• Clinical and Experimental Reproductive Medicine
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• v.33 no.4
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• pp.265-272
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• 2006

Objective: This study was carried out to evaluate the effect of the isolation methods of inner cell mass from mouse blastocyst, types of feeder cells and treatment time of mitomycin C on the formation rate of ICM colony. Methods: The inner cells were isolated by conventional immunosurgery, partial trophoblast dissection with syringe needles and whole blastocyst co-culture method. Commercially available STO and primary cultured mouse embryonic fibroblast (pMEF) feeder cells were used, and mitomycin C was treated for 1, 2 or 3 hours, respectively. The formation rate of ICM colony was observed after isolation of ICM and culture of ICM on the feeder cells for 7 days. Result: The ICM colony formation rate on STO were significantly higher in partial trophoblast dissection group (58%) than that in immunosurgery (12%) or whole blastocyst culture (16%) group (p<0.05). The formation rate on pMEF feeder layer was higher in partial trophoblast dissection (88%) and whole blastocyst culture (82%) group than that in immunosurgery (16%) group (p<0.05). When mitomycin C treated to pMEF for 2 hours, the formation rate of 88% was significantly higher than those of other conditions. Conclusion: Above results showed that the efficient isolation method of ICM from blastocyst was the partial trophoblast dissection and the appropriate treatment time of mitomycin C was 2 hours. However, the subculture of ICM colony and characterization of stem cells should be carried out to confirm the efficacy of the partial trophoblast dissection method.

• 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.

• 대한생식의학회:학술대회논문집
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• 2004.11a
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• pp.39-39
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• 2004

• Development and Reproduction
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• v.12 no.1
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• pp.77-86
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• 2008

This study was carried out to investigate the effect of leukemia inhibitory factor (LIF) on the derivation of mouse ES cells from isolated blastomeres. Two-cell stage mouse embryos were obtained from superovulated BDF1 female mice. Collected embryos were cultured to blastocyst stage in culture medium supplemented with 0, 1,000, 2,500 or 5,000 U/mL of LIF. Cultured blastocysts were examined by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE) using differential staining method. When 2-cell embryos were cultured with 2,500 U/ml of LIF, the cell numbers of ICM significantly increased in comparing with those of the control($21.0{\pm}4.0$ vs. $15.9{\pm}5.0$, P<0.01) and 1,000 U/mL of LIF-containing group ($21.0{\pm}4.0$ vs. $16.6{\pm}4.9$, P<0.05). We used an ES cell establishment medium with 20% Knockout Serum Replacement and 0.01 mg/mL ACTH instead of fetal bovine serum. Establishing efficacy of ES cell lines were the highest in 2,500 U/mL of LIF-containing group as 36.7% (11/30). This culture medium was applied to the culture of isolated blastomeres and to derivate ES cell lines. Three ES cell lines (21.4%) from isolated blastomeres of 2-cell stage embryos were established. In further experiments, we could establish one ES cell line (4.0%) from single blastomere of 4-cell stage embryo. The subcultured ES cells and their embryoid bodies were characterized by analyzing gene expression for undifferentiation and differentiation marker gene using immunocytochemistry and RT-PCR. In conclusion, LIF supplementation in culture medium could increase the cell number in ICM of blastocysts and support derivation of ES cell lines from isolated blastomeres.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.49-60
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• 2008

Objective: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-${\beta}1$ on the development of 2-cell mouse embryos. Methods: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-${\beta}1$, its receptor-1, -2, integrin-${\beta}3$ and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. Results: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-${\beta}1$, its receptor-1, integrin-${\beta}3$ and prolactin except TGF-${\beta}1$-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). Conclusion: The administration of progesterone, estrogen and TGF-$\beta$ could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.25-33
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• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.