Figure 4. Medium conditioned by hES cells prevents lengthening of the G1 phase.

(A) Experimental design. (B) Human ES cells were plated without feeder layer at high density (3:1) (first row) and low density (1:3) (second and third rows), and maintained with differentiation media for 72 h. Media changes were applied every 12 h. Control cells grown in high density (first row) or low density (second row) received fresh media, but cells in grown in low density (1:3) (third row) received the conditioned medium (CM) supernatant of cells grown in high density (3:1, first row) every 12 h. After 72 h, cultures were synchronized for an additional 16 h using nocodazole. Upon release from mitotic block, BrdU incorporation was determined at 0 h, 2 h and 4 h to determine the duration of G1 and the onset of S phase (scale bar = 100 μm).