Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Establishment and Maintenance of Embryonic Stem-like Cell Lines from In Vitro Produced Bovine Blastocysts

체외수정 유래 소 배반포로부터 유사 배아 줄기 세포의 확립 및 유지

  • Lee, Yu-Yeon (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Kim, Sun-Uk (Disease Model research Center, Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Kim, Ji-Su (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Song, Bong-Seok (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Cho, Yoon-Jeong (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Park, Jung-Sun (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Yu, Dae-Yeul (Disease Model research Center, Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Jin, Dong-Il (Division of Animal Science and Resources, Research Center for Transgenic and Cloned Pigs, Chungnam National University) ;
  • Lee, Kyung-Kwang (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB)) ;
  • Koo, Deog-Bon (Center for Regenerative Medicine Korea Research Institute of Bioscience and Biotechnology(KRIBB))
  • 이유연 (한국생명공학연구원 재생의학연구센터) ;
  • 김선욱 (한국생명공학연구원 질환모델연구센터) ;
  • 김지수 (한국생명공학연구원 재생의학연구센터) ;
  • 송봉석 (한국생명공학연구원 재생의학연구센터) ;
  • 조윤정 (한국생명공학연구원 재생의학연구센터) ;
  • 박정선 (한국생명공학연구원 재생의학연구센터) ;
  • 유대열 (한국생명공학연구원 질환모델연구센터) ;
  • 진동일 (충남대학교 축산학과) ;
  • 이경광 (한국생명공학연구원 재생의학연구센터) ;
  • 구덕본 (한국생명공학연구원 재생의학연구센터)
  • Published : 2007.09.30
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Abstract

This study was conducted to examine the establishment of bovine ES-like cells having pluripotency. The hatched blastocysts derived from culture of in vitro fertilized embryos for 10 to 12 days dissociated mechanically into ICM-and trophectoderm-rich clumps using needle, and cultured onto mitotically-inactivated MEF feeder layer. The primary colonies originated from ICM cells were detached mechanically 7 days after seeding and subsequent subculture was conducted at intervals of every 5 to 7 days. Two ES -like cell lines were established and maintained over 40 passages. Self-renewal of the established lines was confirmed by examining the alkaline phosphatase activity, stem cell-specific marker profiles including SSEA isotopes, Oct-4 and STAT3. Moreover, the established cell lines could produce anchorage-independent embryoid bodies (EBs) with gradual decrease of Oct-4 transcript level in time-dependent manner.

본 연구는 소 배반포의 내부 세포괴로부터 다능성(pluripotency)을 지닌 배아 줄기 세포(embryonic stem cell) 또는 그 유사 세포를 분리 및 배양함으로써 줄기 세포 관련 분야의 기반 기술을 확립하고자 하였다. 소 체외수정란을 $10{\sim}12$일간 체외배양하여 생산된 부화 배반포를 세포분열이 불활성화된 생쥐 태아 섬유아 세포(mouse embryonic fibroblast, MEF) 위에서 배양하여 콜로니 형성을 유도하였으며, 이들로부터 내부 세포괴 유래의 형태를 지닌 것만을 광학현미경 하에서 물리적으로 분리하여 약 $5{\sim}7$일 간격으로 계대배양을 실시하였다. 이러한 방법을 통하여 배아 줄기 유사 세포의 특성을 40계대 이상 유지하는 2개의 세포주를 확립하였다. 각각의 세포주들은 높은 alkaline phosphatase(AP) 활성을 지니고 있었으며, 형광 면역 염색법과 PCR 기법을 사용하여 Oct-4, Nanog, STAT3, SSEA3 및 SSEA4의 발현을 관찰할 수 있었다. 이러한 결과를 종합하여 볼 때, 본 연구에서는 소 배반포로부터 배아 줄기 세포주를 확립하는 제반 기술이 확립되었다고 판단되며, 향후 관련 분야 연구에 활용될 수 있을 것으로 기대된다.

Keywords

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