Wong Yu Qian, Xu Hongyan, Wu Qiang, Liu Xinyu, Lufei Chengchen, Xu Xiu Qin, Fu Xin-Yuan
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597.
Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore 117599.
Stem Cells Int. 2018 Sep 4;2018:8632950. doi: 10.1155/2018/8632950. eCollection 2018.
Studies have demonstrated that STAT3 is essential in maintaining self-renewal of embryonic stem cells (ESCs) and modulates ESC differentiation. However, there is still lack of direct evidence on STAT3 functions in ESCs and embryogenesis because constitutive STAT3 knockout (KO) mouse is embryonic lethal at E6.5-E7.5, prior to potential functional role in early development can be assessed. Therefore, in this study, two inducible STAT3 ESC lines were established, including the STAT3 knockout (InSTAT3 KO) and pSTAT3 overexpressed (InSTAT3 CA) using Tet-on inducible system in which STAT3 expression can be strictly controlled by doxycycline (Dox) stimulation. Through genotyping, deletion of STAT3 alleles was detected in InSTAT3 KO ESCs following 24 hours Dox stimulation. Western blot also showed that pSTAT3 and STAT3 protein levels were significantly reduced in InSTAT3 KO ESCs while dominantly elevated in InSTAT3 CA ECSs upon Dox stimulation. Likewise, it was found that STAT3-null ESCs would affect the differentiation of ESCs into mesoderm and cardiac lineage. Taken together, the findings of this study indicated that InSTAT3 KO and InSTAT3 CA ESCs could provide a new tool to clarify the direct targets of STAT3 and its role in ESC maintenance, which will facilitate the elaboration of the mechanisms whereby STAT3 maintains ESC pluripotency and regulates ESC differentiation during mammalian embryogenesis.
研究表明，信号转导和转录激活因子3（STAT3）在维持胚胎干细胞（ESC）的自我更新以及调节ESC分化过程中至关重要。然而，目前仍缺乏关于STAT3在ESC和胚胎发生中功能的直接证据，因为组成型STAT3基因敲除（KO）小鼠在胚胎发育第6.5至7.5天就会胚胎致死，在此之前无法评估其在早期发育中的潜在功能作用。因此，在本研究中，我们建立了两种可诱导的STAT3 ESC系，包括使用Tet-on诱导系统建立的STAT3基因敲除（InSTAT3 KO）和pSTAT3过表达（InSTAT3 CA）的ESC系，在该系统中，STAT3的表达可通过强力霉素（Dox）刺激得到严格控制。通过基因分型，在Dox刺激24小时后，InSTAT3 KO ESC中检测到STAT3等位基因的缺失。蛋白质免疫印迹法也显示，在Dox刺激后，InSTAT3 KO ESC中pSTAT3和STAT3蛋白水平显著降低，而在InSTAT3 CA ESC中则显著升高。同样，研究发现STAT3缺失的ESC会影响ESC向中胚层和心脏谱系的分化。综上所述，本研究结果表明，InSTAT3 KO和InSTAT3 CA ESC可为阐明STAT3的直接靶点及其在ESC维持中的作用提供新工具，这将有助于详细阐述STAT3在哺乳动物胚胎发生过程中维持ESC多能性和调节ESC分化的机制。
