Maintenance and Differentiation of Pluripotential Embryonic Cell Lines from Mouse Blastocysts

BCF1 생쥐 배반포기 유래 배아간세포 작성에 관한 연구

The present study was designed to demonstrate that ES cell lines efficiently could be isolated from explanted blastocysts of hybrid BCF1 mouse when grown on STO feeder layer derived from mouse fibroblasts in culture medium supplemented with leukemia inhibitory factor (LIF). The expanded blastocysts were attached to mitomycin C-inactivated STO feeder layer and were cultured for 4 days. Four days later the ICM was disaggregated by a short term trypsin treatment (0.25% trypsin / 0.04% EDT A for 2-3 min). The resulting cell suspension was seeded on a new STO feeder layer and covered with DMEM supplemented with 10% FCS, 0.1 mM nonessential amino acid, 0.1 mM sodium pyruvate, 0.1 mM mercaptoethanol and 1,000 U/ml LIF. Colonies of ES-like cells were observed after the second passage. These colonies were repeatedly passaged at approximately 5 day intervals. In this study, five ES-like celllines were isolated by directly explanting blastocysts, but three lines were lost after the 5th passage, possibly due to toxic effects of a new FCS batch. The characterization of developmental potential of isolated cell lines was performed with respect to in vitro differentiation and specific activity of alkaline phosphatase (AP). When cells were cultured in suspension, the aggregates of cell lines were capable of forming simple embryoid bodies (EB), and showed the capacity for forming cystic multilayer EBs. In addition, the cell lines were positive for AP staining, a biochemical marker characteristic of mouse ES cells.

생쥐 배반포기 내부세포괴를 체외에서 분리 배양하여 분화가 억제된 내부세포괴 유래 증식세포를 미분화 상태에서 무한히 증식할 수 있는 전능성을 지닌 배아간세포(embryonic stem cell : ES cell)로 확립하고자 본 연구를 실시하였다. BCF1 생쥐 배반포를 10% FCS, 0.1mM nonessential amino acid, 0.1mM sodium pyruvate, 0.1mM 2-mercaptoethanol과 1,000U/ml LIF(세포분확억제인자)가 첨가된 DMEM 기초배양액에 mitomycin-C를 처리한 STO 단층배양세포에서 배양하여 분화가 억제된 내부세포괴 유래의 배아간세포를 분리하였다. 배반포를 STO 단층배양세포에서 4일간 배양하여 내부세포괴세포를 신선한 STO 단층배양세포에서 약 5일 간격으로 반복하여 계대배양을 실시하였다. 5차 계대배양후 뚜렷한 분화 양상없이 배양된 미분화 세포군에 대한 alkaline phosphatase (AP)염색과 체외분화능 검색을 실시한 결과 적색의 미분화 AP 양성반응이 확인되었으며 체외에서 배분화 형성이 유도됨에 따라 배양된 배아간세포주의 다능성 배아간세포 특성을 확인할 수 있었다.