Fig. 2. Differentiation course of Gsc⁺ mesendoderm cells analyzed by FACS. (A) Expression of ECD and GFP from day 4 to day 6. The earliest GFP⁺ cells were detected on day 3.5 and co-expressed ECD (data not shown). On day 4, more than 90% of GFP⁺ cells are ECD⁺. Two major populations of GFP⁺ cells, ECD⁺ and ECD^-, were observed from day 5. (B) Expression pattern of ECD and R of GFP⁺ cells. The two molecules are co-expressed in the day 4 GFP⁺ cells but segregate into ECD⁺R^- and ECD^-R⁺ cells on day 6. (C) The FACS pattern of ECD, Vegfr2 and ßR of GFP⁺ cells on day 5. Vegfr2 and ßR are expressed in the ECD^- population exclusively. (D) Cell sorting and re-culture experiments of Gsc⁺ cells. ECD⁺ and ECD^low populations in day 4 GFP⁺ cells were purified and re-cultured for 1 day in SFM containing activin. ECD^high population could give rise to both ECD⁺ and ECD^- cells.