294 NAÏVE STATE-LIKE PLURIPOTENT STEM CELL LINES DERIVED FROM PORCINE EMBRYONIC FIBROBLASTS

Abstract

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is the “naïve” state, which is characterised by small, round or dome-shaped colony morphologies, LIF and BMP4 signalling pathways, and 2 active X chromosomes in females; mouse embryonic stem cells (mESC) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSC) or human embryonic stem cells (hESC). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and nodal/activin signalling pathways, and X chromosome inactivation in females. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions, and upregulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEF) via various previously reported factors. We were able to successfully induce PEF into a naïve state-like pluripotent stem cell line by viral infection using FUW-tetO-hOCT4, FUW-tetO-hSOX2, FUW-tetO-hKlf4, FUW-tetO-hMYC, and FUW-M2rtTA obtained from Addgene and addition of 2i and LIF. These naive state-like pluripotent stem cells display mESC-like morphologies, clonogenicity by trypsin, and expression of Oct4, Sox2, Nanog, and SSEA1 using PCR, immunocytochemistry, and fluorescence-activated cell sorting. All cell lines maintained stemness characteristics and stable morphology for more than 30 passages. In addition, naïve state-like pluripotent stem cells could be induced to differentiate to fibroblast-like cells by withdrawal of doxycycline, lif, and 2i. These differentiated cells could be regenerated into naïve state-like pluripotent stem cells by addition of doxycycline, lif, and 2i. We suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines and needs various exogenous factors, including continuous transgene expression, GSK3β and MEK inhibitors (2i), and LIF to be induced into naïve state-like pluripotent stem cells.

This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.