Identification and Characterization of Hemoangiogenic Progenitors during Cynomolgus Monkey ES Cell Differentiation.

Previous reports about the close association of development of hematopoietic (HCs) and endothelial cells (ECs) have suggested the presence of the hemangioblast, a putative common precursor of both lineage cells in mouse. The presence of putative ancestors is suggested by the fact that both HCs and ECs share common markers such as vascular endothelial growth factor receptor (VEGFR)-2, CD34, and CD31. The precise analysis about the relationship of HCs and ECs in human embryogenesis, however, remains unclear because of obstacle including the ethical restrictions on experiments using their embryo. To further understand the mechanisms that regulate the differentiation of HCs and ECs in humans, it is currently necessary to employ primate (human and monkey) ES cells. In this study, we identified the intermediate-stage cells with HC and EC differentiation potentials from nonhuman primate ES cells by coculture with OP9 stromal cells. Sequential FACS analysis demonstrated that during in vitro HC and EC differentiation, the expression of common surface markers such as CD34 and CD31 could first be detected, followed by the expression of lineage-specific markers (CD45, CD41 and VE-cadherin) in order. Sequential FACS and immunostaining analyses showed that both lineage developments concomitantly occurred, which were preceded by emergence of two hemoangiogenic progenitors, VEGFR-2^high CD34⁻ and VEGFR-2^high CD34⁺ cells. While both VEGFR-2 ^high cells could differentiate into primitive and definitive HCs as well as ECs, VEGFR-2^high CD34⁺ cells possessed higher hemoangiogenic potential than VEGFR-2^high CD34⁻ cells. In contrast, HC and EC production cluster was rare in the VEGFR-2^{low or −} fractions. During HC and EC differentiation, most of VEGFR-2^high cells first expressed CD34, and then diverged into both lineage cells at the VEGFR-2^high CD34⁺ stage. We found that the VEGFR-2^high cells can generate both HCs and ECs at the single cell level, which strongly supports the existence of hemangioblasts in primates. To our knowledge, this is the first time that hemangioblasts have been shown to be present during primate ES cell differentiation. Furthermore, differentiation pathway into HC and EC lineages can be defined by surface markers, which facilitates further investigations in vitro and in vivo.