In transgenic pig production for generating animal models of human diseases, apoptosis of early implantation embryo disturbs the transgenic pig production. In general, epithelial-mesenchymal transition (EMT) is considered important in embryo development and apoptosis. In addition, it was reported that 17β-oestradiol (E2), among hormones that participate in early implantation of embryo, could induce EMT and neural differentiation in mouse embryonic stem cells. Therefore, in this study, we examined the effects of the steroid hormone, E2, in the changes of EMT and apoptotic markers in porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC). During the study, we cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (b-FGF) and leukemia inhibitory factor (LIF) and performed RT-PCR and an alkaline phophatase (AP) test to measure pluripotent and undifferentiation markers of these porcine stem cells. The RT-PCR results showed that OCT4, NANOG, and SOX2 were expressed in these pESC and piPSC, indicating their pluripotency as stem cells. Also, these porcine stem cells showed positive AP activity, demonstrating undifferentiation. Additionally, we treated pESC and piPSC with E2 to examine effects of steroid hormone on the changes of EMT and apoptotic markers (i.e. bcl-2, bax, E-cad, and vimentin). The E2 treatment increased the expression of vimentin and bcl-2, while decreased the expression of E-cadherin and bax. By using immunocytochemistry (ICC), we examined the protein expression of EMT markers, which are vimentin and E-cadherin at the translational level, and found that expression of vimentin protein was increased while E-cadherin protein level was reduced at periphery of the colonies in pESC and piPSC. In conclusion, these results indicate that E2 can promote EMT process and reverse apoptosis in these pESC and iPSC. In a future study, we will further examine the effects of progesterone on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate the underlying mechanisms of EMT and apoptosis controlled by steroid hormones in porcine stem cells.

In transgenic pig production for generating animal models of human diseases, apoptosis of an early implantation embryo disturbs the transgenic pig production. Porcine embryonic stem cells (pESC) and porcine induced pluripotent stem cells (piPSC) have an advantage for the generation of transgenic pigs; however, porcine stem cells have not yet been established. In addition, epithelial–mesenchymal transition (EMT) may play a critical role in embryo development and apoptosis. Thus, in this study we generated pESC and pIPSC and further examined the changes in EMT and apoptotic markers. We cultured pESC and piPSC in pESC media containing basic fibroblast growth factor (bFGF), doxicyclin, and leukemia inhibitory factor (LIF), and performed RT-PCR and alkaline phosphatase (AP) test to measure pluripotency markers. The RT-PCR results show that OCT-4, NANOG, and SOX2 were expressed in these stem cells, characteristic of stem cells. AP-positive cells were observed in pESCs and piPSC. In addition, we performed immunocytochemistry (ICC) to examine the expression of surface markers, such as SSEA-1 and SSEA-4. We found that pESC and piPSC expressed these markers, indicating that they have a stem cell property similar to rodent and human stem cells. Second, we treated pESC and piPSC with transforming growth factor beta (TGF-β) to examine the relationship between EMT and apoptotic markers, and confirmed a significant variation of EMT and apoptotic markers, i.e. bcl-2, bax, E-cadherin, and vimentin, by Western blot analysis. In a future study, we will examine the effect(s) of various endocrine hormones secreted by the ovary, such as E2 or P4, on the expressions of EMT and apoptotic markers in pESC and piPSC. Consequently, this study will contribute to elucidate underlying mechanism(s) of EMT and apoptosis by endocrine factors to prevent early apoptosis of pig embryos in these porcine stem cells. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009599), Rural Development Administration, Republic of Korea.