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Korean Journal of Fertility and Sterility 2005;32(2):133-147.
Published online June 1, 2005.
Establishment of Human Embryonic Stem Cells using Mouse Embryonic Fibroblasts and Human Fetal Fibroblasts as Feeder Cells.
Hye Won Cho, Kyoung Rae Ko, Mi Kyoung Kim, Jae Ik Lee, Su Il Sin, Dong Hyung Lee, Ki Hyung Kim, Kyu Sup Lee
1Department of Obstetrics and Gynecology, College of Medicine, Pusan National University, Korea.
2Yalemari Wemen's Clinic, Korea.
3Infertility Clinic, Pusan National University Hospital, Busan, Korea.
Abstract
OBJECTIVE
S: 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.
Key Words: Inner cell mass (ICM); Embryonic stem cell; Fibroblast; Feeder cell
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