• Development and Reproduction
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• v.19 no.3
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• pp.119-126
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• 2015

The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF ($72.8{\pm}7.69$ and $81.2{\pm}3.56$) than D3/STO ($32.0{\pm}4.30$ and $56.0{\pm}4.90$) or D3/- ($55.0{\pm}4.64$ and $62.0{\pm}6.20$). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.4
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• pp.275-290
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• 2023

Background: Porcine pluripotent stem cells (pPSCs) would provide enormous potential for agriculture and biomedicine. However, authentic pPSCs have not established yet because standards for pPSCs-specific markers and culture conditions are not clear. Therefore, the present study reports comparative pluripotency characteristics in porcine induced pluripotent stem cells (piPSCs) derived from different viral transduction and reprogramming factors [Lenti-iPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM)]. Methods: Porcine fibroblasts were induced into Lenti-iPSCs (OSKM) and Lenti-iPSCs (OSKMNL) by using Lentiviral vector and Sev-iPSCs (OSKM) by using Sendaiviral vector. Expressions of endogenous or exogenous pluripotency-associated genes, surface marker and in vitro differentiation in between Lenti-piPSCs (OSKM), Lenti-iPSCs (OSKMNL) and Sev-piPSCs (OSKM) were compared. Results: Colonial morphology of Lenti-iPSCs (OSKMNL) closely resembles the naïve mouse embryonic stem cells colony for culture, whereas Sev-iPSCs (OSKM) colony is similar to the primed hESCs. Also, the activity of AP shows a distinct different in piPSCs (AP-positive (+) Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but AP-negative (-) Lenti-iPSCs (OSKM)). mRNAs expression of several marker genes (OCT-3/4, NANOG and SOX2) for pluripotency was increased in Lenti-iPSCs (OSKMNL) and Sev-iPSCs (OSKM), but Sev-iPSCs (OSKM). Interestingly, SSEA-1 of surface markers was expressed only in Sev-iPSCs (OSKM), whereas SSEA-4, Tra-1-60 and Tra-1-81 were positively expressed in Lenti-iPSCs (OSKMNL). Exogenous reprogramming factors continuously expressed in Lenti-iPSCs (OSKMNL) for passage 20, whereas Sev-iPSCs (OSKM) did not express any exogenous transcription factors. Finally, only Lenti-iPSCs (OSKMNL) express the three germ layers and primordial germ cells markers in aggregated EBs. Conclusions: These results indicate that the viral transduction system of reprograming factors into porcine differentiated cells display different pluripotency characteristics in piPSCs.

• Animal Bioscience
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• v.36 no.12
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• pp.1905-1917
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• 2023

Objective: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. Methods: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. Results: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. Conclusion: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

• International Journal of Oral Biology
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• v.35 no.4
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• pp.209-214
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• 2010

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.109-120
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• 2023

Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.91-95
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• 2008

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and $\beta$-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.65-70
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• 2004

In mammals, male and female germline stem cells are derived from primodial germ cells. Despite many efforts to identify stem cells from gonads, there has been little successe to identify germline stem cells yet. In this study, we isolate and characterized porcine germline stem cells using only stem cell markers that are prevalently expressed in various tissues. Gonadal cells derived from both male and female formed colonies and showed AP activities and different lectin binding properties. Pluripotency of germline stem cells was also identified by positive signals against putative stem cells markers such as SSEA-1 and SSEA-3. In addition, nestin was also found in primary gonad cells that have a similar morphology to the AP-positive cells. The nestin expression suggests that the germline stem cells may have similar expression of the prevalent stem cell markers found in other tissues. The demonstration of nestin expression together with pluripotent cell markers calls further investigation of the possible differentiation of nestin-positive cells into neurons.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.215-220
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• 2007

This study was conducted to examine the establishment of bovine ES-like cells having pluripotency. The hatched blastocysts derived from culture of in vitro fertilized embryos for 10 to 12 days dissociated mechanically into ICM-and trophectoderm-rich clumps using needle, and cultured onto mitotically-inactivated MEF feeder layer. The primary colonies originated from ICM cells were detached mechanically 7 days after seeding and subsequent subculture was conducted at intervals of every 5 to 7 days. Two ES -like cell lines were established and maintained over 40 passages. Self-renewal of the established lines was confirmed by examining the alkaline phosphatase activity, stem cell-specific marker profiles including SSEA isotopes, Oct-4 and STAT3. Moreover, the established cell lines could produce anchorage-independent embryoid bodies (EBs) with gradual decrease of Oct-4 transcript level in time-dependent manner.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.143-148
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• 2013

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-$34^{TM}$ cell culture media at $37^{\circ}C$. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin ${\alpha}6$ and ${\beta}1$, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.