Cultured Human Keratocytes from the Limbus and Cornea Both Express Epithelial Cytokeratin 3: Possible Mesenchymal-Epithelial Transition

Abstract

The corneal limbus is the repository of epithelial stem cells (SC) that sustain the turnover of corneal epithelial cells. The limbus stroma contains mesenchymal SC that generates stromal keratocytes. Mesenchymal-epithelial transition is a phenomenon wherein cells of mesenchymal phenotype can transdifferentiate to epithelial phenotype. Our aim was to study whether limbal keratocytes, cytokeratin 3 (CK3) negative, could be induced to transdifferentiate into CK3 positive cells. Human keratocytes were isolated from the limbus and cornea of cadaver donors, cultured and evaluated for CD34, CK3 and vimentin expression by immunofluorescence and RTPCR and for keratocan by RT-PCR. All cells regardless of site expressed vimentin and some also expressed CD34 and CK3. Double immunofluorescence revealed three subpopulations: CK3−/CD34+, CK3+/CD34+ and CK3+/CD34−. Total CD34 cell yield was higher in the limbus with a peak time to confluence (TTC) of more than 30 days. Total CK3 cell yield was greater in the cornea with a peak TTC of less than 30 days. Increasing donor age corresponded to a decreased CD34 yield and an increased CK3 yield. CK3−/CD34+ and CK3+/CD34− cells behaved similarly to total CD34 and CK3 cells in relation to age, site and TTC while CK3+/CD34+ cells showed intermediate features. Keratocan was present in corneal samples.