Department of Systems Biology, Agricultural Biotechnology, Research Institute of Iran , Karaj, Iran.
Department of Developmental Biology, University of Science and Culture, Academic Center for Education, Culture and Research (ACECR) , Tehran, Iran.
J Proteome Res. 2017 Oct 6;16(10):3585-3595. doi: 10.1021/acs.jproteome.7b00322. Epub 2017 Sep 13.
Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state when cultured with 2 inhibitors (2i) of extracellular signal-regulated kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors (R2i) of FGF4 and TGFβ. The molecular mechanisms that control ESC self-renewal and pluripotency are more important for translating stem cell technologies to clinical applications. In this study, we used the shotgun proteomics technique to compare the proteome of the ground state condition (R2i- and 2i-grown cells) to that of serum. Out of 1749 proteins identified, 171 proteins were differentially expressed (p < 0.05) in the 2i, R2i, and serum samples. Gene ontology (GO) analysis of differentially abundant proteins showed that the focal adhesion signaling pathway significantly down-regulated under ground state conditions. mESCs had highly adhesive attachment under the serum condition, whereas in the 2i and R2i culture conditions, a loss of adhesion was observed and the cells were rounded and grew in compact colonies on gelatin. Quantitative RT-PCR showed reduced expression of the integrins family in the 2i and R2i conditions. The serum culture had more prominent phosphorylation of focal adhesion kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures compared to serum. Activation of integrins by Mn in the 2i and R2i cultures resulted in reduced Nanog and increased the expression of lineage marker genes. In this study, we demonstrated that reduced focal adhesion enabled mESCs to be maintained in an undifferentiated and pluripotent state.
当用细胞外信号调节激酶 (MEK) 和糖原合成酶激酶-3 (GSK3) 的 2 种抑制剂 (2i) 和 Royan FGF4 和 TGFβ 的 2 种抑制剂 (R2i) 培养时，小鼠胚胎干细胞 (mESC) 可以保持多能状态。控制 ESC 自我更新和多能性的分子机制对于将干细胞技术转化为临床应用更为重要。在这项研究中，我们使用鸟枪法蛋白质组学技术比较了基础状态（R2i 和 2i 培养的细胞）和血清的蛋白质组。在鉴定出的 1749 种蛋白质中，有 171 种蛋白质在 2i、R2i 和血清样品中差异表达（p < 0.05）。差异丰富蛋白的基因本体论（GO）分析表明，在基础状态下，焦点粘附信号通路显著下调。mESC 在血清条件下具有高度粘附性，而在 2i 和 R2i 培养条件下，观察到粘附丧失，细胞呈圆形并在明胶上密集生长。定量 RT-PCR 显示 2i 和 R2i 条件下整合素家族的表达降低。与 2i 和 R2i 培养相比，血清培养中焦点粘附激酶 (FAK) 的磷酸化更为明显。与血清培养相比，2i 和 R2i 培养中细胞外信号调节激酶 (ERK)1/2 的活性降低。在 2i 和 R2i 培养中用 Mn 激活整合素导致 Nanog 减少，谱系标记基因表达增加。在这项研究中，我们证明了减少焦点粘附使 mESC 能够保持未分化和多能状态。
