Figure 6. Human ES cells have the intrinsic ability to enter two consecutive S phases.

(A) Experimental design. (B) Human ES cells were synchronized using nocodazole and mitotically blocked colonies were separated from the inactivated MEF feeder layer by gentle mechanical disruption and dispase treatment. Colonies were then seeded on plastic coverslips (i.e., without feeder layer) and cultures were supplemented under different culture conditions: regular human ES cell media (Medium 1) (first row), medium in which FGF2 is omitted (Medium 2) (second row) and medium consisting of DMEM/F12 only (Medium 3) (third row). After adherence (< 1 h), BrdU incorporation was determined for multiple time-points at increasing temporal intervals as indicated (columns) to cover cell cycle progression through two consecutive cell cycles (up to 24 h). In parallel, we treated a control group of cells at18 h after mitotic release (i.e., after one cell cycle) with aphidicolin to prevent the initiation of a second S phase (24 h Control) (scale bar = 200 μm). The bottom panel shows a line graph that quantifies BrdU incorporation as a function of time after mitotic release and reflects the oscillating behavior expected from progression into two consecutive S phases. (C) Shows the quantification of results depicted in (B); the error-bars represent SEM for four independent determinations of 400 each (n=1600).