Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
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Establishment of Mouse Embryonic Stem Cell-like Cells from In Vitro Fertilized Embryos

체외수정 생쥐 배아에서의 배아 줄기세포 확립

  • Shin, Yong-Moon (Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Park, Yong-Bin (Institute of Reproductive Medicine and Population, Medical Research Cente) ;
  • Kim, Hee-Sun (Institute of Reproductive Medicine and Population, Medical Research Cente) ;
  • Oh, Sun-Kyung (Institute of Reproductive Medicine and Population, Medical Research Cente) ;
  • Chun, Dae-Woo (Institute of Reproductive Medicine and Population, Medical Research Cente) ;
  • Suh, Chang-Suk (Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Choe, Young-Min (Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Kim, Jung-Gu (Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Lee, Jin-Yong (Department of Obstetrics and Gynecology, College of Medicine, Seoul National University) ;
  • Kim, Seok-Hyun (Department of Obstetrics and Gynecology, College of Medicine, Seoul National University)
  • 문신용 (서울대학교 의과대학 산부인과학교실) ;
  • 박용빈 (의학연구원 인구의학연구소) ;
  • 김희선 (의학연구원 인구의학연구소) ;
  • 오선경 (의학연구원 인구의학연구소) ;
  • 천대우 (의학연구원 인구의학연구소) ;
  • 서창석 (서울대학교 의과대학 산부인과학교실) ;
  • 최영민 (서울대학교 의과대학 산부인과학교실) ;
  • 김정구 (서울대학교 의과대학 산부인과학교실) ;
  • 이진용 (서울대학교 의과대학 산부인과학교실) ;
  • 김석현 (서울대학교 의과대학 산부인과학교실)
  • Published : 2002.03.30
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Abstract

Objective: In order to acquire the technique for the establishment of human embryonic stem cells (ESe) 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 Fl hybrid (C57BL female $\times$ CBA mael) 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.

Keywords

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