• Clinical and Experimental Reproductive Medicine
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• v.29 no.1
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• pp.1-12
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• 2002

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. Materials and Methods: After Fl hybrid (C57BL female $\times$ CBA mael) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). Results: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. Conclusion: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.