• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.129-138
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• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

• Asian-Australasian Journal of Animal Sciences
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• 제23권9호
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• 한국발생생물학회지:발생과생식
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• 제12권3호
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• pp.297-303
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• 2008

본 논문은 인공적인 단백질인 MCL-1ES BH3M에 관한 것으로 MCL-1ES BH3M를 과발현시 세포사멸을 유도한다. MCL-1L을 주형으로 재조합 PCR을 통해서 MCL-1ES BH3M를 클로닝하였다. 새롭게 클로닝한 단백질인 MCL-1ES BH3M 단백질은 안정성을 유지하기 위해서 PEST 도메인이 제거되어 있으며, 다른 BCL-2 패밀리 단백질과의 결합을 조절하기 위해서 BH3도메인의 Leu-Arg-Arg-Val-Gly-Asp-Gly 서열을 7개의 Ala 잔기로 인위적으로 돌연변이를 유도하였다. MCL-1ES BH3M를 293T 세포에서 과발현할 경우 세포사멸을 유도하였고, 항-세포사멸 단백질인 MCL-1L을 같이 과발현하더라도 세포사멸을 유도하였다. 또한, 과발현시 Caspase 9과 3를 활성화하였으며 면역염색법을 통해서 MCL-1ES BH3M 과발현시 미토콘드리아에 MCL-1ES BH3M 단백질이 부분적으로 위치하는 것을 확인하였다. 이상의 결과로 MCL-1ES BH3M는 Caspase 9과 3의 활성을 통해서 세포사멸을 유도한다. 결론적으로 본 연구는 세포사멸을 유도하는 새로운 molecule을 클로닝하였고, 이 molecule에 의한 세포사멸 기능을 확인하였다.

• 한국가축번식학회지
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• 제24권4호
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• pp.451-455
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• 2000

본 연구는 TT2 embryonic stem(ES) cell을 이용하여 chimeric mouse를 생산하는데 있어서 더욱 간편한 공배양방법 개발하기 위하여 수행되었다. 유전자 적중 생쥐의 개발은 유전자의 기능을 연구하는데 매우 중요한 수단으로 이용되고 있다. 이러한 생쥐의 개발에 있어서 chimeric mouse를 생산하는 과정은 ES cell의 종류의 차이는 있지만 주로 배반포기의 수정란에 ES cell을 주입하고 있다. 이 기술은 고가의 미세조작장치 뿐만 아니라 고도의 기술을 요하고 있다. 그러므로 본 연구에서는 TT2 ES cell를 8세포기 수정란과 공배양할 때의 필요로 하는 적절한 ES cell의 수를 검증함으로써 chimeric mouse의 생산 효율을 높일 수 있었다. 각각 0.5$\times$$10^{6}$, 1$\times$$10^{6}$과 2$\times$$10^{6}$$m\ell$의 ES cell을 8 세포기의 수정란과 공배양하였을때 0.5$\times$$10^{6}$과 1$\times$$10^{6}$$m\ell$에서 높은 배반포기로의 발달율을 나타내었다. 또한 가임신된 생쥐에 이들 배반포기를 이식한 결과 1$\times$$10^{6}$$m\ell$에서 높은 chimeric mouse 생산 효율을 나타내었다. 이러한 결과는 적절한 수의 ES cell과 수정란을 공배양함으로써 매우 간단하게 효율 좋은 chimeric mouse을 얻을 수 있음을 제시하고 있다.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.139-147
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• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.83-83
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• 2002

This study was to compare the characteristics of parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Mouse oocytes were recovered from superovulated 4wks hybrid F1 (C57BL/6xCBA/N) female mice. The oocytes were treated with 7% ethanol for 5 min and 5 ㎍/㎖ cytochalasin-B for 4 h. For IVF, the oocytes were inseminated with epididymal sperm of hybrid Fl male mice (1×10/sup 6//㎖). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count in blastocysts was carried out differential labelling using propidium iodide (red) and bisbenzimide(blue). (omitted)

• 한국발생생물학회지:발생과생식
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• 제12권1호
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• pp.77-86
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• 2008

