• 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.

• Journal of Electrochemical Science and Technology
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• v.14 no.2
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• pp.145-151
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• 2023

Planer-type electrolyte substrates are often utilized for stack manufacturing of electrolyte-supported solid oxide fuel cells (ES-SOFCs) to fulfill necessary requirements such as a high mechanical strength and redox stability. This work did an electrochemical analysis of ES-SOFC with different NiO-YSZ anode thicknesses to find the optimal value for the high performance of the fuel cell. The cell resistivities were constant at anode thickness between 25-58 ㎛, but a thick anode (74 ㎛) caused a high electrode resistivity leading to a dramatic reduction in cell performance. A stability test was performed for 50 hours at 700℃, and the results showed a degradation rate of 0.3% per 1000 h by extrapolated fitting.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.272-272
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• 2004

The objective of this study is to test whether human embryonic stem cells expressing Nurr1 (Nurr1-transfected hES cells) could be expressed TH according to neuronal differentiation. As an effort to direct differentiation of hES (MB03 registered in NIH) cells to dopamine-producing neuronal cells, Nurr1 was transfected using conventional transfection protocol into MB03 cell and examined the expression of tyrosine hydroxylase (TH) after differentiation induced by retinoic acid (RA) and ascorbic acid (AA). (omitted)

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.27 no.3
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• pp.84-95
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• 2014

Objective : Ephedra sinica (ES) has has been used as remedy of allergic diseases for a long time in Korea. In the present study, we investigated the anti-allergic effects of ES on experimental allergic asthma mouse model using ovalbumin (OVA). Methods : BALB/c mice were sensitized and challenged with 100 ug of OVA and 1 mg of aluminum potassium sulfate of 0.2 ml phosphate-buffered saline(PBS) intraperitoneally on day 1 and 15. mice were challenged on 3 consecutive days with 5% OVA and AHR was assessed 24 h after the last challenge. we examined the lung histology, airway hyper sensitivity, total inflammatory cell count in bronchoaveloar lavage fluid(BALF), Th2-associated cytokines production and IgE production. Results : ES potently inhibited the lung damage and the development of Penh. ES also reduced the number of BAL cells during OVA-induced allergic asthma. Furthermore, ES inhibited cytokines production such as IL-4, IL-13 productions, and IgE level of serum. Conclusion : These results suggest that ES may inhibit the production of IL-4, IL-13, IgE and infiltration of inflammatory cell and be beneficial oriental medicine for allergic asthma.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.293-298
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• 2003

목 적: 인간의 배아줄기세포는 전분화능과 영속성을 가지고 있어 발생 및 분화에 관련된 기초 연구 뿐 만 아니라 재생의학, 약물검색 등에서도 매우 유용한 재료로 이용될 수 있다.본 연구에서는 유전체의 변형이 배아줄기세포주의 확립 효율에 미치는 영향을 살펴보고자 비정상적인 포배기 배아에서 내세포괴를 분리하여 배양하였다. 연구 방법: 인간의 체외수정 및 배아이식술에서 공여 받은1개 또는3개의 전핵이 관찰되는 비정상 수정란 (n=20)과 착상전 유전진단에서 이수성이 확인된 배아 (n=27)를 대상으로 하였다. 일반적인 immunosurgery 방법으로 영양배엽세포들을 제거하고 내세포괴를 분리한 후 PMEF 혹은 STO feeder 세포위에서 배양하였다. 배아줄기세포의 배양시스템을 검증하기 위해서 이미 확립된 Miz-hES1 cell line을 동시에 같은 조건 하에서 계대배양하였다. 결 과: 비정상 수정란에서 발생된 포배기 배아에서 분리한 1개의 내세포괴가 배아줄기세포와 유사한 colony를 형성하였으나, 계대배양에는 실패하였다. 이수성 배아에서 발생된 포배기 배아의 내세포괴 배양에서는 두개의 colony가 계대배양 중에 영양배엽세포의 형태로 분화되어 미분화 상태를 유지하지 못하였다. 동일한 시기와 조건 하에서 계대배양된 Miz-hES1 cell line이 미분화상태로 유지됨을 karyotyping (46, XY)과 immunophenotyping (positive in SSEA-3 and -4)으로 확인하였다. 결 론: 본 연구의 결과에서 비정상 수정란과 이수성 배아에서 발생된 포배기 배아에서 유래한 내세포괴는 배아줄기세포주 확립 및 미분화 상태 유지 능력이 매우 저조한 것으로 여겨진다. 따라서, 인간의 배아줄기세포주를 확립하는데 있어 배아의 정상여부가 중요한 요소로 작용할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1267-1272
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• 2005

The effects of coexpression of GroEL/ES and DnaK/DnaJ/GrpE chaperones on the productivity of the soluble form of human granulocyte colony stimulating factor (hG-CSF) in E. coli were examined. Recombinant hG-CSF protein was coexpressed with DnaK/DnaJ/GrpE or GroEL/ES chaperones under the control of the araB or Pzt-1 promoter, respectively. The optimal concentration of L-arabinose for the expression of DnaK/DnaJ/GrpE was found to be 1 mg/ml. When L-arabinose was added at $OD_{600}$=0.2 (early-exponential phase), soluble hG-CSF production was greatly increased. In addition, it was observed that the DnaK/DnaJ/GrpE and GroEL/ES chaperones had no synergistic effects on preventing aggregation of hG-CSF protein. Consequently, by coexpression of the DnaK/DnaJ/GrpE chaperone, the signal intensity of the hG-CSF protein band in the soluble fraction of cell lysate was increased from $3.5\%\;to\;13.9\%$, and Western blot analysis also revealed about a 4-5-fold increase of production of soluble hG-CSF over the non-induction case of DnaK/DnaJ/GrpE.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.60-60
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• 2003

