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Stefan Schrader, Stephen J. Tuft, Michele Beaconsfield, Gerd Geerling, Julie T. Daniels; Wnt Signalling In An In Vitro Niche Model For Conjunctival Progenitor Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1927.
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© ARVO (1962-2015); The Authors (2016-present)
Our group has recently shown, that co-culture of mitotically active conjunctival fibroblasts (HCF) with human conjunctival epithelial cells (HCEC) improves the maintenance of epithelial cells with progenitor cell characteristics during in vitro expansion. The aim of this study was to examine differences in gene expression between this co-culture system and routine culture conditions by microarray gene analysis.
Human conjunctival epithelial cells were expanded from bulbar biopsies and then transferred to the serum free co-culture system (HCEC-HCF) and a culture system in which growth arrested 3T3 feeder cells and feotal calf serum are used (HCEC-3T3). Differences in gene expression between both culture systems were explored using a genome level microarray platform (Affymetrix GeneChip® Human Gene 1.0 ST Array) and the results of selected genes were confirmed by quantitative RT-PCR.
The microarray analysis revealed a significant upregulation of 1372 and downregulation of 1334 genes in the HCEC-HCF compared to the HCEC-3T3 system. The gene clustering showed 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 of the Wnt signalling pathway by Q-PCR array revealed significant downregulation of the Wnt ligands Wnt3, Wnt4, Wnt7B, Wnt10A, Wnt receptor proteins FZD1, LRP5, LRP6, ß-Catenin and important Wnt target genes like CCND1.
Our 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.
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