As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.

Stem cells are progenitor cells that are capable of self-renewal and differentiation into various cells. Especially, pluripotent stem cells (PSCs) have in vivo and in vitro differentiation capacity into three germ layers and can proliferate infinitely. The differentiation ability of PSCs can be applied for regenerative medicine and tissue engineering. In domestic animals, their PSCs have a potential for preclinical therapy as well as the production of transgenic animals and agricultural usage such as cultured meat. Among several domestic animals, a pig is considered as an ideal model for biomedical and agricultural purposes mentioned above. In this reason, studies for pig PSCs including embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs) have been conducted for decades. Therefore, this review will discuss the history of PSCs derived from various origins and recent progress in pig PSC research field.

Because of the physiological and immunological similarities between pigs and humans, porcine embryonic stem cells (ESCs) have been identified as important candidates in preliminary studies on human disease. A comparative understanding of pig ESCs with the human is required to achieve these goals. To gain insights into pig stem cells, the transcriptome of pig ES-like cells were compared with pig preimplantation embryos and human/mouse pluripotent stem cells by RNA-seq analysis. As a result, pig stem cells were more similar to late epiblasts of pig preimplantation embryos than early ICM as revealed by transcriptome analysis, suggesting that pig stem cells are in a developmentally primed state. Moreover, the physiological and biological functions of pig ESCs were more similar to those of human PSCs than to those of mouse PSCs, as determined by direct differentiation and GO/KEGG term analysis. Overall, our data indicate that pig ESCs are in a primed pluripotent state resembling human PSCs. Our findings will facilitate both the development of large animal models for human stem cell therapy and the generation of pluripotent stem cells from other domestic animals for agricultural use. This work was supported by the Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry, and fisheries (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-1-HD020), and partially supported by the grants from the Agenda Program of Rural Development Administration, Republic of Korea (No. PJ01362402)

Because the pig is a valuable candidate for a preclinical model of human cell therapy as well as an important food source, understanding a physiology of pig myogenic progenitors such as skeletal muscle satellite cells and myoblasts is required for cure of muscular diseases and improvement of meat production. For these reasons, we tried to isolate and culture the pig progenitor cells from skeletal muscle. Pig satellite cells, known as muscle stem cells, were isolated from biceps femoris of neonatal pigs by enzymatic digestion method. Muscle satellite cells are quiescent in uninjured muscle. Upon injury, they are activated into proliferating state, known as myoblasts, by growth factors and, in turn, differentiated forward to myocytes and myotubes. To trigger proliferation in vitro, the isolated satellite cells were cultured with epidermal growth factor (EGF) and dexamethasone (BMP4 inhibitor). As a result, the pig satellite cells were efficiently converted into proliferating myoblasts and stably maintained over an extended period. The myoblasts were confirmed by markers of PAX7, MYF5, and MYOD1. Our finding showed that modulating EGF and BMP4 signaling are essential for maintaining muscle stem cells. This culture method could be applied for a production of cultured meat and further basic research of muscle development. This work was supported by the Korea Institute of Planning and Evaluation for Technology in food, agriculture, forestry, and fisheries (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-1-HD020).

The transcription factor POU5F1, also known as OCT4 plays critical roles in maintaining pluripotency during early mammalian embryonic development and in embryonic stem cells. It is important to establish an OCT4 promoter region-based reporter system to study pluripotency. However, there is still a lack of information about the porcine OCT4 upstream region. To improve our understanding of the porcine OCT4 regulatory region, we identified conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. The similarity of nucleotide sequences in the 5' upstream region was low among mammalian species. However, the OCT4 promoter and four regulatory regions, including distal and proximal enhancer elements, had high similarity. The putative transcription factor binding sites in the Oct4 5' upstream region nucleotide sequences from mice and pigs also differed. Some of these genes are related to pluripotency, and further research will allow us to better understand the differences in species-specific pluripotency. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay results indicated that the porcine OCT4 distal enhancer and proximal enhancer were highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively. Similar to OCT4 upstream-based reporter systems derived from other species, the porcine OCT4 upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work provides basic information about the porcine OCT4 upstream region and various porcine OCT4 fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and the establishment of embryonic stem cells in pigs. This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2017R1D1A1B03032256).

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.

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.

Several studies have been conducted with the aim of establishing embryonic stem cell lines from porcine embryos. However, most researchers to date have found it difficult to maintain an ES-like state in derived cell lines, with the cells showing a strong tendency to differentiate into an epithelial or EpiSC-like state. We have also been able to derive cell lines of an EpiSC-like state and a differentiated non-ES-like state from porcine embryos of various origins, including invitro fertilized(IVF), in vivo derived, IVF aggregated and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells(piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) into porcine fibroblast cells. X chromosome inactivation (XCI) have recently been addressed as a hallmark to determine whether pluripotent cell is naïve or primed state. In this study, we could confirm the X chromosome inactivation status in female cell lines as well as marker expression, pluripotency and of our Epi- SC-like pESC lines along with our piPSC line. All of our cell lines showed AP activity and expressions of the genes Oct4, Sox2, Nanog, Rex, TDGF1, bFGF, FGFR1, FGFR2, Nodal and Activin-A involved in pluripotency and signaling pathways, XCI in female cell lines, in vitro differentiation potential and a normal karyotype, thus displaying similarities to epiblast stem cells or hES cells. Therefore, it may be inferred that, as a non-permissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines.

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 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.