Phenotypic change and induction of cytokeratin expression during in vitro culture of corneal stromal cells

Sidney, Laura E.; McIntosh, Owen D.; Hopkinson, Andrew

Abstract

Purpose: Cells of the corneal epithelium and stroma can be distinguished in vivo by different intermediate filaments, cytokeratins for corneal epithelial cells (CEC) and vimentin for keratocytes. Isolated and cultured keratocytes change phenotype, losing expression of keratocyte markers and gaining markers associated with mesenchymal stromal cells (MSC). This study investigates this change in phenotype in relation to intermediate filament expression in cultured corneal stromal cells (CSC) compared to CEC.

Methods: Expression of epithelial markers (CK3, CK12, CK19, pan cytokeratin, E-cadherin), keratocyte markers (CD34, vimentin) and MSC markers (CD73, CD90 and CD105) were compared in CEC and CSC by immunocytochemistry and RT-qPCR. Expression was evaluated at different stages of CSC culture and compared to another stromal cell type, extracted from Wharton’s jelly (WJ-MSC).


Results: In vivo keratocytes did not express cytokeratins. However, cultured CSC expressed epithelial-associated CK3, CK12 and CK19 but other cytokeratins. Expression of cytokeratins increased as CSC were passaged and decreased as CSC were induced to become quiescent. Comparatively, WJ-MSC, expressed lower levels of CK3, CK12 and CK19, but also stained for pan cytokeratin and expressed KRT5.


Conclusions: Cultured CSC undergo phenotypic change during culture, expressing specific cytokeratin filaments normally associated with CEC. Cytokeratin expression begins as cells are cultured on plastic and increases with passage. This discovery may influence the way that differences are discerned between cultured CEC and CSC. Investigators need to be aware that the expression of cytokeratins does not necessarily represent epithelial contamination, and that CEC and CSC may be more related than previously recognised.

Citation

Sidney, L. E., McIntosh, O. D., & Hopkinson, A. (2015). Phenotypic change and induction of cytokeratin expression during in vitro culture of corneal stromal cells. Investigative Ophthalmology & Visual Science, 56(12), https://doi.org/10.1167/iovs.15-17810

• Journal Article Type: Article
• Acceptance Date: Oct 8, 2015
• Publication Date: Nov 1, 2015
• Deposit Date: Jan 14, 2016
• Publicly Available Date: Jan 14, 2016
• Journal: Investigative Ophthalmology and Visual Science
• Print ISSN: 0146-0404
• Electronic ISSN: 1552-5783
• Publisher: Association for Research in Vision and Ophthalmology
• Peer Reviewed: Peer Reviewed
• Volume: 56
• Issue: 12
• DOI: https://doi.org/10.1167/iovs.15-17810
• Keywords: Cornea, Corneal stromal cells, Cytokeratins, Corneal epithelial cells, Mesenchymal stem cells
• Public URL: https://nottingham-repository.worktribe.com/output/981558
• Publisher URL: http://iovs.arvojournals.org/article.aspx?articleid=2469548
• Contract Date: Jan 14, 2016