Embryonic stem (ES) cells have property of self-renewal and can differentiate into the cells of all three primary germ layers. Recently, many growth factors, alteration of culture condition and gene modifications have been used to differentiate mouse and human ES cells into specific cell types. This study was performed to evaluate the differentiation protocol for human ES cells to the endodermal lineage cells. Human ES cells (Miz-hESl ) were cultured on STO feeder layer mitotically inactivated with mitemycin C, and embryoid bodies (EBs) were formed by suspension culture. Differentiation protocol of EBs consisted of three steps: stage I, culture of EBs for 6 days with ITSFn medium; stage II, culture of stage I cells for 8 days with N2 medium ; stage III, culture of stage II cells for 22 days with N2 medium. mRNA levels of the endodermal lineage differentiation genes were analyzed by semi- quantitative RT-PCR. The Oct-4 expression, a marker of the pluripotent state, was detected in undifferentiated human ES cells but progressively decreased after EBs formation. Differentiating human ES cells expressed marker genes of endodermal differentiation and pancreatic islet cells. GATA4, a-fetoprotein, Glut-2, and Ngn3 were expressed in all stages. However, albumin and insulin were expressed in only stage III cells. The human ES cells can be differentiated into endodermal lineage cells by multiple step culture system using various supplements. We are developing the more effective protocols for guided differentiation of human ES cells.

• Journal of Embryo Transfer
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• v.32 no.1
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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 Embryo Transfer
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• v.9 no.3
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• pp.243-247
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• 1994

최근 의약적으로 유용한 단백질을 대량 생산키 위한 실현 가능한 방법이 유전자변환 가축의 이용과 관련되어 발전되어 왔다. 이러한 유전자 변환동물은 이종의 단백질을 유즙속으로 분비시키는 생체반응기로서 이용되고 있다. 이러한 전략적 목적을 위해 현재 유전자 변환동물의 생산을 위한 이용에 있어 여러 가지 방법들이 보고되고 있다. 그러나 ES 세포의 사용이 이러한 방법들 사이에서 가장 실질적인 것으로 추정되고 있다. 본 실험에서는 유전자 구축을 위해 사람 황체 호르몬(human luteinizing hormone; hLH)의 전사를 유도하기 위해 각각 2.2 및 0.5 kb의 토끼 $\beta$-casein pronoter 단편을 이용하여 생쥐의 유선에 hLH를 발현시키도록 조절하고 발현이 thynidine kinase(TK) pronoter에 의해 좌우되는 neo 유전자를 selectable marker로서 plasnid속에 삽입하였다. 그 결과 생긴 구축 유전자는 각각 pCas 2.2와 pCas 0.5로 명명하였다. 구축된 유전자로 2$\times$107의 TT-2 ES세포를 170V, 550$\mu$F로 100$\mu$g의 선상 plasmid에 의해 electroporation 시켰다. 감염된 colony들은 250$\mu$g/$m\ell$ G418을 함유하는 ESM 배양액에서 선별 7일 이후에 회수하여 성공적으로 감염된 ES세포는 PCR 및 Southern blot에 의해 확인되었고 그들 중 나머지는 trypsin 처리 후 각각 미세조작과 공배양 기술을 사용하여 ICR 생쥐의 8세포기 수정란 속에 도입하였다. 결국 24시간 동안 37$^{\circ}C$, 5% $CO_2$에서 배양된 배반포를 chimera의 생산을 위해 위임신 유기된 G418 선발처리 이후 400 및 275개의 ES 세포 colony가 생존하였으며, 3개의 ES 세포으 colony 의 genome 속에 임의적으로 plamid가 삽입된 것을 Southern blot에 의해 확인되었다. 총 13 chimera 생쥐가 3 colony로부터 생산되었으나 germ-line chimera는 현재 조사중이다. chimera 생산빈도는 공배양 기술보다 주입방법에서 현저히 높았다.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.4
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• pp.261-271
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• 2004

Objective: This study was performed to evaluate the possibility of prolonged culture of human embryonic stem cells (hESC; SNUhES2) on human amniotic fluid cells (hAFC), which had been storaged after karyotyping. Method: The hAFC was prepared for feeder layer in the presence of Chang's medium and STO medium (90% DMEM, 10% FBS) at $37^{circ}C$ in a 5% $CO_2$ in air atmosphere. Prior to use as a feeder layer, hAFC was mitotically inactivated by mitomycin C. The hESCs on hAFC were passaged mechanically every seven days with ES culture medium (80% DMEM/F12, 20% SR, bFGF). Results: The hAFC feeder layer support the growth of undifferentiated state of SNUhES2 for at least 59 passages thus far. SNUhES2 colonies on hAFC feeder appeared slightly angular and flatter shape as compared with circular and thicker colonies observed with STO feeder layer and showed higher level with complete undifferentiation in seven days. Like hESC cultured on STO feeders, SNUhES2 grown on hAFC expressed normal karyotype, positive for alkaline phosphatase activity, high telomerase activity, Oct-4, SSEA-3, SSEA-4, Tra-1-60 and Tra-1-81 and formed embryoid bodies (EBs). Conclusion: The hAFC supports undifferentiated growth of hESC. Therefore, these results may help to provide a clinically practicable method for expansion of hESC for cell therapies.