Schrader S, O'Callaghan A R, Tuft S J, Beaconsfield M, Geerling G, Daniels J T
Cells for Sight Transplantation and Research Programme Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK; Moorfields Eye Hospital NHS Foundation Trust, London, UK; Department of Ophthalmology, University of Düsseldorf, Germany.
J Tissue Eng Regen Med. 2014 Dec;8(12):969-77. doi: 10.1002/term.1599. Epub 2012 Aug 30.
Mimicking an environment in vitro that is more similar to the stem cell niche in vivo, by co-culture of mitotically active conjunctival fibroblasts (HCF) with human conjunctival epithelial cells (HCECs), improves the maintenance of epithelial cells with progenitor cell characteristics during in vitro expansion. However, little is known about the pathways controlling the fate of the epithelial progenitor cells during in vitro culture. In this study, differences in gene expression between this in vitro 'niche' model and standard culture conditions, in which growth-arrested 3 T3 feeder cells and fetal calf serum are used, were explored using a genome level microarray platform, quantitative (q)RT-PCR and western blot. The microarray analysis revealed significant alterations of biological processes involved in cell proliferation, differentiation and cell death. The analysis of stem cell-related pathways indicated changes in expression of genes involved in the Wnt signalling pathway, and further investigation by qPCR revealed significant downregulation of the Wnt ligands Wnt3, Wnt4, Wnt7B and Wnt10A, Wnt receptor proteins FZD1, LRP5, LRP6, ß-catenin and TCF7L1 and important Wnt target genes, such as CCND1, also confirmed by western blot and immunocytochemistry. The results indicate that epithelial cell expansion in the HCEC-HCF co-culture system is accompanied by significant changes in expression of genes involved in the Wnt signalling pathway. This altered pathway activation might be involved in the enhanced maintenance of epithelial progenitor cells in this in vitro 'niche' model.
通过将有丝分裂活跃的结膜成纤维细胞（HCF）与人结膜上皮细胞（HCEC）共培养，在体外模拟更类似于体内干细胞微环境的环境，可改善体外扩增过程中具有祖细胞特征的上皮细胞的维持。然而，关于体外培养过程中控制上皮祖细胞命运的途径知之甚少。在本研究中，使用基因组水平的微阵列平台、定量（q）RT-PCR和蛋白质印迹法，探索了这种体外“微环境”模型与使用生长停滞的3T3饲养细胞和胎牛血清的标准培养条件之间基因表达的差异。微阵列分析揭示了细胞增殖、分化和细胞死亡相关生物学过程的显著改变。对干细胞相关途径的分析表明，Wnt信号通路相关基因的表达发生了变化，qPCR进一步研究显示Wnt配体Wnt3、Wnt4、Wnt7B和Wnt10A、Wnt受体蛋白FZD1、LRP5、LRP6、β-连环蛋白和TCF7L1以及重要的Wnt靶基因如CCND1显著下调，蛋白质印迹法和免疫细胞化学也证实了这一点。结果表明，HCEC-HCF共培养系统中上皮细胞的扩增伴随着Wnt信号通路相关基因表达的显著变化。这种改变的信号通路激活可能参与了这种体外“微环境”模型中上皮祖细胞维持的增强。
