• Korean Journal of Clinical Oncology
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• v.14 no.2
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• pp.76-82
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• 2018

Purpose: Lgr5 is a well-known stem cell marker in colorectal cancer (CRC). This retrospective study evaluated the expressions of Lgr5 in CRC specimens, and examined whether these expressions were associated with survival outcomes. Methods: We used immunohistochemistry to retrospectively examine expressions of Lgr5 in paraffin-embedded specimens from 337 patients with CRC between January 2009 and December 2013. All clinicopathologic data were collected by retrospective review based on medical records. The correlation between its expression and clinicopathological data as well as clinical outcomes of patients was analyzed. Results: Low expression and high expression of Lgr5 in 337 patients were 175 (51.9%) and 162 (48.1%), respectively. There was no statistically significant difference in the association of Lgr5 expression with clinicopathologic factors (age, tumor location, lymphatic invasion, vascular invasion, perineural invasion, TNM stage, and differentiation). In the survival analysis, the high expression group of Lgr5 showed a better prognosis than the low expression group in disease-free survival (P=0.044). However, overall survival was not significantly different between the two groups (P=0.087). In multivariate analysis, we found that high expression of Lgr5 was independent prognostic factor for tumor relapse (hazard ratio, 0.601; 95% confidence interval, 0.388-0.929; P=0.022). Conclusion: In present study, high expression of Lgr5 is an independent predictor of favorable prognosis in patients with CRC. So, further well designed, prospective, large scale studies are needed to examine the value of Lgr5 as a prognostic biomarker for CRC.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.193-200
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• 2017

The purpose of this study is to confirm whether spontaneous adipocyte generation during chondrogenic induction culture affects the chondrogenic differentiation of porcine skin-derived stem cells (pSSCs). For this purpose, chondrogenic differentiation characteristics and specific marker gene expression were analyzed using cell lines showing different characteristics of spontaneous adipocyte formation. Of the four different lines of pSSCs, the pSSCs-IV line showed higher Oil red O (ORO) and glycosaminoglycan (GAG) extraction levels. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the levels of adipogenic markers peroxisome proliferator-activated receptor gamma 2 ($PPAR{\gamma}2$) and adipocyte Protein 2 (aP2) mRNAs were significantly higher in pSSCs-IV than those of the other pSSC lines (P<0.05). Among three chondrogenic markers, collagen type II (Col II) and sex determining region Y-box (Sox9) mRNAs were strongly expressed in pSSCs-IV (P<0.05), but not in aggrecan (Agg), which was significantly higher in pSSCs-II (P<0.05). These results demonstrate that the spontaneous adipocyte generation during chondrogenic differentiation has a positive effect on the chondrogenesis of pSSCs. More research is needed on the correlation between adipocyte generation and cartilage formation.

• BMB Reports
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• v.53 no.12
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• pp.622-627
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• 2020

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166+ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166+ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anticancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.39-48
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• 2008

Objective: The aim of this study was to investigate whether multipotent germline stem cells (MGSCs) can be established from neonatal mouse testis. Methods: Various cells containing MGSCs were collected from neonatal testis of ICR mice and allocated to plates for in vitro culture. After 7 days in culture, the cells were passed to a fresh culture plate and continuously cultured. From the third or fourth passage, the presumed MGSCs were cultured and maintained on mitomycin C-inactivated STO feeder cells. The MGSCs were cultured in a condition where mouse embryonic stem cells (ESCs) are cultured. Characteristics of the MGSCs were evaluated by RT-PCR, immunocytochemistry, alkaline phosphatase activity, karyotyping, and transmission electron microscopy. Results: Two MGSCs lines were established from 9 pooled sets of neonatal testicular cells. MGSCs colonies were morphologically undistinguishable from ESCs colonies and both MGSC lines as well as ESCs expressed undifferentiated stem cell markers, such as Thy-1, Oct-4, Nanog, Sox2 and alkaline phosphatase. Fine structure of undifferentiated MGSCs were similar to those of ESCs and 60% of MGSCs (12/20) had normal karyotype at passage 10. They were able to form embryoid bodies (EBs) and MGSC-derived EBs expressed marker genes of three germ layers. Conclusion: We could establish the MGSCs from neonatal mouse testis and they were differentiated to multipotent lineages of three germ layers. Molecular characteristics of MGSCs were similar to those of ESCs. Our results suggest a possibility that multipotent stem cells derived from testis, the MGSCs, could replace the ESCs in biotechnology and regenerative medicine.

• Journal of Korean Ophthalmic Optics Society
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• v.18 no.4
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• pp.541-548
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• 2013

Purpose: Marrow stromal cells (MSCs) have been known for their potential to trans-differentiate into neural and glial cells in vitro and in vivo. To investigate the influence of the developing host environment on the survival and morphological and molecular differentiation, murine MSCs transplanted into the eye of Brazilian opossum (Monodelphis domestica). Methods: Enhanced green fluorescent protein (GFP) - expressing MSCs were transplanted into developing Brazilian opossums. Animals were allowed to survive for up to 4 weeks after transplantation, at which time the eyes were prepared for immunohistochemical analysis. Results: Some transplanted MSCs survived and showed morphological differentiation into neural cells with some processes within the host vitreous chamber. Some transplanted cells expressed class III ${\beta}$-tubulin (TuJ1, a marker for neuronal cells) or glial fibrillary acid protein (GFAP, a marker for glial cells) or Nestin (a marker for neural stem cells). In addition, some transplanted cells were located in ganglion cell layer but did not show morphological and molecular differentiation. Conclusions: Our result show that the most effective stage of development for transplantation into the retina was postnatal day 16, which retinas developmentally corresponded to postnatal day 4-5 days mouse retina based on cell differentiation and lamination patterns. The present findings suggest that the age of the host appears to play a key role in determining cell fate in vivo.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.209-215
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• 2004

