mESCs differentiate toward meso-endoderm lineage spontaneously during long-term culture. (A) Morphology of E14 cells cultured in mES medium containing 1 000 U/ml LIF without passage for 1-8 days. Medium was changed every day. Scale bar, 50 μm. (B-F) qRT-PCR analysis of pluripotency genes (B), lineage-specific genes (mesoderm: Brachyury and SMA, endoderm: GATA4, Sox17, ectoderm: Nestin and Sox1) (C), genes involved in EMT (D), vascular marker genes (E) and VEGF genes (F) in mESCs described in A on days 1, 3, 5 and 8. (G) ELISA analysis of VEGF secretion from mESCs described in A. (H) VEGF-induced morphology and alkaline phosphatase (AP) change in E14 cells. Scale bar, 50 μm. E14 cells were cultured in mES medium containing 1 000 U/ml LIF for 48 h, then vehicle or 1, 3, 10 ng/ml mVEGF164 was added into the medium for another 48 h prior to analysis. Data are mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs day 1. Independent experiments were repeated at least three times.