Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
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Establishment of Embryonic Stem Cell Line from Isolated Blastomeres from Mouse Preimplantation Embryos

생쥐 초기 배아에서 분리한 할구를 이용한 배아줄기세포주 확립

  • Lim, Chun Kyu (Laboratory of Reproductive Biology & Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Sung, Ji Hye (Laboratory of Reproductive Biology & Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Choi, Hye Won (Laboratory of Reproductive Biology & Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Cho, Jae Won (Laboratory of Reproductive Biology & Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Shin, Mi Ra (Laboratory of Reproductive Biology & Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine) ;
  • Jun, Jin Hyun (Laboratory of Reproductive Biology & Infertility, Cheil General Hospital and Women's Healthcare Center, Sungkyunkwan University School of Medicine)
  • 임천규 (성균관대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 성지혜 (성균관대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 최혜원 (성균관대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 조재원 (성균관대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 신미라 (성균관대학교 의과대학 제일병원 생식생물학 및 불임연구실) ;
  • 전진현 (성균관대학교 의과대학 제일병원 생식생물학 및 불임연구실)
  • Published : 2006.03.31
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Abstract

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

목 적: 본 연구에서는 착상전 생쥐 배아에서 분리한 할구를 이용하여 배아줄기세포주를 확립하고 그 효용성과 특성을 살펴보고자 하였다. 연구방법: 생쥐 (C57BL/6J)의 2- 또는 4-세포기 배아에서 투명대를 제거하고 할구를 분리하여 지지세포와 공동배양한 후 할구로부터 형성된 내세포괴를 분리하여 계대배양을 실시하였다. 계대배양 중인 세포주의 특성을 확인하기 위해 alkaline phosphatase 활성도와 표지 인자 및 관련 유전자 발현을 세포면역화학적 염색과 RT-PCR 방법으로 살펴보았다. 또한, 계대배양 중인 배아줄기 세포주의 염색체 분석을 실시하였다. 결 과: 전체적으로 2-세포기에서 분리한 할구와 4-세포기에서 분리한 할구에서 각각 3.0% (1/33)와 4.0% (1/25)의 효율로 배아줄기세포주를 확립할 수 있었다. 이는 4-세포기의 배아를 사용하였을 때의 16.7% (5/30)에 비해 현저하게 낮았다. 분리된 할구로부터 확립된 배아줄기세포주에서 SSEA-l 과 Oct-4의 발현을 관찰하였고, 이들에서 분화된 배아체에서 삼배엽성 분화 관련 유전자들의 발현도 확인할 수 있었다. 결 론: 본 연구에서는 동물모델을 이용하여 착상전 초기 배아에서 분리한 할구를 이용하여 배이줄기세포를 확립할 수 있음을 확인하였다. 지속적인 관련 연구를 통해 인간의 체외수정 및 배아이식술에서 배아의 파괴 또는 발생 능력에 손상을 주지 않고 새로운 인간 배아줄기세포주를 생산할 수 있는 방법을 개발하고 실용화할 수 있을 것으로 사료된다.

Keywords

Acknowledgement

Supported by : 제일의료장학재단

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