Prolonged culture of ES-D3 cells in the absence of LIF leads to an enrichment of epithelial self-renewing cells with an ES cell-like transcription profile. A) ES-D3 cells were cultured as indicated in Figure 2 and total RNA samples were collected at indicated time point. The mRNA expression levels of self-renewal markers (Nanog, Sox2, and Oct3/4), ES cell markers (Rex1, Klf4 and Tbx3) and a differentiation marker (FGF5) were determined using qPCR analysis. The data show means +/−STD of replicates from 2–3 independent samples. P-values (student’s t-test) < 0.005 are marked with an asterisk (*). B) ES-D3 cells were grown as above and lysed at indicated time points followed by SDS-PAGE and western blot analysis using Nanog, Sox2, Oct3/4 and Stella antibodies to study the protein expression levels of these stem cell markers. β-actin was used as a loading control. The data shown is representative of 2–3 experiments with similar results.