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.

最终的造血系统会产生造血干/祖细胞（HSPC），这些造血干/祖细胞会产生所有成熟的血液和免疫细胞，但在人类中仍然定义不清。在这里，我们通过单细胞RNA-seq在最早的造血阶段解析人类造血种群。我们表征人类胚胎干细胞（hESC）分化和早期胚胎发育中早期原始和确定性造血之间的独特的分子谱。我们鉴定出CD44可以特异性地区分确定性造血功能并从hESC生成确定性HSPC。通过单细胞克隆试验验证了hESCs衍生的HSPC对各种血液和免疫细胞的多能性。令人惊讶的是，这些hESCs衍生的HSPC在体内会产生血液和淋巴样谱系。最后，我们表征基因表达动力学在确定性和原始造血作用中的作用，并揭示了ROCK抑制作用在增强人类确定性造血作用中未报道的作用。我们的研究为理解人类早期造血提供了前景，并为临床产生血液和免疫细胞奠定了坚实的基础。