Establishment of Human Embryonic Stem Cells using Mouse Embryonic Fibroblasts and Human Fetal Fibroblasts as Feeder Cells / 대한불임학회지

OBJECTIVES: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. METHODS: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. RESULTS: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46, XX karyotype. CONCLUSIONS: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

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.

Effects of Medium on Blastocyst Formation, Cell Number and Percentage of ICM in Mice / 대한불임학회지

OBJECTIVE: The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. MATERIALS AND METHODS: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values<0.05 were accepted as statistically significant. RESULTS: The blastulation rate in MEM (64.9+/-4.95%) was significantly higher (p=0.0031) than that in TCM (57.2+/-5.22%). No differences were found in the number of ZiB and ZeB between MEM (31.9+/-2.62, 33.0+/-4.58%), and TCM (27.2+/-4.28, 30.1+/-4.58%). A total 314 blastocysts (MEM=166; TCM= 148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM (73.1+/-3.3) than in MEM (61.7+/-2.5). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM (20.9+/-1.3 vs. 17.1+/-1.2%, p=0.0281). The ICM:TE ratio was higher in TCM than in MEM (1 : 4.85+/-0.68 vs. 1 : 3.78+/-0.78, NS). CONCLUSION: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

Effects of Medium on Blastocyst Formation, Cell Number and Percentage of ICM in Mice / 대한불임학회지

OBJECTIVE: The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. MATERIALS AND METHODS: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values<0.05 were accepted as statistically significant. RESULTS: The blastulation rate in MEM (64.9+/-4.95%) was significantly higher (p=0.0031) than that in TCM (57.2+/-5.22%). No differences were found in the number of ZiB and ZeB between MEM (31.9+/-2.62, 33.0+/-4.58%), and TCM (27.2+/-4.28, 30.1+/-4.58%). A total 314 blastocysts (MEM=166; TCM= 148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM (73.1+/-3.3) than in MEM (61.7+/-2.5). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM (20.9+/-1.3 vs. 17.1+/-1.2%, p=0.0281). The ICM:TE ratio was higher in TCM than in MEM (1 : 4.85+/-0.68 vs. 1 : 3.78+/-0.78, NS). CONCLUSION: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

Establishment of Human Embryonic Stem Cells Derived from Frozen-Thawed Blastocysts / 대한불임학회지

OBJECTIVE: This study was to establish the human embryonic stem (ES) cells derived from frozenthawed 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.