Attempting to Convert Primed Porcine Embryonic Stem Cells into 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 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), or Myc, which are known reprogramming factors, were transduced into pESCs. And pluripotency genes were analyzed in one or two reprogramming factor-expressed pESCs. When cultured without DOX, pESCs were stably maintained in basic fibroblast growth factor-supplemented media. However, when treated with DOX, the cells lost their alkaline phosphatase (AP) activity and differentiated within 2 weeks. Subsequently, we investigated the expression of genes related to pluripotency in DOX-treated pESCs using quantitative reverse transcription-polymerase chain reaction (PCR). Expression levels of Oct4, E-cadherin, and Fut4 were significantly increased by Oct4 overexpression, and Oct4 and Fut4 were upregulated in the Sox2-infected group. When a combination of two reprogramming factors, including Oct4 or Sox2, was introduced, weak AP activity remained. In addition, several of the two reprogramming factor transduction groups could be maintained after subculturing with transgene activation. Although 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 PCR confirmed that the expression of intermediate reprogramming markers was not maintained. Given these results, additional methods are needed to support the completion of each reprogramming phase to succeed in the conversion of the pluripotent state of pESCs. This study improves our understanding of pluripotent networks and can be used to aid in the establishment of bona fide pig pluripotent stem cells.