Establishment of putative ground state rabbit embryonic stem cell lines using molecular inhibitors of cell signaling pathways

• Main Authors: Yu-Hsuan Lin, 林鈺瑄
• Other Authors: Jyh-Cherng Ju
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/75534743293215124212

碩士 === 國立中興大學 === 動物科學系所 === 102 === Naïve and primed pluripotent cells differ in morphology, proliferative pattern, regulatory signaling pathway, gene expression profile, and particularly the germline transmissible capacity. The aim of this study was to establish naïve rabbit ES (rbES) cell lines using small molecule inhibitors of cell signaling pathways. In Experiment 1, FBS-based culture system supplemented with LIF, bFGF and various concentrations of inhibitors against GSK3β, MEK1 or MEK proteins was tested. Results showed that media supplemented with 2 µM GSK3β inhibitor (iGSK3β), 0.5 µM MEK1 inhibitor (iMEK1) or 0.5 µM MEK inhibitor (iMEK) alone had higher percentages of deriving stable rbES cell lines than those with other concentrations of inhibitors. When two optimized concentrations of inhibitors were used, stable cell lines were established in the FBS-based culture medium supplemented with LIF, bFGF and 2 µM iGSK3β + 0.5 µM iMEK, but not in the group with 2 µM iGSK3β + 0.5 µM iMEK1. Although all rbES cell lines, both treated with one and two inhibitors expressed the pluripotent marker Oct4, their colonies morphology remained flat-shaped. In Experiment 2, various concentrations of iGSK3β, iMEK1 or iMEK were tested in KSR-based culture systems supplemented with LIF and bFGF. When ES cell media were supplemented with either one or two inhibitors, the morphology of colony was changed from flat-shaped to dome-shaped, but no stable dome-shaped rbES cell lines were established. All were differentiated after subcultures. In media supplemented with 2 µM iGSK3β, 0.5 µM iMEK1 or 0.5 µM iMEK, significantly higher percentages of dome-shaped colonies were observed compared to those in control groups (24.2％ vs. 5.4％, 26.7％ vs. 5.4％, 36.1％ vs. 5.6％, P＜0.05). Upon treating with two inhibitors, rbES cells derived from the 2 μM iGSK3β + 0.5 μM iMEK group had higher percentages of dome-shaped colonies than those from the 2 μM iGSK3β + 0.5 μM iMEK1 treatment group (39.8% vs. 34.1%, P < 0.05). In Experiment 3, the mTeSRTM1-based culture medium supplemented with LIF and various concentrations of iGSK3β、iMEK1 or iMEK was tested. Results showed that percentages of dome-shaped colonies increased in the 3 µM iGSK3β (53.2％), 0.5 μM iMEK1 (36.6％) and 1 μM iMEK (74.9％) groups (53.6 %) rather than other treatment groups (12.7％- 63.3％, P＜0.05). However, no stable rbES cell lines were estabilished from those single inhibitor treated groups. In contrast, only the combined treatment group（3 μM iGSK3β + 1 μM iMEK）had established and maintained stable rbES cell lines, in addition to having a higher percentage of dome-shaped colonies, compared to the 3 μM iGSK3β + 1 μM iMEK1 treatment group (84.2% vs. 24.6, P <0.05). The established ES cell lines all fully expressed pluripotency markers including alkaline phosphatase (AP), Oct-4, TRA-1-60, TRA-1-81 and Nanog. These cells also had the capacity of forming embryoid bodies (EBs) and teratomas with the expression of marker genes of three germlayers. Western-blot analysis showed that the phosphorylated Akt, β-catenin and Oct4 were all increased, the phosphorylated Erk decreased. Based on this study, combined inhibition to GSK3β and MEK enhances formation of dome-shaped colonies and sustains stemness of rabbit ES cells, whose naïve status and germline differentiation capacity warrant further investigation.