days at stage 3 (days 24–60). Cells at 60 days after differentiation were transferred onto Matrigel (1:60 dilution)-coated 10-cm dishes and cultured in DMEM/Ham's F12 supplemented with N2 supplement (Life Technologies) and refreshed every 3 days at stage 4 (days 60–120). Cells at 120 days after differentiation were passaged on non-coated polystyrene 10-cm dishes for astrocyte culture, because neurons weakly attach to non-coated polystyrene 10-cm dishes whereas astrocytes attach strongly and proliferate. By repeated passage in the same manner at days 140, 160, and 180, astrocytes increased their own abundance ratio and showed positive GFAP immunostaining (more than 80%) at stage 5 (days 120–180).
