MicroRNAs (miRNAs) are approximately 22 nucleotides of small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The miRNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, metabolism, imprinting, and differentiation. Recently, a paper has shown that expression of the miRNA-302/367 cluster expressed abundantly in mouse and human embryonic stem cells (ESCs) can directly reprogram mouse and human somatic cells to induced pluripotent stem cells (iPSCs) efficiently in the absence of any of the four factors, Oct4, Sox2, c-Myc, and Klf4. To apply this efficient method to porcine, we analyzed porcine genomic sequence containing predicted porcine miRNA-302/367 cluster through ENSEMBL database, generated a non-replicative episomal vector system including miRNA-302/367 cluster originated from porcine embryonic fibro-blasts (PEF), and tried to make porcine iPSCs by transfection of the miRNA-302/367 cluster. Colonies expressing EGFP and forming compact shape were found, but they were not established as iPSC lines. Our data in this study show that pig miRNA-302/367 cluster could not satisfy requirement of PEF reprogramming conditions for pluripotency. To make pig iPSC lines by miRNA, further studies on the role of miRNAs in pluripotency and new trials of transfection with conventional reprogramming factors are needed.

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is a “naïve” state, which is characterized by small, round or dome-shaped colony morphologies, LIF and BMP4 signaling pathways and two active X chromosomes in female; mouse ES cells (mESCs) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSCs) or human ES cells (hESCs). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and Nodal/Activin signaling pathways and X chromosome inactivation in female. It has been suggested that, as a non-permissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. 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, LIF, hypoxic conditions and up-regulation 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(PEFs) via doxycycline (dox)-inducible reprogramming factors and LIF. In this study, we have been able to successfully induce PEFs into a LIF-dependent naïve pluripotent-like cell line showing a mESC-like morphology and the expression of pluripotent markers. Our results suggest the possibility of reprogramming to naive pluripotent- like stem cells from PEFs in porcine species. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

XIST has been known to long-non coding RNA which regulate X-chromosome inactivation in female mammal and the gene has been suggested to having important role in early embryo development and embryonic stem cell. However, its coding region has been unclear in pig. To determine the coding region of XIST in pig, we have examined candidate site of XIST coding region in pig by BLAST, PCR, and sequencing. By comparing pig whole genome sequence (Sus scrofa 10.2) with human, murine, and bovine XIST transcript sequence using BLAST, we selected candidate coding region of XIST in pig. The result showed XIST is coded on the minus strand of NW_003612825 contig and its length was nearly 32kb which was similar to the length of human and bovine XIST gene. With the candidate model, we performed RT-PCR to confirm the coding region of XIST with 24 primer pairs and they were expressed only female porcine embryonic fibroblast (PEF) but not in male PEF. By designing candidate intron spaning primer we could confirmed candidate intron is present between first and last exon (distance, 9.2kb vs product size, 2kb). The seqeucne of amplicon was analyzed and we could confirmed there were 5 small exons (less than 400 bp) like XIST coding region of other species which have 4 to 5 small exon between first and last exon. To confirm coding strand in pig, we conducted strand specific reverse transcription. We confirmed candidate XIST was coded on the negative strand of contig on X-chromosome as the result of homology analysis by BLAST. With the candidate pig XIST sequence, we aligned the sequence with XIST sequences of 3 species, human, mouse, and bull by clustalW. These result showed candidate sequence of pig XIST is most similar to that of bovine and the homology between pig and human was higher than result between mouse and human. These results could support for X chromosome inactivation analysis and the function of XIST in pig preimplantation embryos. * This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012006276)

Pluripotent stem cells are cells that have a self-renewal capacity and the ability to differentiate into all lineages. These cells can be divided into naive- and primed-state pluripotent stem cells according to their pluripotent state. Only the naive state comprises a full pluripotency or ground state that contributes to germ-line transmission. Naive states are found in specific permissive strains or species, such as 129, C57BL/6 and BALB/C in mice. However, a number of attempts have been made to derive naive-state pluripotent stem cell lines from non-permissive species, including humans and pigs, using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions and up-regulation of Oct4 or Klf4. Therefore, in this study we investigated whether a naive pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEFs) via previously reported factors. Our mouse embryonic stem cell (mESC)-like cell lines expressed the pluripotency markers Oct4, Sox2 and Nanog and a stable mESC-like morphology for more than 50 passages. In addition, these cell lines could be sequentially reprogrammed into mESC-like induced pluripotent stem (iPS) cells from secondary or tertiary fibroblast-like cells differentiated from mESC-like iPS cells by addition of doxycycline (DOX), LIF and 2i. Our results suggest that, as a non-permissive species, porcine stem cells can be induced into mESC-like iPS cells from PEFs by various exogenous factors, including continuous transgene expression, 2i and LIF. However, further work that aims to effectively induce the activation of endogenous transcription factors is necessary to derive authentic naive-state pluripotent porcine stem cells.