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.

To have a better understanding of pluripotency, whole gene expression of embryo-derived stem cells (EdSCs) in bovine species was investigated. EdSCs were established from the embryos produced by in vitro fertilization, parthenogenesis and somatic cell nuclear transfer. Then, the microarray was performed and analyzed. Differently expressed genes (DEGs) were also confirmed by Real-time PCR. Among 10,203 DEGs, little difference was found in gene expression among three kinds of EdSCs. Conversely, all EdSCs have an immensely different gene expression when compared with somatic cells, consistent with scatter plat results. To investigate shared pathways for pluripotency in all EdSCs, 2,415 co-DEGs were identified which compared with somatic cells. By KEGG database, there were 54 signaling pathways in co-DEGs and some of them were related with pluripotency maintenance such as TGFβ, WNT and JAK-STAT signaling. In TGFβ signaling, BMP family and SMAD family were involved in co-up-regulated DEGs. In WNT signaling, WNT family and receptors were included in co-up-regulated DEGs, while inhibitors of WNT signaling were associated with co-down-regulated DEGs. In JAK-STAT signaling, STAT3 belonged to co-down-regulated DEGs. These DEGs were also confirmed by Real-time PCR. Taken together, BMP and WNT pathways may be activated and paly central roles to retain pluripotency in bovine EdSCs, whereas the LIF/STAT3 pathway may not be operated well. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and No. 2015048003 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.