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Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways
Cell and Bioscience ( IF 7.5 ) Pub Date : 2021-08-04 , DOI: 10.1186/s13578-021-00669-3
Xianglin Mei 1, 2 , Hanhan Zhao 1, 2 , Huihan Ai 1 , Shuyue Wang 1 , Zhenbo Song 2 , Lihua Zheng 2 , Guannan Wang 2 , Ying Sun 2 , Yongli Bao 1
Affiliation  

Stem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells. In the present study, we used the previously constructed luciferase reporters driven by the promoter and 3’-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways. We screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways. These findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

中文翻译:

硫酸莫达林通过 JAK/STAT3 和 Wnt 信号通路促进 Oct4 表达并维持干细胞的自我更新和多能性

干细胞已被广泛用于各种再生医学应用,并且它们在许多疾病的临床治疗中发挥着重要作用。然而,有限数量的干细胞及其在体外扩展繁殖时发生自发分化的趋势限制了它们的实际应用。八聚体结合转录因子 4 (Oct4) 是一种属于 POU 转录因子家族 V 类的转录因子,是维持干细胞自我更新能力和多能性的基础。在本研究中,我们使用先前构建的荧光素酶报告基因分别由 Oct4 的启动子和 3'-UTR 驱动来筛选 Oct4 的潜在激活剂。集落形成试验,球形成能力试验,碱性磷酸酶 (AP) 活性测定和畸胎瘤形成测定用于评估硫酸莫代尔 (MDLS) 在促进 P19 细胞自我更新和增强多能性中的作用。免疫荧光、RT-PCR和蛋白质印迹法用于测量茎相关基因的表达变化和相关信号通路的激活。我们筛选了 480 种市售小分子化合物,发现 MDLS 在 mRNA 和蛋白质水平上都极大地促进了 Oct4 的表达。此外,MDLS显着促进了P19细胞的自我更新能力。此外,我们观察到多能性标志物和碱性磷酸酶 (AP) 的表达在 MDLS 处理的菌落中显着增加。此外,MDLS 可以促进畸胎瘤形成并增强体内 P19 细胞的分化潜能。此外,我们发现在LIF存在的情况下,MDLS可以替代饲养细胞来维持OG2-mES细胞(Oct4-GFP报告基因小鼠胚胎干细胞系)的未分化状态,并且MDLS扩增的OG2-mES细胞表现出升高的表达体外多能性标志物的水平。最后,我们发现 MDLS 通过激活 JAK/STAT3 和经典 Wnt 信号通路来促进 Oct4 表达,并且这些作用通过用相应信号通路的抑制剂处理而逆转。这些发现首次证明了 MDLS 可以保持干细胞的自我更新和多能性。MDLS 扩增的 OG2-mES 细胞在体外表现出多能性标志物的表达水平升高。最后,我们发现 MDLS 通过激活 JAK/STAT3 和经典 Wnt 信号通路来促进 Oct4 表达,并且这些作用通过用相应信号通路的抑制剂处理而逆转。这些发现首次证明了 MDLS 可以保持干细胞的自我更新和多能性。MDLS 扩增的 OG2-mES 细胞在体外表现出多能性标志物的表达水平升高。最后,我们发现 MDLS 通过激活 JAK/STAT3 和经典 Wnt 信号通路来促进 Oct4 表达,并且这些作用通过用相应信号通路的抑制剂处理而逆转。这些发现首次证明了 MDLS 可以保持干细胞的自我更新和多能性。
更新日期:2021-08-04
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