Effects of IWR-1 on the transcriptome of bovine embryonic stem cells and mouse embryonic fibroblasts

The use of tankyrase inhibitors is essential for capturing livestock embryonic stem cells (ESC), yet their mechanisms of action remain largely uncharacterized. Previous studies indicate that their roles extend beyond the suppression of canonical WNT signaling. This study investigates the effects of the tankyrase inhibitor IWR-1 on the transcriptome of bovine embryonic stem cells (bESC) and mouse embryonic fibroblasts (MEF). We performed RNA-seq analysis on day 1 treated bESC and the co-cultured MEF. Comparing the transcriptomes of cells cultured in complete medium (IWR) and IWR-1-removed medium (IWRR), we identified small fractions of exclusively expressed genes in both bESC and MEF. Regulators of HIPPO pathways TEAD4, AMOTL2, and AJUBA and HIPPO targeted gene CYR61 were differentially expressed in bESC. We also found that IWR-1 altered expression of 8 genes in MEF associated with signaling pathways activated by TGF-ß ligands, including Gdf6, Inhbb, Bmp4, Fst, Inhba, Thbs1, Tgfb1, and Bmp2. Overall design: Five bESC, bESC1, bESC2, bESC4, bESC8, and bESC11, were derived from day 7.5 blastocysts and maintained on MEF. Cells were cultured with a medium supplemented with IWR-1. Each line of bESC were passaged into 2 wells in 6-well plates. On the day 0 of the experiment, culture medium was removed and rinsed with DPBS 3 times. Then, each line of bESC were treated with either the complete medium that supplemented with IWR-1 (IWR) or the IWR-1 removed medium (IWRR). The co-cultured bESC and MEF in each well were collected for RNA-seq on day 1.