LIF를 첨가한 배양액을 이용한 할구 유래 생쥐 배아줄기세포주의 확립
Derivation of mouse ES cells from isolated blastomeresin media supplemented LIF
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본 연구에서는 LIF의 첨가가 분리된 할구 유래의 생쥐 배아줄기세포 확립에 미치는 영향을 살펴보았다. 과배란 유도된 BDF1 생쥐로부터 2-세포기의 배아를 회수하여 각기 LIF가 첨가되지 않은 배양액과 1,000, 2,500, 5,000 U/ml의 LIF가 첨가된 배양액에서 포배기 배아까지 배양하였다. 배양된 포배기배아는 차별화 염색 방법을 이용하여 내세포괴와 영양외배엽의 수를 계수하였다. 2,500 U/ml의 LIF 첨가 시 대조군(21.0±4.0 vs. 15.9±5.0, P < 0.01)과 1,000 U/ml의 LIF를 처리한 군(21.0±4.0 vs. 16.6±4.9, P < 0.05)에 비해서 내세포괴의 수가 유의하게 (P<0.05) 증가하였다. 배아줄기세포주 확립 배양액으로는 FBS 대신 20% KSR과 0.01 mg/ml의 ACTH를 사용하였다. 2,500 U/ml의 LIF를 첨가 시 배아줄기세포 확립 효율이 36.7% (11/30)로 가장 높은 효율을 나타내었다. 이러한 배양조건을 기본으로 하여 2-와 4-세포기 배아의 단일 할구를 분리하여 각기 21.4% (3/14)와 4.0% (1/20)의 효율로 단일 할구 배아줄기세포주를 확립할 수 있었다. 단일 할구로부터 확립된 배아줄기세포주와 이들에서 분화된 배아체는 세포면역학적 염색 방법과 RT-PCR 방법을 통해 그들의 미분화 특성과 삼배엽성 분화 특성을 확인할 수 있었다. 결론적으로 배양액에 LIF를 첨가하여 포배기 배아의 내세포괴 수를 증가시킬 수 있었으며, 분리된 할구의 배아줄기세포주 확립 효율을 향상시킬 수 있었다.
Abstract
This study was carried out to investigate the effect of leukemia inhibitory factor (LIF) on the derivation of mouse ES cells from isolated blastomeres. Two-cell stage mouse embryos were obtained from superovulated BDF1 female mice. Collected embryos were cultured to blastocyst stage in culture medium supplemented with 0, 1,000, 2,500 or 5,000 U/ml of LIF. Cultured blastocysts were examined by counting the number of cells in the inner cell mass (ICM) and trophectoderm (TE) using differential staining method. When 2-cell embryos were cultured with 2,500 U/ml of LIF, the cell numbers of ICM increased significantly in comparing with those of the control (21.04.0 vs. 15.95.0, P < 0.01) and 1,000 U/ml of LIF-containing group (21.04.0 vs. 16.64.9, P < 0.05). We used an ES cell establishment medium with 20% Knockout Serum Replacement (KSR) and 0.01 mg/ml ACTH instead of fetal bovine serum (FBS). Establishing efficacy of ES cell lines were the highest in 2,500 U/ml of LIF-containing group as 36.7% (11/30). We applied this culture medium to the cultivation of isolated blastomeres and to derivate ES cell lines. Three ES cell lines (21.4%) from isolated blastomeres of 2-cell stage embryos were established. In further experiments, we could establish one ES cell line (4.0%) from single blastomere of 4-cell stage embryo. Growing ES cells and their embryoid bodies were characterized by analyzing gene expression for undifferentiation and differentiation marker gene using immunocytochemistry and RT-PCR. In conclusion, LIF supplementation in culture medium could increase the cell number in ICM of blastocysts and support derivation of ES cell lines from isolated blastomeres.
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