• Journal of dental hygiene science
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• v.15 no.4
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• pp.488-494
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• 2015

A recent report showed that nuclear factor of activated T cell (NFATc) 1 is a member of the NFAT family and is strictly implicated osteoblast differentiation and bone formation. Furthermore, the precise expression and function of NFATc1 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the function of NFATc1 in osteoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs). NFATc1 messenger RNA (mRNA) and protein levels were accessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assay, respectively. Cell proliferation determined using MTT assay. Differentiation was evaluated by alkaline phosphatase activity and formation of calcium nodule with alizarin red S staining. The mRNA expression of osteoblastic differentiation related genes were examined by RT-PCR. Marked upregulation of NFATc1 mRNA and protein was observed in cells grown in osteogenic medium (OS). NFATc1 transactivation was detected in hPDLCs that had been incubated in OS for 14 days. Treatment with $10{\mu}M$ cyclosporine A (CsA), a known calcineurin inhibitor, reduced the proliferation of hPDLCs, while $5{\mu}M$ CsA had no effect. Inhibition of the calcineurin/NFATc1 pathway by CsA, attenuated OS-induced osteoblastic differentiation in hPDLCs. In summary, this study demonstrates for the first time that NFATc1 plays a key role in osteoblastic differentiation of hPDLCs and activation of NFATc1 could provide a novel mechanism for periodontal bone regeneration.

• Development and Reproduction
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• v.9 no.2
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• pp.105-114
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• 2005

Embryonic stem(ES) cells have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decision and to develop methods for getting enough cell numbers for clinical applications. Hematopoiesis has been widely studied, and hematopoietic differentiation from ES cells is a good model to study lineage commitment. In this study, we investigated stemness and compared the efficiency of hematopoietic differentiation using two different mouse embryonic stem cell lines TC-1 and B6-1. Although the two cell lines showed known stem cell properties with minor differences, the embryoid body formation efficiency in methylcellulose was much higher in TC-1 than B6-1. When measured potentials of hematopoietic differentiation using functional(colony-forming cell) and phenotypic(specific marker expression) assays, we found that TC-1 can differentiate into hematopoietic cells in methylcellulose culture but B6-1 cannot. These results imply that we can improve the efficiency of hematopoietic cell differentiation by selection of proper cell lines and this may be also applied in the differentiation of human embryonic stem cells.

• Development and Reproduction
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• v.12 no.1
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• pp.31-39
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• 2008

Neural tissue has limited intrinsic capacity of repair after injury, and the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesechymal-like stem cells from human adipose tissues (AT-MSCs), and studied on transdifferentiation-promoting conditions in neural cells. Dopaminergic and cholinergic neuron induction of AT-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulphoxide (DMSO) and butylated hydroxyanisole(BHA) in N2 Medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. AT-MSCs treated with bFGF, SHH and FGF8 were differentiatied into dopaminergic neurons that were immunopositive for TH antibody. Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor (bFGF), retinoic acid (RA) and sonic hedgehog (Shh). AT-MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including neuro D1, $\beta$-tubulin III, GFAP and nestinwas markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after preinduction medium culture, we confirmed the differentiation of dopaminergic and cholinergic neurons by TH/$\beta$-tubulin III or ChAT/ $\beta$-tubulin III positive cells. Conclusively, AT-MSCs can be differentiated into dopaminergic and cholinergic neuronsand these findings suggest that AT-MSCs are alternative cell source of treatment for neurodegenerative diseases.

• Journal of the Korean Society of Food Science and Nutrition
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• v.42 no.7
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• pp.1015-1021
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• 2013

Glycyrrhiza inflata Batal, an important species of licorice, is one of the most widely used medicinal plants for over 4000 years. Glycyrrhiza plant species has been well known for its various therapeutic activities such as anti-inflammatory, anti-allergic, and anti-ulcer. The purpose of this study was to determine the effects of Glycyrrhiza inflata Batal ethanol extracts (GBE) on adipocyte and osteoblast differentiation. Mesenchymal C3H10T1/2 cells were treated with sub-cytotoxic doses of GBE, and its effects on adipocyte differentiation were assessed. We found that GBE dose-dependently increased lipid accumulation and also induced the expression of adipocyte markers, such as $PPAR{\gamma}$ and its target genes, aP2, and adiponectin, in C3H10T1/2 cells. Consistently, similar effects of GBE on lipid accumulation were also observed in preadipocyte 3T3-L1 cells that further supports the pro-adipogenic activities of GBE. We also investigated the effects of GBE on osteoblast differentiation of mesenchymal C3H10T1/2 cells. As a results, we found that GBE increased the activity of alkaline phosphatase in a dose-dependent manner and also promoted the expression of osteoblast markers, such as ALP and RUNX2, during osteoblast differentiation of C3H10T1/2 cells. Similar pro-osteogenic effects of GBE were also observed in preosteoblast MC3T3-E1 cells. Finally, our data show that a major bioactive compound found in Glycyrrhiza inflata Batal, licochalcone A (LA) but not glycyrrhizic acid (GA), can mediate the pro-adipogenic and pro-osteogenic effects of GBE. Taken together, this study provides data to show the possibility of GBE and its bioactive component LA as putative strategies for type 2 diabetes and bone diseases.