본 연구에서는 LIF의 첨가가 분리된 할구 유래의 생쥐 배아줄기세포 확립에 미치는 영향을 살펴보았다. 과배란 유도된 BDF1 생쥐로부터 2-세포기의 배아를 회수하여 각기 LIF가 첨가되지 않은 배양액과 1,000, 2,500, 5,000 U/mL의 LIF가 첨가된 배양액에서 포배기 배아까지 배양하였다. 배양된 포배기 배아는 차별화 염색 방법을 이용하여 내세포괴와 영양외배엽의 수를 계수하였다. 2,500 U/mL의 LIF 첨가 시 대조군($21.0{\pm}4.0$ vs. $15.9{\pm}5.0$, P<0.01)과 1,000 U/mL의 LIF를 처리한 군($21.0{\pm}4.0$ vs. $16.6{\pm}4.9$, P<0.05)에 비해서 내세포괴의 수가 유의하게(P<0.05) 증가하였다. 배아줄기세포주 확립 배양액으로는 FBS 대신 20% KSR과 0.01 mg/mL의 ACTH를 사용하였다. 2,500 U/mL의 LIF를 첨가 시 배아줄기세포 확립 효율이 36.7%(11/30)로 가장 높은 효율을 나타내었다. 이러한 배양 조건을 기본으로 하여 2-와 4-세포기 배아의 단일 할구를 분리하여 각기 21.4%(3/14)와 4.0%(1/20)의 효율로 단일 할구 배아줄기세포주를 확립할 수 있었다. 단일 할구로부터 확립된 배아줄기세포주와 이들에서 분화된 배아체는 세포면역학적 염색 방법과 RT-PCR 방법을 통해 그들의 미분화 특성과 삼배엽성 분화 특성을 확인할 수 있었다. 결론적으로 배양액에 LIF를 첨가하여 포배기 배아의 내세포괴 수를 증가시킬 수 있었으며, 분리된 할구의 배아줄기세포주 확립 효율을 향상시킬 수 있었다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.97-97
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• 2003

Embryonic stem (ES) cells, derived from preimplantation embryos, are able to differentiate into various types of cells consisting the whole body, or pluripotency. 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 (hES, MB03) cells and terminally differentiated cells (HeLa), we examined the ability of these two types of cells in confronting a severe oxidative insult, that is $H_2 O_2$. Ratio of dying cells as determined by the relative amount of dye neutral red entrapped within the cells after the exposures. Cell death rates were not significantly different when either MB03 or HeLa were exposed up to 0.4 mM $H_2 O_2$. However, relative amount of dye entrapped within the cells sharply decreased down to 0.12% in HeLa cells when the cells were exposed to 0.8 mM $H_2 O_2$, while it was approximately 54% in MB03. Pretreatment of cells with BSO (GSH chelator) and measurement of GSH content results suggest that cellular GSH is the major defensive mechanism of hES cells. Induction of apoptosis in hES cell was confirmed by DNA laddering, induction of Bax, and chromatin condensation. In summary, hES cells 1) are extremely resistant to oxidative stress, 2) utilize GSH as a major defensive mechanism. and 3) experience apoptosis upon exposure to oxidative stress.

• 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.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.45-51
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• 1999

소 배반포로부터 배아주 (embryonic stem, ES) 유사세포를 분리하기 위해서는 영양외배엽 (trophectoderm, TE) 세포를 제거하는 것이 효과적이다. 따라서 본 실험은 효과적으로 TE를 제거하기 위한 calcium ionophore A23187 (CIPA) 처리조건을 확립하고, 분리해낸 ES 유사세포의 in vitro 다능성 (pluripotency)을 검증하고자 수행하였다. CIPA 농도 및 처리시간을 달리 하였을 때 50 $\mu$M에서 25분간 처리한 군이 colony 형성율 (51%)및 10 passage 까지의 배양성적 (4.8%)에서 가장 좋은 결과를 나타내었다. 또한 CIPA를 처리하지 않은 군과의 비교에서도 약 5배의 높은 결과를 보임으로서 본 실험에서 확립된 CIPA 처리조건은 가시적인 toxicity 없이 ES 유사세포의 확립에 이용될 수 있음을 시사하였다. 확립된 ES 유사세포는 heterogeneous한 alkaline phosphatase (AP) 활성을 보여 소 ES 유사세포에 대한 타 보고들과 유사한 결과를 보였다. In vitro 부양배양 (suspension culture)에서는 embryoid body로 분화가 가능하였으며, 약 70% 정도의 euploidism을 보였다. 따라서 본 실험에서 확립된 CIPA의 처리조건이 소 배반포로부터 ES 유사세포를 확립하는데 효과적으로 이용될 수 있음을 확인할 수 있었다.