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。

Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways.

作者信息

Mei Xianglin, Zhao Hanhan, Ai Huihan, Wang Shuyue, Song Zhenbo, Zheng Lihua, Wang Guannan, Sun Ying, Bao Yongli

机构信息

National Engineering Laboratory for Druggable Gene and Protein Screening, Northeast Normal University, Changchun, 130117, China.

NMPA Key Laboratory for Quality Control of Cell and Gene Therapy Medicine Products, Northeast Normal University, Changchun, 130024, China.

出版信息

Cell Biosci. 2021 Aug 4;11(1):156. doi: 10.1186/s13578-021-00669-3.

DOI:10.1186/s13578-021-00669-3

PMID:34348786

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8336387/

Abstract

BACKGROUND

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

These findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

摘要

背景

干细胞已被广泛探索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，干细胞数量有限以及它们在体外长期传代培养时易于自发分化的倾向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，对于维持干细胞的自我更新能力和多能性至关重要。

方法

在本研究中，我们分别使用先前构建的由Oct4启动子和3'-UTR驱动的荧光素酶报告基因来筛选Oct4的潜在激活剂。采用集落形成试验、成球能力试验、碱性磷酸酶（AP）活性试验和畸胎瘤形成试验来评估硫酸莫达林（MDLS）在促进P19细胞自我更新和增强多能性方面的作用。采用免疫荧光、RT-PCR和蛋白质印迹法来检测干细胞相关基因的表达变化以及相关信号通路的激活情况。

结果

我们筛选了480种市售小分子化合物，发现MDLS在mRNA和蛋白质水平上均极大地促进了Oct4的表达。此外，MDLS显著促进了P19细胞的自我更新能力。而且，我们观察到在MDLS处理的集落中多能性标志物和碱性磷酸酶（AP）的表达显著增加。此外，MDLS可促进畸胎瘤形成并增强P19细胞在体内的分化潜能。另外，我们发现，在白血病抑制因子（LIF）存在的情况下，MDLS可替代饲养层细胞来维持OG2-mES细胞（Oct4-GFP报告基因小鼠胚胎干细胞系）的未分化状态，并且经MDLS扩增的OG2-mES细胞在体外显示出多能性标志物表达水平升高。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，并且这些效应可通过相应信号通路抑制剂的处理而逆转。