ABSTRACT Vascular formation in vivo involves several processes and signal cascades subsequently occurring in the embryo. Several models by ES cells have been reported for analysis in vitro. We show here a 3D culture system using collagen gel (AteloCell) as a simple and useful system for investigating vascular formations and analyzing the roles of factors in vivo. Although VEGF and PDGF are growth factors with multi-potentials for vascular formation, their sequential roles have not been elucidated. We investigated the effects of VEGF and PDGF B signals for vascular formation by a 3D culture system that embedded embryoid bodies (EBs) from ES cells into a collagen gel. After embedding EBs in the collagen gel with a medium containing VEGF, EBs gave off CD105 immunopositive vessels as the initial step of vasculogenesis. When the factor in the culture medium for EBs was switched from VEGF to PDGF B after 5 days of culture, the morphological features of vessels varied, suggesting the occurrence of vascular-type differentiation. After 11 days of 3D culture, vessels in both groups cultured with VEGF alone and switching to VEGF B at day 5 showed Flk-1 immunoreactivity. Some blood vessels cultured with PDGF B after day 5 expressed either EphrinB2 (arteriole marker) or Flt-4 (lymphatic marker) immunoreactivity, but vessels cultured with VEGF alone exhibited neither of them. Vessels cultured with these two factors could not differentiate into a venous type. The present study indicates that VEGF is the initial signal for vasculogenesis, and that PDGF B is probably involved in vascular diversification.
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