• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.141-149
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• 2008

This study was conducted to develop an efficient cryopreservation method of human embryonic stem (ES) cells using vitrification. In an initial experiment, sub-clumps of human ES cells (CHA-hES3 and CHA-hES4) were vitrified using grids after incubation with STO feeder cells for 1 or 16 h (Groups 1-1 and 1-2, respectively). After storage for $2{\sim}4$ months, thawed clumps were re-plated on a fresh feeder layer. The survival rates of warmed CHA-hES3 and CHA-hES4 cells of Group 1-2 were significantly higher than those of the corresponding Group 1-1 cells. In the second experiment, human ES cells were vitrified after incubation with feeder or feeder-conditioned medium (Groups 2-1 to -7). Relative mRNA expression of BM proteins and survival rates were increased following incubation of ES cells with fresh feeder cells for 16 h. In conclusion, increasing of tight adhesion between ES cells by extended incubation with feeder could reduce cryoinjury after vitrifying/warming.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.80-80
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• 2003

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by marked cell death in the striatum and cortex. Stereotaxic injection of quinolinic acid (QA) into striatum results in a degeneration of GABAergic neurons and exhibits abnormal motor behaviors typical of the illness. The objective of this study was carried out to obtain basic information about whether parthenogenetic mouse embryonic stem (PmES) cells are suitable for cell replacement therapy of HD. To establish PmES cell lines, hybrid F1 (C57BL/6xCBA/N) mouse oocytes were treated with 7% ethanol for 5 min and cytochalasin-B for 4 hr to initiate spontaneous cleavage. Thus established PmES cells were induced to differentiate using bFGF (20ng/ml) followed by selection of neuronal precursor cells for 8 days in N2 medium. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days, then a final differentiation step in N2 medium for 7 days. To establish recipient animal models of HD, young adult mice (7 weeks age ICR mice) were lesioned unilaterally with a stereotaxic injection of QA (60 nM) into the striatum and the rotational behavior of the animals was tested using apomorphine (0.1mg/kg, IP) 7 days after the induction of lesion. Animals rotating more than 120 turns per hour were selected and the differentiated PmES cells (1$\times$10$^4$cells/ul) were implanted into striatum. Four weeks after the graft, immunohistochemical studies revealed the presence of cells reactive to anti-NeuN antibody. However, only a slight improvement of motor behavior was observed. By Nissl staining, cell mass resembling tumor was found at the graft site and near cortex which may explain the slight behavioral improvement. Detailed experiment on cell viability, differentiation and migration explanted in vivo is currently being studied.

• Journal of Biosystems Engineering
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• 제38권2호
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• pp.113-120
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• 2013

Purpose: Stem cells provide new opportunities in the regenerative medicine for human or animal tissue regeneration. In this study, we report an efficient method for the modulating behaviors of electro-active stem cells by micro-electric current stimulation (mES) without using chemical agents, such as serum or induction chemicals. Methods: Dental pulp stem cells (DPSCs) were cultured on the tissue culture dish in the mES system. To find a suitable mES condition to promote the DPSC functions, the response surface analysis was used. Results: We found that a working micro-current of 38 ${\mu}A$ showed higher DPSC proliferation compared with other working conditions. The mES altered the expressions of intracellular and extracellular proteins compared to those in unstimulated cells. The mES with 38 ${\mu}A$ significantly increased osteogenesis of DPSCs compared with ones without mES. Conclusions: Our findings indicate that mES may induce DPSC proliferation and differentiation, resulting in applying to DPSCs-based human or animal tissue regeneration.

• Reproductive and Developmental Biology
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• 제32권2호
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• pp.105-110
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• 2008

The endogenous retrovirus-like elements (HERVs) found on several human chromosomes are somehow involved in gene regulation, especially during the transcription level. HERV-H, located on chromosome Xp22, may regulate gastrin-releasing peptide receptor (GRPR) in connection with diverse diseases. By suppression subtractive hybridization screen on SV40-immortalized lung fibroblast (WI-38 VA-13), we discovered that expression of HERV-HX2, a clustered HERV-H sequence on chromosome X, was upregulated in immortalized lung cells, compared to that of normal cells. Expression of HERV-HX2 was then analyzed in various cell lines, including normal somatic cells, cancer cells, SV40-immortalized cells, and undifferentiated and differentiated human embryonic stem cells. Expression of HERV-HX2 was specifically upregulated in continuously-dividing cells, such as cancer cells and SV40-immortalized cells. Especially, HERV-HX2 in HeLa cells was highly upregulated during the S phase of the cell cycle. Similar results were obtained in hES cells, in which undifferentiated cells expressed more HERV-HX2 mRNA than differentiated hES cells, including neural precursor and endothelial progenitor cells. Taken together, our results suggest that HERV-HX2 is upregulated in cancer cells and undifferentiated hES cells, whereas downregulated as differentiation progress. Therefore, we assume that HERV-HX2 may playa role on proliferation of cancer cells as well as differentiation of hES cells in the transcriptional level.