Objective: Embryonic stem (ES) cells could be differentiated into the specific cell types by alternation of culture condition and modification of gene expression. This study was performed to evaluate the differentiation protocol for mouse and human ES cells to insulin secreting cells. Methods: Undifferentiated mouse (JH-I) and human (Miz-hESI) ES cells were cultured on STO feeder layer, and embryoid bodies (EBs) were formed by suspension culture. For the differentiation, EBs were cultured by sequential system with three stage protocol. The differentiating ES cells were collected and marker gene expressions were analyzed by seIni-quantitative RT-PCR in each stage. Amount of secreted insulin levels in culture media of human ES cells were measured by human insulin specific RIA kit. Results: During the differentiation process of human ES cells, GATA-4, a-fetoprotein, glucose transporter-2 and Ngn-3 expression were increased whereas OctA was decreased progressively. Insulin and albuInin mRNAs were expressed from stage IT in mouse ES cells and from stage III in human ES cells. We detected 3.0~7.9 IlU/rnl secretion of insulin from differentiated human ES cells by in vitro culture for 36 days. Conclusion: The sequential culture system could induce the differentiation of mouse and human ES cells into insulin secreting cells. This is the fIrst report of differentiation of human ES cells into insulin secreting cells by in vitro culture with serum and insulin free medium.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.648-656
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• 2008

The objective of this study was to establish conditions for transfection of a foreign gene into somatic cells using cationic lipid reagents and to evaluate the effects of transfection on in vitro development of somatic cell nuclear transfer (SCNT) embryos. Green fluorescent protein (GFP) gene was used as a foreign gene and a non-transfected somatic cell was utilized as a control karyoplast. Monolayers of porcine cells were established and subsequently transfected with a GFP-expressing gene (pEGFP-N1) using three types of transfection reagents (LipofectAMINE PLUS, FuGENE 6 or ExGen500). Donor cells used for SCNT included transfected fetal or adult fibroblasts and oviduct epithelial cells, either serum-fed or serum-starved. Oocytes matured in vitro for 42 h were reconstructed with either transfected or non-transfected porcine somatic cells by electric fusion and activation using a single DC pulse of 1.8 kV/cm for $30{\mu}s$ in $Ca^{2+}$ and $Mg^{2+}-containing$ 0.26 M mannitol solution. Reconstructed oocytes were subsequently cultured in NCSU-23 medium for 168 h and the developmental competence and cell number in blastocyst were compared. There were no significant differences (P>0.05) in fusion, cleavage rates or development to the blastocyst stage between non-transfected, transfected, serum-fed and serum-starved cells. However, the rates of GFP-expressing blastocysts were higher in the FuGENE 6 group (71.4%) among transfection reagents and in the fetal fibroblasts group (70.4%) for donor cells. These results indicate that fetal fibroblasts transfected with FuGENE 6 can be used as donor cells for porcine SCNT and that GFP gene can be safely used as a marker of foreign genes in porcine transgenesis.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.463-472
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• 2020

Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods. In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.207-213
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• 2013

This study was carried out to examine a molecular marker system for parentage test in Jeju Black cattle (JBC). Based on the preliminarily studies, we finally selected for construction of a novel genetic marker system for molecular traceability, identity test, breed certification, and parentage test in JBC and its related industrial populations. The genetic marker system had eight MS markers, five indel markers, and two single nucleotide polymorphisms (SNPs; g.G299T and g.del310G) within MC1R gene which is critical to verify the breed specific genotypes for coat color of JBC differing from those of exotic black cattle breeds such as Holstein and Angus. The results showed lower level of a combined non-exclusion probability for second parent (NE-P2) of $4.1202{\times}10^{-4}$ than those previously recommended by International Society of Animal Genetics (ISAG) of $5.000{\times}10^{-4}$ for parentage, and a combined non-exclusion probability for sib identity (NE-SI) of $2.679{\times}10^{-5}$. Parentage analysis has been successfully identified the JBC offspring in the indigenous population and cattle farms used the certified AI semens for production using the JBC-derived offspring for commercial beef. This combined molecular marker system will be helpful to supply genetic information for parentage test and traceability and to develop the molecular breeding system for improvement of animal productivity in JBC population.

• Development and Reproduction
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• v.17 no.4
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• pp.451-459
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• 2013

The biological activity of tissue specific stem cell is under the control of their specific microenvironment and the exogenous chemicals derived from digestive tract can be one of the constructing factors of that. It is suggested that the extract of brown algae Ishige okamurae has antioxidant-, apoptosis induction-, and antiinflammatory-effects. On the other hand, a few studies have shown that antioxidant assist inhibition of accumulation of fat. So we studied the effect of the extract of I. okamura on the cellular activity and differentiation of 3T3-L1 preadipocyte to adipose cell. The viability of cell was analyzed using 3-[4,5-dimethylthiazo-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Adipogenesis of 3T3-L1 cell was analyzed after induction in the induction medium containing the I. okamurae extract. The cellular activity was high compared with the vehicle and 0.05 mM caffeine in all groups of I. okamurae extract treated cells. The extract of I. okamura inhibited accumulation of lipids in 10 and $50{\mu}g/ml$. The expression of the marker genes for adipocyte differentiation coincided with cytochemical results. These results suggest that the extract of I. okamurae increases the cellular viability of adipose precursor cells. On the other hand, it suppresses the differentiation of preadipocyte to adipocyte and accumulation of lipids in concentration-dependent manners. It may be possible that the major component of the extract can be applied in the control of adipose tissuegenesis.