Fig. 3. Differentiation of mesendoderm cells into definitive endoderm and mesoderm lineages. (A) RT-PCR analysis of gene expression in GFP⁺ECD⁺ and GFP⁺ECD^- cells on day 5. (B) Multicolour immunofluorescent staining of cells in day 5 cultures. Gsc⁺ECD⁺Foxa2⁺ cells are within the epithelial sheet, whereas Gsc⁺ECD^-Foxa2^- cells disperse around the sheet. (C) RT-PCR analysis of gene expression in differentiated Gsc⁺ECD⁺ cells. Gsc⁺ECD⁺ cells were purified from cultures with activin on day 6 and re-cultured in SCM. Expression level of Gsc, Mixl1 and Afp changes during culture. Gsc is positive only in the day 5 culture (A), Mixl1 is in the day 5 (A) and 12 cultures, and AFP is only in the day 12 culture. (D) Albumin-producing cells are generated from Gsc⁺ECD⁺ population. (i) Gsc⁺ cells were induced in SFM containing activin. On day 6, Gsc⁺ECD⁺ cells were purified by cell-sorter and re-cultured for 3 days on collagen I-coated dish in the presence of Egf, Bmp4, acid Fgf and basic Fgf. (ii) The expression of albumin in the Gsc⁺ECD⁺-derived cells detected by immunostaining. (iii) The expression of albumin in the Gsc⁺ECD⁺-derived cells detected by RT-PCR. Hnf6, a marker of precursor of liver and pancreatic cells, was stimulated to be expressed.