• Journal of Life Science
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• v.21 no.3
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• pp.358-367
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• 2011

Soybean is known to contain various phytochemicals that are related to anti-oxidant, anti-inflammatory and anti-obesity effects in mice and humans. The anti-obesity effect of by-product extracts from soybean on the differentiation of 3T3-L1 pre-adipocytes to adipocytes was investigated by suppressing adipocyte differentiation and lipid accumulation with Oil Red-O assay and quantitative PCR. In inducing differentiation of 3T3-L1 pre-adipocytes in the presence of an adipogenic cocktail, isobutylmethylanthine (IBMX), dexamathasone, and insulin, treatment with filtrated soybean soaked water, soybean milk, and soycurd residue from soybean curd processing significantly decreased mRNA expression of obesity-related gene such as PPAR${\gamma}$, Fabp4, and Scd1, adipsin, apolipoprotein (APOE) and adiponectin (ADIPOQ) without any significant cytotoxicity. We also determined the well-known isoflavones in soybean, such as daidzein and genistein, in the by-product extracts. Taken together, we suggest that soybean by-product extract showed anti-obesity effect by suppressing adipocyte related gene expression, and that by-products collected during soybean curd processing may be a good candidate as an ingredient in health care products.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.247-265
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• 2008

Objectives: Many types of liver diseases can damage regenerative potential of mature hepatocytes, hepatic progenitor cells or oval cells. In such cases, a stem cell-based therapy can be an alternative therapeutic option. We examined whether human amnion-derived mesenchymal stem cells (HAM) and human umbilical cord-derived stem cells (HUC) could differentiate into hepatocyte-like cells as therapeutic cells for the liver diseases. Methods: HAM and HUC were isolated from the amnion and umbilical cord of the volunteers after a caesarean section with informed consent. In order to differentiate these cells into hepatocyte-like cells, cells were cultivated in hepatogenic medium using culture plates coated with fibronectin. Effects of hepatocyte growth factor, L-ascorbic acid 2-phosphate, insulin premixture fibroblast growth gactor 4, dimethylsulfoxide, oncostatin M and/or dexamethasone were examined on the hepatic differentiation. After differentiation, the cells were analyzed by RT-PCR, immunocytochemistry, immunoblotting, albumin ELISA, urea assay and periodic acid-schiffs staining. Results: Initial fibroblast-like appearance of HAM and HUC changed to a round shape during culture in the hepatogenic medium. However, in all hepatogenic conditions examined, HUC secreted more amounts of albumin or urea into medium than HAM. Expression of some of hepatocyte-specific genes increased and expression of new genes were observed in HUC following cultivation in hepatogenic medium. Results of immunocytochemistry and immunoblotting analyses demonstrated that HUC secreted albumin into the culture medium. PAS staining further demonstrated that HUC could store glycogen inside of the cells. Conclusions: Both HUC and HAM could differentiate into albumin-secreting, hepatocyte-like cells. Under the same hepatogenic conditions examined, HUC more efficiently differentiated into hepatocyte-like cells compared with the HAM. The results suggest that HUC and HAM could be used as sources of stem cells for the cell-based therapeutics such as in liver diseases.

• Journal of Life Science
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• v.17 no.7 s.87
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• pp.976-982
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• 2007

The purpose of this study was to determine the modulating effects of histone deacetylase inhibitor, trichostatin A, on the differentiation of mouse C2C12 myoblasts. We demonstrated that trichostatin A induced morphological changes of C2C12 myoblasts into smooth muscles and significantly increased the gene expression of smooth muscle markers including smooth muscle ${\alpha}-actin$ and transgelin. These results were due to the change in the expression level of cell cycle regulators in trichostatin A-treated C2C12 cells. Real-time PCR data revealed that cyclin dependent kinase inhibitor, p21, mRNA expression was significantly increased in trichostatin A-treated C2C12 cells. However, trichostaDn A rapidly decreased cyclin Dl mRNA expression necessary for cell cycle progression in 24hr after treatment. In conclusion, the strong inhibitory effects of trichostatin A on histone deacetylation induced transdifferentiation of C2C12 myoblasts into smooth muscle cells and these results are partly due to the changes in the expression of cell cycle regulators such as p21 and cyclin D1.

