• 한국가금학회지
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• 제27권1호
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• pp.73-78
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• 2000

Embryonic stem (ES) cells are pluripotent cell lines, which derived from preimplantation embryo. These cells have been used as a vehicle of foreign DNA for production of transgenic mammals. this experiment was performed to examined the possible use of blastodermal cells derived from hen's egg for germline manipulation. Stage X blsdtodermal cells isolated from fertilized eggs were cultured in DMEM containing 15% fetal calf serum. Blastodermal cells wre co-cultured on the chicken embryonic fibroblast (CEF) or mouse embryonic fibroblast(MEF) cells. to examine the effects of growth factors on stem cell growth, bFGF and LIF were added. There was no significant difference in colony formation of putative ES cells between CEF and MEF as a feederlayer, but the addition of growth factors enhanced the proliferation and inhibited differentiation of blastodermal cells. To characterize the cell colonies as a putative ES cells, putative embryonic cell colonies were stained by periodic acid Schiffs (PAS) reagent. The putative ES cell colonies showed intensive positive reaction similar to the property of undifferentiated PGC upto 20days in vitro, but not in other cell types. this result demonstrates that PAS-positive cell colonies may be used for the study of establishment of chicken ES cell lines for the production of transgenic chicken.

• 한국수정란이식학회지
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• 제9권3호
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• pp.213-220
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• 1994

생쥐 배반포로부터 내부세포괴(inner cell mass, ICM)를 outgrowth로 분리하여 증식 시킴으로써 배아주(embryonic stem, ES)세포를 확립하고자 본 실험을 실시하였다. 과배란처리와 교미에 의해 생산된 ICR 생쥐의 3.5일 배반포를 sDMEM내의 배아성 섬유아단흥배양층에 배양하여 ICM세포의 증식을 조사한 결과, 3.5일부터 분리한 ICM세포들은 배양 7, 8일에 각각 1,500 및 3,200세포의 미분화세포로 증식하였다. 이들 세포의 계대배양에 의해 잠정적인 ES세포 colony를 얻었으며 10회의 계대배양후에도 그 형태가 변하지 않았다. 이들 세포는 다능성의 분화능을 보여 전형적인 ES세포 형태를 보였다. 이 같은 결과는 ICR배반포에서 outgrowth로 분리한 ICM으로부터 ES세포 확립이 가능함을 보여준 것이다.

• Clinical and Experimental Reproductive Medicine
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• 제28권1호
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• pp.33-40
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• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

• 한국수정란이식학회지
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• 제18권3호
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• pp.179-186
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• 2003

Human embryonic stem (hES) cell lines have been derived from human blastocysts and are expected to have far-reaching applications in regenerative medicine. The objective of this study is to improve freezing method with less cryo-injuries and best survival rates in hES cells by comparing various vitrification conditions. For the vitrifications, ES cells are exposed to the 4 different cryoprotectants, ethylene glycol (EG), 1,2-propanediol (PROH), EG with dime-thylsulfoxide (DMSO) and EG with PROH. We compared to types of vehicles, such as open pulled straw (OPS) or electron microscopic cooper grids (EM grids). Thawed hES cells were dipped into sequentially holding media with 0.2 M sucrose for 1 min, 0.1 M sucrose for 5 min and holding media for 5 min twice and plated onto a fresh feeder layer. Survival rates of vitrified hES cells were assessed by counting of undifferentiated colonies. It shows high survival rates of hES cells frozen with EG and DMSO (60.8％), or EG and PROH(65.8％) on EM grids better than those of OPS, compared to those frozen with EG alone (2.4％) or PROH alone (0％) alone. The hES cells vitrified with EM grid showed relatively constant colony forming efficiency and survival rates, compared to those of unverified hES cells. The vitrified hES cells retained the normal morphology, alkaline phosphates activity, and the expression of SSEA-3 and 4. Through RT-PCR analysis showed Oct-4 gene expression was down-regulated and embryonic germ layer markers were up-regulated in the vitrified hES cells during spontaneous differentiation. These results show that vitrification method by using EM grid supplemented with EG and PROH in hES cells may be most efficient at present to minimize cyto-toxicity and cellular damage derived by ice crystal formation and furthermore may be employed for clinical application.

• Clinical and Experimental Reproductive Medicine
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• 제44권4호
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• pp.193-200
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• 2017

Objective: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. Methods: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. Results: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p< 0.05). In the control-8 group ($22.1{\pm}4.6$), the cell number of the inner cell mass was higher than in the LAH groups (p< 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group ($92.8{\pm}8.9$) than in the others (p< 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group ($19.5{\pm}5.1$, p< 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group ($73.2{\pm}12.1$) than in the other groups, but without statistical significance. Conclusion: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.102-102
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• 2003

Embryonic stem (ES) cells proliferate extensively in the undifferentiated state and have the potential to differentiate into a variety of cell types in response to various environmental cues. The generation of functional dopaminergic neurons from ES cells is promising for cell replacement therapy to treat Parkinson's disease. We compared the in vitro differentiation potential of pluripotent human embryonic stem (hES, MB03) cells induced with basic fibroblast growth factor (bFGF) or retinoic acid (RA). Both types of treatment resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)- $\alpha$ during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression, compared to control (P<0.05). In contrast, no effect was observed on the rate of mature or glutamic acid decarboxylase-positive neurons. Immunostaining and HPLC analyses revealed the higher levels of TH (20.3%) and dopamine in bFGF and TGF-$\alpha$ treated hES cells than in RA or BDNF treated hES cells. The results indicate that TGF-$\alpha$ may be successfully used in the bFGF induction protocol to yield higher numbers of functional dopaminergic neurons from hES cells.

