Cheng Yin, Lin Kai-Hsuan, Young Tai-Horng, Cheng Nai-Chen
Department of Surgery, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
Department of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, Taiwan.
Stem Cells Transl Med. 2020 Apr;9(4):518-530. doi: 10.1002/sctm.19-0234. Epub 2019 Dec 16.
Adipose-derived mesenchymal stem cells (ASCs) exhibit great potential in regenerative medicine, and in vitro expansion is frequently necessary to obtain a sufficient number of ASCs for clinical use. Fibroblast growth factor 2 (FGF2) is a common supplement in the ASC culture medium to enhance cell proliferation. To achieve clinical applicability of ASC-based products, prolonged culture of ASCs is sometimes required to obtain sufficient quantity of ASCs. However, the effect of FGF2 on ASCs during prolonged culture has not been previously determined. In this study, ASCs were subjected to prolonged in vitro culture with or without FGF2. FGF2 maintained the small cell morphology and expedited proliferation kinetics in early ASC passages. After prolonged in vitro expansion, FGF2-treated ASCs exhibited increased cell size, arrested cell proliferation, and increased cellular senescence relative to the control ASCs. We observed an upregulation of FGFR1c and enhanced expression of downstream STAT3 in the initial passages of FGF2-treated ASCs. The application of an FGFR1 or STAT3 inhibitor effectively blocked the enhanced proliferation of ASCs induced by FGF2 treatment. FGFR1c upregulation and enhanced STAT3 expression were lost in the later passages of FGF2-treated ASCs, suggesting that the continuous stimulation of FGF2 becomes ineffective because of the refractory downstream FGFR1 and the STAT3 signaling pathway. In addition, no evidence of tumorigenicity was noted in vitro and in vivo after prolonged expansion of FGF2-cultured ASCs. Our data indicate that ASCs have evolved a STAT3-dependent response to continuous FGF2 stimulation which promotes the initial expansion but limits their long-term proliferation.
脂肪来源的间充质干细胞（ASCs）在再生医学中展现出巨大潜力，体外扩增通常是获得足够数量的ASCs用于临床的必要手段。成纤维细胞生长因子2（FGF2）是ASC培养基中常用的添加剂，用于增强细胞增殖。为了使基于ASC的产品具有临床适用性，有时需要对ASCs进行长时间培养以获得足够数量的细胞。然而，FGF2在长时间培养过程中对ASCs的影响此前尚未确定。在本研究中，ASCs在有或无FGF2的条件下进行长时间体外培养。FGF2在ASC传代早期维持了细胞的小形态并加快了增殖动力学。长时间体外扩增后，与对照ASCs相比，FGF2处理的ASCs细胞尺寸增大、细胞增殖停滞且细胞衰老增加。我们观察到FGF2处理的ASCs初始传代中FGFR1c上调且下游STAT3表达增强。应用FGFR1或STAT3抑制剂可有效阻断FGF2处理诱导的ASCs增殖增强。在FGF2处理的ASCs后期传代中，FGFR1c上调和STAT3表达增强消失，这表明由于下游FGFR1和STAT3信号通路的不应性，FGF2的持续刺激变得无效。此外，FGF2培养的ASCs长时间扩增后，在体外和体内均未发现致瘤性证据。我们的数据表明，ASCs对持续FGF2刺激进化出了一种依赖STAT3的反应，这种反应促进了初始扩增，但限制了它们的长期增殖。
