| 학회지 검색 | HOME > 논문 및 학회지 > 학회지 검색 |
대한생식의학회지 제32권 제2호 2010년
인간태아 섬유아세포와 생쥐배아 섬유아세포를 기저세포로 활용한 인간 배아줄기세포의 확립
부산대학교 의과대학 산부인과학교실1, 예일마리 산부인과2, 부산대학교병원 불임크리닉3
조혜원1,, 고경래2,김미경3,이재익3,신수일1,이동형1,김기형1,이규섭1
Establishment of Human Embryonic Stem Cells using Mouse Embryonic Fibroblasts and Human Fetal Fibroblasts as Feeder Cells
Hye Won Cho1, Kyoung Rae Ko2, Mi Kyoung Kim3, Jae Ik Lee3, Su Il Sin1, Dong Hyung Lee1, Ki Hyung Kim1, Kyu Sup Lee1
1Department of Obstetrics and Gynecology, College of Medicine, Pusan National University, 2Yalemari Wemen's Clinic, 3Infertility Clinic, Pusan National University Hospital, Busan, Korea
.
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.
키워드 : Inner cell mass (ICM), Embryonic stem cell, Fibroblast, Feeder cell
교신저자 : kuslee@pusan.ac.kr
전문 파일 : 
