It is still challenging to establish pESCs due to differences in the genetic backgrounds of mouse, human, and pig. So it is required to find pig specific pluripotency markers and cellular signaling. In this experiments, doxycycline-inducible vectors carrying OCT4, SOX2, NANOG, KLF4 and MYC known as reprogramming factors, were infected into pig stem cells for analyzing gene expression pattern. When cultured without doxycycline, pig stem cells were stably maintained in bFGF supplemented media. However, when treated with doxycycline, pig stem cells lost alkaline phosphatase activity and were differentiated within two weeks. And then, we investigated the expression of genes related to pluripotency in doxycycline-treated pig stem cells by using qRT-PCR. The qRT-PCR data revealed that expression of OCT4, CDH1 and FUT4 were significantly increased by OCT4 overexpression and OCT4 and FUT4 were also upregulated in SOX2-infected group. When infected with combination of two factors including OCT4 or SOX2, some groups could stably maintain at LIF supplemented media, having alkaline phosphatase activity. Given these data, although ectopic gene expression induced differentiation in pig stem cells, ectopic expression of OCT4 and SOX2 could upregulate pluripotent genes and overexpreession of two factors help pig stem cells adapt LIF-contained media. This study could improve understanding of pluripotent networks as well as aid in establishing bona fide pluripotent stem cells in pig.

MicroRNAs are ~22nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. Micro RNAs are phylogenetically conserved and have been shown to be instrumental in a wide variety of key biological processes including cell cycle regulation, apoptosis, control of metabolic pathways, imprinting and differentiation. The expression of miRNAs is often regulated in tissue specific and developmental stage‐specific manners. More than 500 miRNAs have been reported in diverse eukaryotic organism so far. One of the biological functions of miRNAs seems to be the regulation of self‐renewal versus differentiation in stem cells. Recent efforts have focused on defining the miRNA expression profile in undifferentiated ESCs as compared to their differentiated progeny. Among the so‐called ES‐specific miRNAs, the 302‐367 cluster stands out due to its intracellular abundance and high cell type specificity. Levels of miRNA 302‐367 correlate with Oct4 transcripts in ESCs and early embryonic development, indicating an important role in ESC homeostasis and maintenance of pluripotency. Several months ago, a paper showed that expression of the miRNA 302‐367 cluster can directly reprogram mouse and human somatic cell to an iPS cell in absence of any of the four factors (Oct4, Sox2, c‐Myc, Klf4) efficiently. To apply this efficient method to porcine, we made an inducible vector system including miRNA 302‐367 cluster originated from porcine embryonic fibroblasts and could make porcine ips by the miRNA 302‐367 cluster.

The four transcription factors Oct4, Sox2, Klf4 and c-Myc have been used for making induced pluripotent stem cells. Many efforts have focused on reducing the number of transcription factors, especially c-Myc and Klf4 known as oncogene, for making induced pluripotent stem cells. Recently it have been demonstrated that Oct4 and Sox2 are able to reprogram human fibroblasts or cord blood cells to induced pluripotent stem cells and Oct4 has the ability to reprogram mouse and human neural stem cell to induced pluripotent stem cells. These researches imply cell types for reprogramming experiments have great influence on selection of reprogramming factors. Here we report that pig kidney cortex fibroblasts need only c-Myc factor when they are used for making induced pluripotent stem cells. We used two vector system including drug-inducible vector system and constitutive expression vector system. The two systems generate induced pluripotent stem cells from pig kidney fibroblasts successfully. These one-factor induced pluripotent stem cells are not only similar but also different to pig embryonic stem-like cells. These two one-factor induced pluripotent stem cell lines can express pluripotency related genes and be differentiated into all three germ layers in vitro. However, these two cell lines can be sub-cultured as a single cell by trypsin. Our results support that single factor, c-Myc, is sufficient to converting pig kidney cortex fibroblasts into induced pluripotent stem cells.

Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing LIF and bFGF. After establishment, this cells were cultured and stabilized in LIF or bFGF contained media. This cell lines displayed a dome-shaped colony morphology in both culture condition. The cell lines were maintained in both condition over an extended time period and were able to differentiate into the three germ layers in vitro. Interestingly, cell lines cultured in LIF or bFGF expressed different pluripotency markers. LIF-dependent pEGCs expressed naive-pluripotency markers such as OCT4, SOX2, NANOG and SSEA1, while bFGF-dependent pEGCs expressed primed-pluripotency markers such as OCT4, SOX2, NANOG and SSEA4. However, as a result of analysis of XCI, two cell lines showed hemi-methylated pattern similarly in XIST promoter regions. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Pluripotent state of pEGCs were regulated by modulation of culture condition. In LIF supplement, pEGCs showed naive-pluripotency expressing SSEA1, while pEGCs show primed-pluripotency expressing SSEA4 in bFGF condition. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.

Pluripotent cells are categorized as either "naive" or "primed" based upon their pluripotent status. According to previous studies, embryonic stem cells and embryonic germ cells are identified as naive pluripotent stem cells and epiblast stem cells are identified as primed pluripotent stem cells. In a permissive species such as the mouse, naive and primed pluripotent stem cells can be derived from embryos without genetic manipulations. In non-permissive species such as humans and pigs, primed pluripotent cells are only established from embryos. However, previous studies have shown that the embryonic germ cells of non-permissive species share similar morphology and features with naive pluripotent cells. For these reasons porcine embryonic germ cells (pEGCs) may provide a useful cell source for comparative studies on naive pluripotent cells in non-permissive species. In this study, we attempted to establish and characterize porcine embryonic germ cells. Consequently, an embryonic germ cell line was derived from the genital ridges of a porcine dpc 30 fetus in media containing bFGF. This cell line displayed a dome-shaped colony morphology. The cell line was maintained in a stable condition over an extended time period and was able to differentiate into the three germ layers in vitro. Pluripotency markers such as OCT4, SOX2, NANOG and SSEA4 were expressed in these pEGCs. Similar with pESCs, Mek/Erk signaling pathway were activated by bFGF in the cultured pEGCs. In conclusion, we were able to successfully derive embryonic germ cells from genital ridges of a porcine fetus. Unlikely naive pluripotent cells such as mESCs, pluripotency of pEGCs were regulated by Mek/Erk signaling pathway activated by bFGF. This cell line could potentially be used as naive pluripotent cell source for comparative study with porcine embryonic stem cells and other pluripotent cell lines. As porcine pluripotent cells, pEGCs could be useful candidates for preliminary studies of human disease as well as a source for generating transgenic animals.