• Reproductive and Developmental Biology
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• 제31권4호
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• pp.273-278
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• 2007

This study was conducted to examine the protein kinase inhibitors, 6-dimethylaminopurine (DMAP) and cycloheximide (CHXM) on the development and chromosome constitution of porcine parthenogenetic embryos. In vitro matured oocytes were activated by electric stimuli (ES) or a combination of ES with culture in 2 mM DMAP or $10{\mu}g/ml$ CHXM for 4 hr. Activated oocytes were cultured in PZM-3 for 6 days. Some 1-cell embryos and blastocysts were fixed by air dry method to analyze the chromosome constitutions and/or total cell number. Blastocyst development of DMAP-treated group (26.7%) was significantly higher (p<0.05) than those of CHXM-treated and ES control groups. Ploidy in 1-cell stage embryos was not different among groups (77.3 to 81.0%), however, proportion of diploid chromosome constitutions was high in DMAP-treated group (61.9%, p<0.05). In the blastocyst stage, proportion of diploid chromosome plates was significantly high in DMAP-treated group (64.2%, p<0.05), and proportion of abnormal chromosome plates was higher in CHXM-treated group (36.6%, p<0.05) than DMAP-treated group (28.3%,). Proportion of embryos with abnormal chromosome constitutions was slightly increased by DMAP (40.0%) and CHXM (42.1%) treatment due to the increasing of mixoploid (47.4 and 52.0%). The present study shows that the DMAP treatment increase the development of porcine parthenotes. However, parthenogenetic activation by ES or combined treatment with ES and DMAP or CHXM detrimentally affects the chromosome constitutions of porcine parthenotes during early embryonic development, leads to increased abnormal ploidy in the blastocyst stage.

• Preventive Nutrition and Food Science
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• 제20권4호
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• pp.221-229
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• 2015

Hesperidin has been shown to possess a potential inhibitory effect on vascular formation in endothelial cells. However, the fundamental mechanism for the anti-angiogenic activity of hesperidin is not fully understood. In the present study, we evaluated whether hesperidin has anti-angiogenic effects in mouse embryonic stem cell (mES)-derived endothelial-like cells, and human umbilical vascular endothelial cells (HUVECs), and evaluated their mechanism via the AKT/mammalian target of rapamycin (mTOR) signaling pathway. The endothelial cells were treated with several doses of hesperidin (12.5, 25, 50, and $100{\mu}M$) for 24 h. Cell viability and vascular formation were analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and tube formation assay, respectively. Alteration of the AKT/mTOR signaling in vascular formation was analyzed by western blot. In addition, a mouse aortic ring assay was used to determine the effect of hesperidin on vascular formation. There were no differences between the viability of mES-derived endothelial-like cells and HUVECs after hesperidin treatment. However, hesperidin significantly inhibited cell migration and tube formation of HUVECs (P<0.05) and suppressed sprouting of microvessels in the mouse aortic ring assay. Moreover, hesperidin suppressed the expression of AKT and mTOR in HUVECs. Taken together, these findings suggest that hesperidin inhibits vascular formation by blocking the AKT/mTOR signaling pathways.

• Asian Pacific Journal of Cancer Prevention
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• 제17권9호
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• pp.4223-4231
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• 2016

Background: Development of new apoptosis-inducing drugs is a promising trend in anticancer therapy. For this purpose several formamidinoderivatives of doxorubicin were synthesized. The aim of our study was to investigate effects of the five formamidinodoxorubicins in the ES-2 human ovarian clear cell carcinoma line, for comparison with data obtained previously for SKOV-3 human ovarian adenocarcinoma cells, to answer the question of whether and to what extent the histological cell type is a possible determinant of sensitivity to tested anthracyclines. Materials and Methods: In our experimental work the following methods were used: spectrophotometric assays with MTT; fluorimetric assays - double staining with Hoechst 33258 and propidium iodide (PI), measurement of caspase-3, -8, -9 activity, intracellular accumulation of DOX and analogues, estimation of drug uptake, mitochondrial transmembrane potential; flow cytometry - phosphatidylserine (PS) externalization with annexin V-FITC and PI fluorochromes. Results: Effects of the derivatives of doxorubicin were partially linked with the specific type of cancer cell although intracellular accumulation and cellular uptake of DOX and derivatives were similar in both. All of the investigated derivatives were considerably more cytotoxic than DOX. Formamidinodoxorubicins were able to induce caspase-dependent apoptotic cell death in both cell types. Conclusions: All new formamidine derivatives of DOX were able to induce caspase - dependent apoptosis in human ovarian cancer cell lines SKOV-3 and ES-2. Obtained results suggested that formamidine derivatives of DOX may be promising candidates for the prospective chemotherapeutic agents for the two different histological subtypes of ovarian cancer.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.78-78
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• 2003

