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Korean Journal of Fertility and Sterility 2001;28(1):33-40.
Published online March 1, 2001.
Establishment of Human Embryonic Stem Cells Derived from Frozen-Thawed Blastocysts.
Eun Young Kim, Hwa Kyung Nam, Keum Sil Lee, Sae Young Park, Eun Mi Park, Ji Yeon Yoon, Young Tae Heo, Hyun Jung Cho, Sepill Park, Kil Saeng Chung, Jin Ho Lim
1Maria Infertility Medical Institute, Seoul, Korea.
2Maria Biotech, Seoul, Korea.
3College of Animal Husbandry, KonKuk University, Seoul, Korea.
4Maria Hospital, Seoul, Korea.
Abstract
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.
Key Words: Frozen-thawed human blastocysts; Inner cell mass (ICM); Embryonic stem (ES) cell; Differentiation


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