Wang Jianyu, Wei Renyue, Bou Gerelchimeg, Liu Zhonghua
College of Life Science, Northeast Agricultural University of China, Harbin 150030, China; Institute of Life Sciences, Chong Qing Medical University, Chong Qing 400016, China.
College of Life Science, Northeast Agricultural University of China, Harbin 150030, China.
Biochem Biophys Res Commun. 2015 Jan 16;456(3):743-9. doi: 10.1016/j.bbrc.2014.12.041. Epub 2014 Dec 13.
Porcine induced pluripotent stem cells (piPSCs) had been reported during the past 5years, but there were few reports on how the cell signaling works in piPSCs. In order to clarify the signaling work that dominated the characteristic difference of two types of piPSCs which were derived from Oct4, Sox2, Klf4 and c-Myc (termed 4F piPSCs) and Oct4, Sox2, Klf4, c-Myc, Tbx3 and Nr5α2 (termed 6F piPSCs) respectively, we performed this study. 4F piPSCs and 6F piPSCs were cultured in medium with or without the ROCK inhibitor Y27632 after dissociating into single cells, the efficiency of a single cell colony and the number of AP positive colonies were assessed. The total RhoA and GTP-bind RhoA were detected in 4F piPSCs and 6F piPSCs before and after digestion into single cells. To explore the relationship between RHO-ROCK-MLC signaling pathway and the two factors Tbx3 and Nr5α2, the 4F piPSCs were infected with lenti-virus Tbx3 and Nr5α2 (termed 4F+TND). Results showed that the viability of cells could be enhanced by Y27632 and the RHO-ROCK-MLC signaling pathway was activated after dissociation into single cells in 4F piPSCs but not in 6F piPSCs. And, the 4F+TND piPSCs could be passaged and keep in high viability after dissociation into single cells, though the morphology of colonies did not change. These results indicated that the Tbx3 and Nr5α2 can improve the viability of piPSCs after dissociation into single cells by inhibiting the RHO-ROCK-MLC signaling pathway. And this provides useful information for establishing porcine pluripotent cells in future study.
在过去5年中已有猪诱导多能干细胞（piPSCs）的相关报道，但关于细胞信号在piPSCs中如何发挥作用的报道却很少。为了阐明主导分别由Oct4、Sox2、Klf4和c-Myc（称为4F piPSCs）以及Oct4、Sox2、Klf4、c-Myc、Tbx3和Nr5α2（称为6F piPSCs）衍生的两种类型piPSCs特征差异的信号传导作用，我们开展了本研究。将4F piPSCs和6F piPSCs解离为单细胞后，在添加或不添加ROCK抑制剂Y27632的培养基中培养，评估单细胞集落形成效率和碱性磷酸酶（AP）阳性集落数量。在将4F piPSCs和6F piPSCs消化为单细胞前后，检测总RhoA和GTP结合型RhoA。为了探究RHO-ROCK-MLC信号通路与Tbx3和Nr5α2这两个因子之间的关系，用慢病毒Tbx3和Nr5α2感染4F piPSCs（称为4F+TND）。结果显示，Y27632可提高细胞活力，4F piPSCs解离为单细胞后RHO-ROCK-MLC信号通路被激活，而6F piPSCs中未激活。并且，4F+TND piPSCs解离为单细胞后能够传代并保持高活力，尽管集落形态没有改变。这些结果表明，Tbx3和Nr5α2可通过抑制RHO-ROCK-MLC信号通路提高piPSCs解离为单细胞后的活力。这为未来研究中建立猪多能细胞提供了有用信息。
