Liu Jiao, Zhu Xiumei, Li Jinshan, Liu Zhihui, Liu Yanhong, Xue Fei, Yang Lan, An Liyou, Chen Chien-Hong, Presicce Giorgio Antonio, Zheng Qiping, Du Fuliang
Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University Nanjing 210046, Jiangsu, P. R. China.
Renova Life, Inc. Jacksonville, Florida 32258, USA.
Am J Transl Res. 2019 Aug 15;11(8):5122-5133. eCollection 2019.
We previously developed pluripotent rabbit embryonic stem cells (rbES) using a culture system supplemented with basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF), noggin and Y-27632 (referred to as iFLY). In present work, we explored multiple approaches to enhance the chance of deriving domed pluripotent rbES cells by inhibition of MEK, GSK, and PKC signaling pathways. Domed stated rbES were derived in defined medium supplemented with 15% KOSR, 10 IU/mL mouse LIF, 10 ng/mL bFGF and three inhibitors to the MEK (PD0325901, 1 µM), GSK3 (CHIR99021, 3 µM) and PKC (Gö6983, 5 µM) (3i). Domed rbES were passaged every 3-4 days till passage 3-4 for the designated experiments. We showed that bFGF and LIF are indispensable for the derivation and maintenance of rbES; whereas the 3i medium containing inhibitors to the MEK (PD0325901), GSK3 (CHIR99021) and PKC (Gö6983) were necessary for deriving domed rbES. Domed rbES possessed naïve ES markers as and in addition to and by RT-PCR. Domed rbES showed positive staining for Rex1, Fgf4, Klf4, Nanog and Oct4 by immunofluorescence chemistry. Further deleting either one factor in 3i medium as CHIR99021, PD0325901, Gö6983 or bFGF resulted in disappearing of domed rbES colonies. The optimal concentrations of 3i contained 0.75 µM PD0325901, 2.25 µM CHIR99021, and 4.5 µM Gö6983. Our work, in combination of different inhibitors for deriving rabbit ES, supports that the network of signal pathways plays an important role in ES self-renew, propagation and maintenance, and sheds light on deriving authentic properties of rbES in an important yet understudied model animal species.
我们之前使用一种添加了碱性成纤维细胞生长因子（bFGF）、白血病抑制因子（LIF）、头蛋白和Y-27632（称为iFLY）的培养系统，成功培育出了多能性兔胚胎干细胞（rbES）。在当前的研究中，我们探索了多种方法，通过抑制MEK、GSK和PKC信号通路来提高获得穹顶状多能性rbES细胞的几率。在添加了15% KnockOut血清替代物（KOSR）、10 IU/mL小鼠LIF、10 ng/mL bFGF以及三种针对MEK（PD0325901，1 µM）、GSK3（CHIR99021，3 µM）和PKC（Gö6983，5 µM）的抑制剂（3i）的限定培养基中，获得了穹顶状rbES。在指定实验中，将穹顶状rbES每3 - 4天传代一次，直至传代3 - 4次。我们发现，bFGF和LIF对于rbES的培育和维持不可或缺；而含有针对MEK（PD0325901）、GSK3（CHIR99021）和PKC（Gö6983）抑制剂的3i培养基对于获得穹顶状rbES是必需的。通过逆转录聚合酶链反应（RT-PCR）检测，穹顶状rbES除了具有 和 外，还具有原始胚胎干细胞标志物 和 。通过免疫荧光化学检测，穹顶状rbES对Rex1、Fgf4、Klf4、Nanog和Oct4呈阳性染色。在3i培养基中进一步去除CHIR99021、PD0325901、Gö6983或bFGF中的任何一种因子，都会导致穹顶状rbES集落消失。3i的最佳浓度为0.75 µM PD0325901、2.25 µM CHIR99021和4.5 µM Gö6983。我们的研究结合了用于培育兔胚胎干细胞的不同抑制剂，证实信号通路网络在胚胎干细胞的自我更新、增殖和维持中起着重要作用，并为在一种重要但研究较少的模式动物物种中获得真正特性的rbES提供了线索。