• Current Photovoltaic Research
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• 제2권2호
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• pp.59-62
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• 2014

Minority carrier diffusion length is one of the most important parameters of solar cells, especially for short circuit current density (Jsc). In this report, we proposed the calculating method of the minority carrier diffusion length ($L_n$) in CIGS solar cells through biased quantum efficiency (QE). To verify this method's reliability, we chose two CIGS samples which have different grain size and calculated $L_n$ for each sample. First of all, we calculated out that $L_n$ was 56nm and 97nm for small and large grain sized-cell through this method, respectively. Second, we found out the large grain sized-cell has about 7 times lower defect density than the small grain sized-cell using drive level capacitance profiling (DLCP) method. Consequently, we confirmed that $L_n$ was mainly affected by the micro-structure and defect density of CIGS layer, and could explain the cause of Jsc difference between two samples having same band gap.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.247-252
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• 2004

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo. Human ES cells have the capacity to differentiate into various types of cells in the body. Human ES cells are indefinite source of cells for cell therapy in various degenerative disorders including neuronal disorders. Directed differentiation of human ES cells is a prerequisite for their clinical application. The objective of this study is to develop the culture condition for the derivation of neural precursor cells from human ES cells. Neural precursor cells were derived from human ES cells in a stepwise culture condition. Neural precursor cells in the form of neural rosette structures developed into neurospheres when cultured in suspension. Suspension culture of neurospheres has been maintained over 4 months. Expressions of nestin, soxl, sox2, pax3 and pax6 transcripts were upregulated during differentiation into neural precursor cells by RT-PCR analysis. In contrast, expression of oct4 was dramatically downregulated in neural precursor cells. Immunocytochemical analyses of neural precursor cells demonstrated expression of nestin and SOX1. When induced to differentiate on an adhesive substrate, neuro-spheres were able to differentiate into three lineages of neural systems, including neurons, astrocytes and oligo-dendrocytes. Transcripts of sox1 and pax6 were downregulated during differentiation of neural precursor cells into neurons. In contrast, expression of map2ab was elevated in the differentiated cells, relative to those in neural precursor cells. Neurons derived from neural precursor cells expressed NCAM, Tuj1, MAP2ab, NeuN and NF200 in immunocytochemical analyses. Presence of astrocytes was confirmed by expression of GFAP immuno-cytochemically. Oligodendrocytes were also observed by positive immuno-reactivities against oligodendrocyte marker O1. Results of this study demonstrate that a stepwise culture condition is developed for the derivation of neural precursor cells from human ES cells.

• 대한물리의학회지
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• 제14권2호
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• pp.63-70
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• 2019

PURPOSE: While electrical stimulation (ES) is known to be a safe and flexible tool in rehabilitation therapy, it has had limited adoption in muscle regeneration. This study was performed to investigate whether ES can induce myogenic differentiation and to clarify the mechanism underlying the effects of ES on myogenic differentiation. METHODS: This study used rat L6 cell lines as myoblasts for myogenic differentiation. Electric stimulation was applied to the cells using a C-Pace EP culture pacer (IonOptix, Westwood, Ma, USA). The gene expressions of myogenic markers were examined using qPCR and immunochemistry. RESULTS: Our study showed that ES increased the thickness and length of myotubes during myogenic differentiation. It was found that ES increased the expression of myogenic markers, such as MyoD and Myogenin, and also activated the fusion of the myoblast cells. In addition, ES suppressed the expression of small GTPases, which can explain why ES promotes myogenic differentiation. CONCLUSION: We found that ES induced myogenic differentiation by suppressing small GTPases, inhibiting cell division. We suggest that ES-based therapies can contribute to the development of safe and efficient muscle regeneration.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.19-19
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• 2002

This study was to examine whether the in vitro differentiated neural cells derived from human embryonic stem (hES, MB03) cells can be survived and expressed tyrosin hydroxylase(TH) in grafted normal or PD rat brain. To differentiate in vitro into neural cells, embryoid bodies (EB: for 5 days, without mitogen) were formed from hES cells, neural progenitor cells(neurosphere, for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) were produced from EB, and then finally neurospheres were differentiated into mature neuron cells in N2 medium(without bFGF) for 2 weeks. In normal rat brain, neural progenitor cells or mature neuron cells (1×10/sup 7/ cells/㎖) were grafted to the striatum of normal rats. After 2 weeks, when the survival of grafted hES cells was examined by immunohistochemical analysis, the neural progenitor cell group indicated higher BrdU, NeuN＋, MAP2＋ and GFAP＋ than mature neuron cell group in grafted sites of normal rats. This result demonstrated that the in vivo differentiation of grafted hES cells be increased simultaneously in both of neuronal and glial cell type. Also, neural progenitor cell grafted normal rats expressed more TH pattern than mature neuron cells. Based on this data, as a preliminary test, when the neural progenitor cells were grafted into the striatum of 6-hydroxydopamine lesioned PD rats, we confirmed the cell survival (by double staining of Nissl and NeuN) and TH expression. This result suggested that in vitro differentiated neural progenitor cells derived from hES cells are more usable than mature neuron cells for the neural cell grafting in animal model and those grafted cells were survived and expressed TH in normal or PD rat brain.