise differentiation to the oligo-, bi- and that conceptually initiate hematopoiesis are very rare in uni-potent progenitors of different types of mature blood adult human blood system9,12,13. Human HSCs and MPPs cells, such as granulocyte-monocyte progenitors (GMPs), are phenotypically known to locate within the − + − megakaryocyte-erythroid progenitors (MEPs), and Lin CD34 CD38 compartment and both belong to a continuum of undifferentiated hematopoietic stem/pro- genitor cells (HSPCs)6,8,9,12,13. Correspondence: Guangjin Pan ([email protected]) 1CAS Key Laboratory of Regenerative Biology, Joint School of Life Sciences, During embryonic development, the multipotent Guangzhou Institutes of Biomedicine and Health, Chinese Academy of HSPCs are originated in early definitive hematopoiesis – Sciences, Guangzhou Medical University, ,Guangzhou Guangdong 510530, through endothelial-to-hematopoietic transition (EHT)14 18. China 2Guangdong Provincial Key Laboratory of Stem Cell and Regenerative In the mouse model, the first wave hematopoiesis starts in Medicine, South China Institute for Stem Cell Biology and Regenerative yolk sac to generate primitive progenitors (mainly myelo- Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy erythroid) and then in aorta-gonad-mesonephros (AGM) of Sciences, ,Guangzhou Guangdong 510530, China 14–19 Full list of author information is available at the end of the article region to generate definitive HSPCs . These two These authors contributed equally: Yanling Zhu, Tianyu Wang, Jiaming Gu © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Zhu et al. Cell Discovery (2020) 6:89 Page 2 of 18 waves of hematopoiesis generate functionally distinct peripheral blood (PB-HSPCs) (Fig. 1a). t-SNE visualiza- progenitors, and the immune cells such as NK, lymphoid tion on single-cell transcription profiling displayed dis- cells are mainly generated from the second wave of defi- tinct clusters between PB-HSPCs and hESCs-HPCs nitive hematopoiesis. To this end, human primitive and (Fig. 1b, c). Consistent with previous findings9,13, most − + − definitive hematopoiesis have not been well-characterized Lin CD34 CD38 PB-HSPCs formed continuous in early embryonic development. Human pluripotent rather than discrete pattern in transcriptional profiling stem cells (PSCs) such as embryonic stem cells (ESCs) and (Fig. 1b, c). A small number of PB-HSPCs formed a induced pluripotent stem cells (iPSCs) are useful models separate cluster and expressed early lymphoid lineage for investigation of human hematopoietic development markers such as CD10 and LTB5 (Fig. 1c, d), indicating – in vitro20 23. In hPSC differentiation, hemogenic endo- they are lymphoid-primed HSPCs localized in − + − thelial cells (HECs) express CD34 and CD31 and then Lin CD34 CD38 compartment (PB-LPCs)6. The FACS- + show upregulated CD43 on the commitment of hemato- sorted CD43 hESCs-HPCs formed an indiscrete cluster – poietic fate22,24 26. CD43 has served as a marker to indi- separated from both PB-HSPCs and PB-LPCs (Fig. 1b, c). cate hPSC-derived hematopoietic progenitors in many Consistent with the FACS sorting, hESCs-HPCs displayed + studies23,24,27,28. However, even though these CD43 a uniform CD43 expression and were negative for most hPSC-derived HPCs could form various blood CFUs known lineage markers of different mature blood cells + (colony-forming units), they have very limited definitive (Fig. 1d). Surprisingly, most of these CD43 hESCs-HPCs potency to give rise to immune cells23,25. Characterization highly expressed GATA1, a known transcription factor for of human primitive and definitive hematopoiesis at the early erythroid lineage (Er)31, and NFE2, a myeloid (My) earliest stage remains unaddressed, thus largely impeding marker gene32 (Fig. 1d), indicating they might be Er/My- the translational progress of human stem cell biased progenitors rather than the multipotent ones. differentiation. Indeed, genes for myeloid development such as MYL4, Here in this study, we resolve human early hemato- DMTN, and MPIG6B and platelet and erythroid devel- poietic populations by single-cell RNA-seq and reveal opment genes like ITGA2B, VCL, GATA1, HBG, and HBD distinct transcriptional profiling in early definitive and were highly enriched in hESC-HPC cluster (Fig. 1e, g). In primitive hematopoiesis both in human embryonic stem contrast, known genes for definitive HSCs and/or lym- cell (hESC) differentiation and early embryonic develop- phoid were highly enriched in PB-HSPC and PB-LPC ment. We identify CD44 to specifically discriminate the clusters, respectively, such as ETV6, CD34, HLADR, LTB, early definitive and primitive hematopoiesis. In addition, HOXA9, and CCR7 (Fig. 1e, g). The top gene ontology we generate definitive HSPCs from hESCs that give rise to (GO) terms enriched in hESC-HPC cluster were related to various blood and immune lineages, including myeloid, mitochondrial regulation, reflecting this cluster is related erythroid/megakaryocyte cells, NK, T lymphocytes etc. to erythroid lineage (Fig. 1g). Consistently, the function of and strikingly differentiate to various blood and lymphoid lymphoid regulation was significantly enriched in PB- cells in vivo. Our studies provide a valuable model to LPCs (Fig. 1g). Together, we reveal that the marker-pure + understand human early hematopoietic development and CD43 hESCs-derived HPCs are mainly Er/My-biased generate various blood and immune cells for clinical progenitors rather than definitive ones based on gene- purposes. expression profiling, which has not been recognized before. Results Single-cell transcriptional profiling of hematopoietic hESCs-derived HPCs contain cells with different populations in hESC differentiation hematopoietic states + Based on reports from multiple groups20,29,30, we pre- To further characterize the CD43 hESCs-derived viously developed a stepwise strategy for hPSC blood HPCs in detail, we applied pseudotime analysis to order differentiation in a totally defined, monolayer condition, all the hESC-HPCs based on their transcriptional profiling + which allows a robust EHT to generate CD43 hemato- (Fig. 2a). Known genes that encode markers or regulators poietic progenitors (hESCs-HPCs) that could form var- for human HSPCs such as CD34, CD90, MYB, RUNX1, + ious blood CFUs28. However, the multi-potency of CD43 GFI1, IKZF1, HOXA9, and GATA2 were downregulated hESCs-HPCs
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