DKO and Otx2KO ESCs: transcriptomic analysis

To evaluate the identity of DKO and Otx2KO cells, we compared transcription levels by RNA-seq: we identified DKO and Otx2KO differentially expressed genes shared with those specific for wt ESCs cultured in LIF plus 2i (LIF+2i), or ESCs in LIF plus serum (FBS+LIF), or primed for 44 hours (hrs) in FGF2 plus Activin A to generate early Epiblast-like Stem Cells (EpiSC (44 hrs)). We first challenged wt EpiSCs (44 hrs) vs wt ESCs (LIF+2i) and vs wt ESCs (FBS+LIF) to identify the transcriptome fraction containing up-regulated and down-regulated EpiSC-specific transcripts; then, we challenged DKO ESCs (FBS+LIF) vs wt ESCs (FBS+LIF) and vs wt ESCs (LIF+2i) to identify the DKO ESC transcriptome fraction containing bona fide EpiSC-specific gene transcripts. Comparative Venn diagrams showed that DKO ESCs (FBS+LIF) exhibited 632 up-regulated and 367 down-regulated transcripts in common those specifically expressed in EpiSCs (44 hrs). A similar analysis was performed to identify up- and down-regulated DKO ESC (FBS+LIF) transcripts in common with those specific for wt ESCs (LIF+FBS)or wt ESCs (LIF+2i). This analysis showed that DKO ESCs although cultured in LIF+FBS exhibited a transcriptomic profile with a marked signature in common with wt EpiSCs (44 hrs) and progressively less evident in wt ESCs (FBS+LIF) and wt ESCs (LIF+2i). A similar analysis performed for the transcriptome of Otx2KO ESCs (LIF+FBS), showed that, although its highest similarity was with wt ESCs (FBS+LIF), it also exhibited a relevant number (n=215) of gene transcripts in common with those specifically down-regulated in ESCs (LIF+2i), or suggests that Otx2 is required to activate genes normally repressed by LIF+2i in wt ESCs.