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.
Lens epithelial cells-induced pluripotent stem cells as a model to study epithelial-mesenchymal transition during posterior capsular opacification
