Background: 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.
Methods: Human keratocytes were isolated from the limbus and cornea of cadaver donors, cultured and evaluated for CD34, CK3 and vimentin expression by immunofluorescence and RT-PCR and for keratocan by RT-PCR.
Results: 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.
Conclusions: Suspension cultured human keratocytes of the limbus behave as progenitor cells of corneal keratocytes being slower cycling and with a greater proportion expressing CD34. Cultured keratocytes both from the limbus and cornea are able to express CK3. This phenomenon may reflect mesenchymal-epithelial transition or, given the loss in vitro of the micro-anatomical features of the limbal-corneal area, may indicate the acquisition by keratocytes of a differentiation and migration pathway similar to that of the overlying epithelium. This suggests that the limbus/ corneal stromal niche may exhibit site-specific modulating abilities that direct the development of site-dependent intermediate filament repertoire of epithelial cells.
