Figure 4. Early differentiation of ES cells is inhibited by ectopic expression of p18.

(A, B) Comparison of the undifferentiated colonies presented in WT and p18-overexpression B6 ES cells analyzed by AP staining. Experiments were performed in triplicate in the presence or absence of leukemia inhibitory factor (LIF) in transduced and non-tranduced ES cells (B6 background). (A) The left panel (+LIF) and right panel (-LIF) represent bright field images of differentiated ES cells generated by cultivating ES cells in the presence or absence of LIF for 5 d. (B) Representative images of the differentiation morphology associated with transduced, and non-transduced, ES cells (B6 background). (C) Comparison of embroid body formation at day 5 after equal numbers of ES cells were plated at day 0 for all of the groups indicated. A mean diameter for these EB was determined from 4 measurements. Data represent the mean ± SD. (D) Representative bright field images, as well as fluorescence images, of EB grown in the presence of LIF for 5 d (E, F and G). Total RNA was extracted from EB at day 0, 3, 5, and 10. Expression of p18, Oct4, Nanog, Sall4, Gata6, Map2, Cdx2, and BRACHYURY were analyzed using real-time RT-PCR. Data were analyzed according to the ΔC_T method. All values were normalized to β-actin and expressed relative to WT levels. Values are expressed as the mean ± SD. In A to G, data represent three independent experiments with similar results.