Figure 2.

The Cell Cycle Directs Differentiation of hESCs

(A–C) Cell-cycle-dependent differentiation of hESCs. qPCR analysis for the expression of germ layer markers in FACS sorted Tra-1-60-positive FUCCI-hESCs incubated for 6 hr in culture condition inductive for three germ layers.

(D and E) Cell cycle regulates the timing of differentiation in hESCs. Fucci-hESCs sorted into early G1 phase, late G1, G1/S transition, or S/G2/M phase cells were differentiated into (D) endoderm or (E) neuroectoderm and analyzed for germ layer and pluripotency marker expression at different time points by qPCR.

(F and G) Early G1 phase directs endoderm whereas late G1 promotes neuroectoderm differentiation. Flow cytometry analysis for the expression of germ layer markers in FACS sorted Fucci-hESCs incubated for up to two days in culture condition inductive for (F) endoderm or (G) neuroectoderm differentiation.

(H) Restricted capacity of differentiation during G1 transition. Left: schematic overview of experimental approach. Right: qPCR analysis of neuroectoderm markers in samples (1–4) treated as shown in schematic overview.

(I) Schematic presentation of pancreatic differentiation from sorted or unsorted cells.

(J) Cell-cycle stage of pluripotent cells affects insulin expression during pancreatic differentiation. Immunostaining of insulin during pancreatic differentiation.

(K) Cell-cycle stage affects foregut marker FoxA2 expression. Immunostaining for FoxA2 at day 8 of hepatic differentiation of hESCs sorted in early G1 or late G1 phase.

(L) Cell-cycle stage of pluripotent cells affects pancreatic differentiation. Early G1 phase cells differentiating into endoderm improves pancreatic differentiation, whereas late G1 phase cells reduces pancreatic differentiation.

(M) Cell-cycle stage affects liver differentiation. Expression of liver markers at day 25 of hepatic differentiation shows variability of developmental potential for cells that were used during initial endoderm differentiation. All data are shown as mean ± SD (n = 3). Student’s t test was performed. ^∗p < 0.05. Scale bar represents 100 μm.

See also Figure S1.