Different Kinetics and Function of Vascular Endothelial Growth Factor Recepotor-1 and −2 during Hemangioblast Development from Primate Embryonic Stem Cells.

The close developmental association between hematopoietic and endothelial cells suggests that both lineage cells share a common precursor, the hemangioblast. The vascular endothelial growth factor (VEGF)-A system has been proven to have roles in the embryonic development of hemangioblast in mouse by the use of embryos or embryonic stem (ES) cells deprived of the genes encoding its ligand or receptors (VEGFR-1 and VEGFR-2). On the other hand, there have been only a few reports on the hemangioblast development during primate (human and monkey) embryogenesis.

We have previously demonstrated that the hemangioblast is highly enriched in the VEGFR-2^high CD34⁺ cell fraction, differentiated from cynomolgus monkey ES cells by 6-day coculture with OP9 stromal cells. In the current study, we examined whether the VEGF-A system was involved in the development of hemangioblast induced from primate ES cells by using the coculture system. VEGFR-1 and −2 were expressed by undifferentiated monkey ES cells at low levels. The VEGFR-2^low cells gradually decreased while VEGFR-2^high cells could be detected on day 6. On the other hand, VEGFR-1 was constantly expressed at low levels on day 6 and thereafter. Exogenous VEGF-A₁₆₅, the action of which is mediated VEGFR-1 and VEGFR-2, increased the proportion of VEGFR-2^high CD34⁺ cells in a dose-dependent manner. Addition of VEGF-E (VEGFR-2-specific agonist) and VEGFR-1 blocking antibody resulted in the increase of VEGFR-2^high CD34⁺ cells in a dose-dependent manner, while VEGFR-2 blocking antibody suppressed their development. Thus, VEGF-A/VEGFR-2 interaction promoted the development of hemangioblast, while VEGF-A/VEGFR-1 interaction acted as a negative regulator.

We then examined when VEGF-related signals work on VEGFR-2^high cell development by changing factors on day 4. The proportion of VEGFR-2^high CD34⁺ cells with the last 2-day VEGF-A₁₆₅ and -E treatment were almost equivalent to that with continuous exposure to the VEGF. On the contrary, initial 4-day VEGF-A₁₆₅ and -E treatment did not any stimulatory effects on VEGFR-2^high CD34⁺ cell development. Our previous results show that during differentiation, Brachyury (early mesodermal marker) is first expressed on day 4, followed by the up-regulation of genes, such as SCL, LMO2, and MYB, representing hematopoietic and/or endothelial potentials on day 6. Collectively, VEGFR-2-mediated signals might contribute to the commitment and/or expansion of the hemangioblast rather than their development.

This coculture system provides an opportunity to better understand the regulative mechanisms on early hematopoietic and endothelial cell development, which remains unresolved by experiments using human embryos.