The effect of serum on germ layer induction in EBs. (A) Bry-GFP ES cells were cultured in the presence (serum) and absence (SF) of serum. The resulting EBs were evaluated for expression of the indicated genes at the times (days) shown at the top of the panel. (B) FACS analysis of GFP-Bry expression in the serum and serum-free EBs. (C) tet-Hex ES cells were cultured for a total of 10 days. EBs were grown from 0 to 6 days in the presence of serum. The numbers refer to the number of days cultured in the presence of serum. At the indicated time point, the EBs were transferred to serum-free conditions and grown until a total of 6 days of culture. At 6 days of culture, EBs were replated, and neurite development was evaluated at day 10. Cells in each condition were cultured in the absence (-) or presence (+) of Dox (1 μg/mL). Day-6 EBs from each group were replated, and the number of EBs with neurite outgrowths was counted 4 days later. (D) Hematopoietic potential of day-6 EBs generated in the presence or absence of serum. Ep indicates primitive erythroid colonies; MAC, macrophage colonies; MIX, multilineage colonies. Data represent means ± SEMs (n = 3).