α4-Integrin⁺ Endothelium Derived from Primate Embryonic Stem Cells Generates Both Primitive and Definitive Hematopoietic Cells.

The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. We previously showed the development of both primitive and definitive hematopoiesis when cynomolgus monkey embryonic stem cells were co-cultured with OP9 stromal cells. In this study, we examined the hematopoietic potential of endothelial cells developing in our coculture system and demonstrated that VE-cadherin⁺CD45⁻ endothelial cells derived from embryonic stem cells were able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. All floating erythrocytes which emerged initially expressed ε- and ζ-globins, while β-globin expression was hardly detected. The percentage of floating erythrocytes positive for β-globin gradually increased thereafter, and almost all erythrocytes were positive by day 40. Meanwhile, expression of ε- and ζ-globins declined gradually. Clonal analysis revealed that single bipotential cells for hematopoietic and endothelial lineages were included in this endothelial cell population. Hemogenic activity of endothelial cells was observed exclusively in the α4-integrin⁺ subpopulation. RT-PCR data showed that Runx1, a transcriptional factor associated with definitive hematopoiesis, was expressed in the hemogenic α4-integrin⁺ subpopulation, but not the non-hemogenic α4-integrin⁻ subpopulation among embryonic stem cell-derived endothelial cells. The kinetics of this hemogenic subpopulation was similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo, in that they emerged only for a limited time. On the other hand, VE-cadherin⁻CD45⁻α4-integrin⁺ cells gave rise to more primitive erythrocytes than VE-cadherin⁺CD45⁻α4-integrin⁺ cells, but hardly contributed to definitive hematopoiesis. These results indicate that VE-cadherin⁺CD45⁻α4-integrin⁺ endothelial cells generate primitive and definitive hematopoietic cells sequentially, while VE-cadherin⁻CD45⁻α4-integrin⁺ cells are primary sources for primitive hematopoiesis. It seems that precursors of primitive and definitive erythropoiesis arise simultaneously but that the definitive precursors require a period of maturation before they differentiated into blood cells. We suggest that a subset of endothelial cells is involved in primitive as well as definitive hematopoiesis during primate embryogenesis, and that α4-integrin marks the hemogenic subpopulation in primates.