Embryonic stem (ES) cells, derived from preimplantation embryo, are able to differentiate into various types of cells consisting the whole body, or pluripotency. In contrast, terminally differentiated cells do not usually alter their nature but frequently die or transform if they are exposed to inappropriate external stimulations. In addition to the plasticity, ES cells are expected to be different from terminally differentiated cells in very many ways, such as patterns of gene expressions, ability and response of the cells in confronting environmental stimulations, metabolism, and growth rate. As a model system to differentiate these two types of cells, human ES cells (MB03) and terminally differentiated cells (HeLa), we examined the ability of these two types of cells in confronting a severe oxidative insult, that is $H_2O$$_2$. Approximately 1$\times$10$^4$ cells were plated in 96 well plate and serum starved for overnight. The conditioned cells were exposed to a various concentration of $H_2O$$_2$ fur 24 hrs and loaded with neutral red (50$\mu\textrm{g}$/ml) for 4 hrs, washed with PBS for 2 min three times, and entrapped dye was dissolved out using acetic ethanol. Cytotoxicity was determined by reading the amount of dye in the medium using microplate reader. equipped with 575 nm filter. Relative amount of the dye entrapped within MB03 or HeLa were not significantly different when cells were exposed up to 0.4 mM $H_2O$$_2$. However, this sharply decreased down to 0.12% in HeLa cells when the cells were exposed to 0.8 mM $H_2O$$_2$, while it was approximately 54% in MB03 suggesting that this concentration of $H_2O$$_2$ is the defensive threshold for HeLa cells. The resistance to oxidative stimulation reversed, however, when cells were co-treated with BSO (L-buthionine- 〔S, R〕-sulfoximine) which chelates intracellular GSH. This result suggests that cellular GSH is the major defensive mechanism of human ES cells. Induction of enzymes involved in GSH metabolism and type of cell death is currently being studied.

• 한국수정란이식학회지
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• 제32권1호
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• pp.25-31
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• 2017

This study was conducted to investigate the effect of activation method on the endoplasmic reticulum (ER) stress induction, apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus (ES) (E), 2) $ES+10{\mu}M$ Ca-ionophore (A23187) treatment (EC), 3) ES+2 mM 6-dimethylaminopurine (6-DMAP) treatment (ED), or 4) ES+A23187 and 6-DMAP treatments (ECD). Parthenogenetic embryos were sampled to analyze x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptosis genes at 3 h after ES and the 1-cell and blastocyst stages. In the EC group, the band intensity of spliced Xbp1 (Xbp1s) mRNA was higher than those of the other groups at the 3 h and 1-cell stage, and higher than that of the E group at the blastocyst stage. Four ER stress-associated genes were expressed at the highest level in the EC group and weakly expressed in the ED group at 3 h after activation. However, most of the genes were highly expressed at the 1-cell and blastocyst stages with some variation in the EC and ECD groups. Expression of Bcl-2-associated X protein (Bax) and caspase-3 mRNA was significantly higher in the EC group than in the other groups at all development stages. The developmental rates to the blastocyst stage were higher in the ED and ECD groups than in the E and EC groups. These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by the activation method and subsequently lead to the apoptosis of embryos.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.216-219
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• 2004

The effect of culture temperature on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli was investigated. E. coli cell was cotransformed with two plasmids (pTCGT1 and pGroll) in which the cgt and groEL/ES genes are under the control of T7 promoter and pzt-1 promoter, respectively. When tetracycline (10 ng/ml) and IPTG (l mM) were added as inducers at the early-exponential phase (2 h) and mid-exponential phase (3h), respectively, the solubilization of the inclusion body CGTase was greatly dependent on the temperature of the culture. At low culture temperature of $25^\circ{C}$, 2- or 3-fold higher activity and specific activity were obtained over $37^\circ{C}$. SDS-PAGE analysis revealed that about 62% of CGTase in the total CGTase protein was found in the soluble fraction by applying overexpression of GroEL/ES chaperone and by cultivation of E. coli at $25^\circ{C}$, whereas 33% of CGTase was detected in the soluble fraction at $37^\circ{C}$. Therefore, the expression of GroEL/ES and cultivation at $25^\circ{C}$ greatly enhanced the soluble production of CGTase in E. coli.