Fig. 1. Transition from ES cells to epiblast-like cells. (A) Flk1 and GFP expression analysis by flow cytometry on mouse day-3 embryoid bodies (EBs) grown in serum-free (left) or serum-supplemented (right) culture. (B) GFP-Bry ES cells were differentiated in serum-free media supplemented with serum or with the indicated combination of factors added at the start of the culture (B, Bmp4; A, activin A; F, bFGF; V, VEGF). At day 5, EB-derived cells were tested for the presence of hematopoietic progenitors in a secondary replating assay. Primitive, primitive erythrocytes; definitive, all definitive colonies (macrophage, macrophage/erythrocyte, mixed colonies and granulo-macrophage colonies). (C) Outline of the experimental design employed in subsequent experiments. (D) Gene expression analysis in ES cells and day 1, 2 or 3 serum-free EB-derived cells by RT-PCR. β-actin expression levels were used for cDNA quantity control. cDNA from day-4 EBs grown in serum was used as positive control for Sox17, brachyury, Gata4; cDNA from day-6 EBs grown in neuronal condition (Li et al., 1998) was used as positive control for Sox1 and Wnt1 PCR. (E) ES cells grown in serum-free media rapidly lose their ability to form EBs. ES cells and day 1, 2 or 3 EB-derived cells were replated in semi-solid conditions allowing the formation and quantification of EBs. ^*A statistically significant difference (P<0.05) compared with ES cell control. All data shown are representative of at least three experiments. For B and E, data are presented as the mean number of colonies from three dishes. Error bars represent s.e.m.