Fig. 3. Activin A and bFGF induce hemangioblast specification. (A) VEGF, activin A or bFGF were added at day 2.5 of serum-free culture supplemented with Bmp4 at 4 ng/ml from day 0. EBs were harvested at day 3 and assessed for their ability to form blast colonies by secondary replating. (B) activin A and bFGF were added at day 2.5 of serum-free culture supplemented with Bmp4 at 4 ng/ml from day 0. EBs were harvested 3, 6 or 12 hours after the induction with activin A and bFGF and assessed for their ability to form blast colonies by secondary replating. ^*A statistically significant difference (P<0.05) compared with Bmp4-induced cultures. (C) Tie2, CD45, CD31 and VE-cadherin expression levels were analyzed on pools of blast colonies. Top panel presents CD45 and Tie2 staining; the level of CD31 and VE-cadherin expression was then assessed on CD45⁺Tie2^- (middle) and CD45^-Tie2⁺ (bottom) cells. (Da-c) Individual blast colonies (examples a, b and c) were tested for their potential to generate endothelium tubule-like structures in Matrigel plugs. Colony in c is stained with CD31 antibody. 10x magnification, bright field. (E) At 3, 6 and 12 hours after activin A and bFGF stimulation, cells were tested for their relative expression of GFP-Bry and Flk1 by flow cytometry. (F) EBs were grown for 2.5 days in serum-free culture with 4 ng/ml Bmp4 and then activin A and bFGF were added. At days 4, 5 and 6 of differentiation, EB-derived cells were tested for the presence of hematopoietic progenitors by colony-forming ability in secondary replating assay. Primitive, primitive erythrocytes; definitive, all definitive colonies (see Fig. 1). Primitive colonies were scored 5 days after replating, definitive colonies after 8 days. ^**A statistically significant difference (P<0.05) compared with the serum control. All data shown are representative of at least three experiments. For A, B and D, data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m.