Wnt signalling in an in vitro niche model for conjunctival progenitor cells

• Published in: Journal of tissue engineering and regenerative medicine Vol. 8; no. 12; pp. 969 - 977
• Main Authors: Schrader, S., O'Callaghan, A. R., Tuft, S. J., Beaconsfield, M., Geerling, G., Daniels, J. T.
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.12.2014; Hindawi Limited
• Subjects: 3T3 Cells; Animals; cell culture; cell interactions; Conjunctiva - cytology; Conjunctiva - metabolism; conjunctival stem cells; fibroblasts; In Vitro Techniques; Mice; ocular surface; Regenerative medicine; Signal Transduction; stem cell niche; Stem Cells - cytology; Tissue engineering; Wnt Proteins - metabolism; Wnt signalling; cell interactions; conjunctival stem cells; fibroblasts; stem cell niche; Wnt signalling; cell culture; ocular surface
• ISSN: 1932-6254; 1932-7005
• DOI: 10.1002/term.1599

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. Copyright © 2012 John Wiley & Sons, Ltd.