Evaluation of Molecular Markers in Corneal Regeneration by Means of Autologous Cultures of Limbal Cells and Keratoplasty

Purpose: To determine the epithelial phenotype in patients with a limbal stem cell deficiency (LSCD) after ocular surface reconstruction with autologous cultured stem cells. To correlate the epithelial phenotype with the clinical outcome. Methods: Six eyes affected by LSCD, verified and graded by impression cytology, were treated with an autologous fibrin-cultured limbal stem cell graft. The clinical outcome was defined as a "success'' or a "failure,'' depending on ocular surface stability. To improve their visual function, 4 patients underwent lamellar or penetrating keratoplasty after the stem cell graft. The phenotype of the regenerated corneal epithelium was determined by immunofluorescence of the corneal button to detect CK12, CK3, CK19, and Muc1 as corneal and conjunctival markers. Results: After a mean follow-up of 24 months, 5 cases were defined as successes; 1 case presented an epithelial defect 4 months after grafting and was defined as a failure. Immunofluorescence performed on 4 patients after lamellar and penetrating keratoplasty confirmed the presence of epithelial corneal markers (CK12 and CK3) in 2 of the success cases and the presence of conjunctival markers (CK19 and Muc1) in the 1 failure case. In one of the success cases, both corneal and conjunctival markers were detected on the corneal button. All success cases showed maintenance of marker accounting for high proliferative potential (Delta Np63 alpha) after transplantation. Conclusions: Autologous cultures of limbal stem cells can regenerate a functional corneal epithelium in patients affected by unilateral LSCD. We showed a correlation between the clinical outcome and the molecular marker expression.