Establishment of Mouse Embryonic Stem Cell-like Cells from In Vitro Fertilized Embryos / 대한불임학회지

OBJECTIVE: In order to acquire the technique for the establishment of human embryonic stem cells (ESC) 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 F1 hybrid (C57BL x CBA) 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.

The Study on Vitrification and Ultrarapid Thawing of Human Embryonic Stem Cells / 대한불임학회지

OBJECTIVE: This study was carried out to establish the effectiveness of the vitrification method and the optimal cryoprotectants in the cryopreservation of human embryonic stem cells (ESC). MATERIALS AND METHODS: Human ESC clumps established at Seoul National University Hospital (SNUhES 1) were cryopreserved with the vitrification method using the EM grid. EDS and EFS40 were used as vitrification solutions. RESULTS: Between the EDS and EFS40 groups, there was no significant difference in the recovery rate after cryopreservation of human ESC. The formation rates of ESC colonies in the vitrified groups were significantly lower than those in the control ESC group (p<0.05, p<0.05). In addition, the formation rate of ESC colonies in the EDS group was significantly higher than that in the EFS40 group (p<0.05). The ESC colonies in the vitrified groups were significantly smaller after culture duration of 2 and 4 days, respectively, compared with the control ESC group (p<0.01, p<0.05). However, these effects could be reduced to nonsignificant level by the additional culture of ESC colonies. The vitrified human ESC retained the properties of pluripotent cells, including the expression of cell surface markers for the undifferentiated cells such as alkaline phosphatase and SSEA-4 (stage-specific embryonic antigen-4), and the expression of transcription factor Oct-4 (octamer-binding transcription factor-4), and the normal karyotype. CONCLUSION: The vitrification method using the EM grid and EDS solution was confirmed to be very effective for the cryopreservation of human ESC.

THE EXPERIMENTAL STUDY OF CHIMERAS CONSTRUCTION FROM EMBRYONIC STEM CELL / 解剖学报

Objective To carry out construction of chimeras from embryonic stem cells of outbred KM mice. Methods We isolated embryonic stem cells from inner cell mass of KM mice blastocysts. Then we transferred embryonic stem cells into blastocoele of C57BL/6J inbred mice by microinjection in order to construct chimeric mice. Results The cells isolated from inner cell mass have typical characteristics,i e positive alkaline phophatase staining,normal karyotype,forming embryoid body. Now,we have constructed one chimeric mice successfully. Conclusion Embryonic stem cells isolated from inner cell mass can be used for the chimeras production successfully,which forms the substantial base of transgenic animal model by the way of using embryonic stem cells.