Conversion of primed porcine embryonic stem cells to a naive state through the overexpression of reprogramming factors

Abstract

Establishing pig embryonic stem cells (pESCs) remains a challenge due to differences in the genetic backgrounds of mouse, human, and pig. Therefore, pig-specific pluripotency markers and their cellular signaling must be identified. In this study, doxycycline (DOX)-inducible vectors carrying Oct4, sex determining region Y-box 2 (Sox2), Nanog, Kruppel-like family 4 (Klf4), and Myc, which are known reprogramming factors, were transduced into pESCs to analyze the pluripotent gene network. Pig ESCs were stably maintained in basic fibroblast growth factor (bFGF)-supplemented media, when cultured without DOX. However, when treated with DOX, the cells lost their alkaline phosphatase activity and differentiated within two weeks. Subsequently, we investigated the expression of genes related to pluripotency in DOX-treated pESCs using quantitative reverse transcription PCR. Expression levels of Oct4, E-cadherin, and Fut4 were significantly increased by Oct4 overexpression, and Fut4 were upregulated in the Sox2 overexpressed group. When a combination of two reprogramming factors, Oct4 or Sox2 was introduced, weak alkaline phosphatase activity remained. In addition, Oct4 and Nanog, Oct4 and Klf4, or Sox2 and Nanog combinations transduction groups could be maintained after subculturing with transgene activation. Although a long-term culture failed, pESCs transduced with Oct4 and Nanog, Oct4 and Klf4, or Sox2 and Nanog combinations could be subcultured even under transgene activation conditions. Analysis of the cause of long-term culture failure by quantitative real-time PCR (qPCR) confirmed that the expression of intermediate reprogramming markers such as Lin28 and Sall4 was not maintained. Given these results, additional strategies are needed to support the completion of each reprogramming phase to succeed in the conversion of the pluripotent state of pESCs. The present study improve our understanding of pluripotent networks and could be used to aid in the establishment of bona fide pig pluripotent stem cells.