Figure 4.

Gene Expression, Cell Signaling, and Morphological Differences of Early Differentiating EpiLCs

(A) Representative immunostainings comparing mESCs cultured in 2i + LIF and after 1 h of induction to EpiLCs (1 h EpiLC Diff). Cells were evaluated for ERK1/2 phosphorylation and for the re-organization of the actin cytoskeleton (phalloidin). Nuclei were stained with DAPI. Scale bars, 50 um.

(B) qRT-PCR analysis of primed and naive pluripotency markers in 2i + LIF or after 1, 2, 24, and 48 h of EpiLC induction. Results are presented displaying the log2 transformed values of the three independent biological replicates relative to time 0 h (2i + LIF), to clearly show the high reproducibility of the results. Letters indicate significant differences between groups (p < 0.05) by randomized block design ANOVA.

(C) Morphological analysis of cell colonies grown in 2i + LIF or after 1 h induction to EpiLCs. Colonies were automatically detected from 20 wide-field images (see Experimental Procedures) and morphological properties were recorded and analyzed. Left images display representative colony segmentations for 2i + LIF and 1 h EpiLCs Diff. Scale bars, 100 μm. Right charts display the distribution density for the indicated morphological variables comparing 2i + LIF and 1 h EpiLC Diff (n = 326 and 291 colonies, respectively). Statistical differences were evaluated using the Kolmogorov-Smirnov and the Mann-Whitney U test. ^∗p values < 0.05 were considered statistically significant.