Chen Guofang, Xu Xinxiu, Zhang Lihong, Fu Yanbin, Wang Min, Gu Haifeng, Xie Xin
CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China.
1] CAS Key Laboratory of Receptor Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Shanghai 201203, China [2] Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
Cell Res. 2014 Sep;24(9):1121-36. doi: 10.1038/cr.2014.112. Epub 2014 Aug 22.
Maintaining the self-renewal of embryonic stem cells (ESCs) could be achieved by activating the extrinsic signaling, i.e., the use of leukemia inhibitory factor (LIF), or blocking the intrinsic differentiation pathways, i.e., the use of GSK3 and MEK inhibitors (2i). Here we found that even in medium supplemented with LIF, mESCs still tend to differentiate toward meso-endoderm lineages after long-term culture and the culture spontaneously secretes vascular endothelial growth factors (VEGFs). Blocking VEGF signaling with sunitinib, an anti-cancer drug and a receptor tyrosine kinase (RTK) inhibitor mainly targeting VEGF receptors (VEGFRs), is capable of maintaining the mESCs in the undifferentiated state without the need for feeder cells or LIF. Sunitinib facilitates the derivation of mESCs from blastocysts, and the mESCs maintained in sunitinib-containing medium remain pluripotent and are able to contribute to chimeric mice. Sunitinib also promotes iPSC generation from MEFs with only Oct4. Knocking down VEGFR2 or blocking it with neutralizing antibody mimicks the effect of sunitinib, indicating that blocking VEGF/VEGFR signaling is indeed beneficial to the self-renewal of mESCs. We also found that hypoxia-inducible factor alpha (HIF1α) and endoplasmic reticulum (ER) stress are involved in the production of VEGF in mESCs. Blocking both pathways inhibits the expression of VEGF and prevents spontaneous differentiation of mESCs. Interestingly, LIF may also exert its effect by downregulating HIF1α and ER stress pathways and subsequent VEGF expression. These results indicate the existence of an intrinsic differentiation pathway in mESCs by activating the autocrine VEGF signaling. Blocking VEGF signaling with sunitinib or other small molecules help to maintain the mESCs in the ground state of pluripotency.
维持胚胎干细胞（ESC）的自我更新可以通过激活外在信号通路来实现，即使用白血病抑制因子（LIF），或者阻断内在分化途径，即使用GSK3和MEK抑制剂（2i）。在这里，我们发现即使在添加了LIF的培养基中，小鼠胚胎干细胞（mESC）在长期培养后仍倾向于向中内胚层谱系分化，并且培养物会自发分泌血管内皮生长因子（VEGF）。用舒尼替尼（一种抗癌药物和主要靶向VEGF受体（VEGFR）的受体酪氨酸激酶（RTK）抑制剂）阻断VEGF信号通路，能够使mESC维持在未分化状态，而无需饲养细胞或LIF。舒尼替尼有助于从囊胚中获得mESC，并且在含有舒尼替尼的培养基中维持的mESC保持多能性，并且能够参与嵌合小鼠的形成。舒尼替尼还能促进仅用Oct4从成纤维细胞中诱导生成诱导多能干细胞（iPSC）。敲低VEGFR2或用中和抗体阻断它会模拟舒尼替尼的作用，表明阻断VEGF/VEGFR信号通路确实有利于mESC的自我更新。我们还发现缺氧诱导因子α（HIF1α）和内质网（ER）应激参与了mESC中VEGF的产生。阻断这两条通路会抑制VEGF的表达并防止mESC的自发分化。有趣的是，LIF也可能通过下调HIF1α和ER应激通路以及随后的VEGF表达来发挥其作用。这些结果表明mESC中存在通过激活自分泌VEGF信号通路的内在分化途径。用舒尼替尼或其他小分子阻断VEGF信号通路有助于将mESC维持在多能性的基础状态。