• Journal of the korean academy of Pediatric Dentistry
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• v.29 no.2
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• pp.278-284
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• 2002

To examine the global gene expression of osteoclastogenesis-related genes in RAW 264.7 and its differentiated OCLs through the use of Atlas Mouse cDNA Array 2.1 membranes printed with 1176 well-characterized mouse genes involved in biology. Both samples were screened in parallel using cDNA expression arrays. The array results were additionally validated using RT-PCR. The results of cDNA arrays showed that 6 genes were up-regulated >2.5-fold (PKC beta II. POMC, PTEN, etc) and 16 genes were down-regulated >2.5-fold (Osteopontin, Cyclin D1, Cathepsin C, PTMA, etc) in both samples at the mRNA level. RT-PCR analysis of PKC beta II of these differentially expressed genes gave result consistent with cDNA array findings. The result of osteoclastogenesis showed that the PKC beta II gene was overexpressed in OCLs compared with RAW264.7 cell line. Osteoclastogenesis-related genes are differentially expressed in RAW264.7 cell line and its differentiated OCLs. its gene overexpression correlates with osteoclast differentiation in RAW264.7 cell line.

• Microbiology and Biotechnology Letters
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• v.45 no.1
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• pp.27-34
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• 2017

$C/EBP{\beta}$ and $C/EBP{\delta}$ are required for the initiation of adipogenesis and induce the expression of key adipogenic regulators, such as $PPAR{\gamma}$ and $C/EBP{\alpha}$. In the present study, we have examined the effects of silibinin and its possible molecular mechanisms in regulating adipocyte differentiation and expression of $C/EBP{\beta}$ and $C/EBP{\delta}$ in the early stage of adipogenesis. Silibinin statistically significantly inhibits intracellular lipid accumulation and the mRNA expression of various genes involved at different stages during adipogenesis. Silibinin also suppresses expression of lipoprotein lipase (LPL), fatty acid binding protein 4 (AP2), and adiponectin in 3T3-L1 adipocytes. Thus, the anti-adipogenic effect of silibinin seems to originate from the ability to inhibit the expression of $C/EBP{\beta}$ and $C/EBP{\delta}$. Furthermore, silibinin decreases cell viability for differentiation period and induces apoptotic cell death through capspase-3 activation.

• Journal of Animal Science and Technology
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• v.54 no.2
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• pp.103-109
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• 2012

Present study was performed to investigate the effect of single and co-culture of adipocyte and muscle cell lines on cell differentiation. 3T3-L1 (adipocyte) and L6 (muscle) cell lines were single-cultured on the condition of 10% fetal bovine serum (FBS)/Dulbeco's modified eagle's medium (DMEM) for 48 h followed by culture within 5% FBS/DMEM as a growth media. Then, the growth media was replaced by differentiation media composed of 2% FBS/DMEM without additives in single- or co-culture of the 3T3-L1 and the L6 cells to induce differentiation of both cell types. In co-culture system, the 3T3-L1 and the L6 cells were grown in separated places by being seeded on a $0.4{\mu}m$ insert membrane and on the bottom of 6 well plate, respectively. Cell differentiation was measured using morphological investigation and cytosolic analysis of glycerol-3-phosphate dehydrogenase (GPDH; for 3T3-L1) and creatine kinase (CK; for L6). Based on the GPDH results, the presence of L6 cells did not stimulate 3T3-L1 differentiation showing more differentiation of 3T3-L1 cells in the single-culture compared to the co-culture condition. In contrast, 3T3-L1 cells in the co-culture promoted differentiation of L6 cells. Enzymatic analysis supported this result showing that 3T3-L1 cells showed statistically (P<0.05) higher GPDH activity in the single-culture than the co-culture, whereas CK results of L6 cells were vice versa (P<0.05). Overall, present results may indicate that co-culture system is more reliable and precise technique compared to single-culture. Further studies on several co-culture trials including different media conditions, supplementation of differentiating substances, molecular biological analysis, etc. should be required to obtain practical and fundamental mass data.