Partial Isolation of Corneal Epithelial Stem Cells by Adhesion to Collagen IV

Abstract

Abstract: : Purpose: The concept of corneal epithelium stem cells has been recognized for a decade, but isolation of the stem cells has never been achieved. The objective of this study was to isolate the corneal epithelial stem cells based on their properties and proposed molecular markers. Methods: Immunofluorescent staining and in situ hybridization of human corneal frozen sections were performed to evaluate expression of basal or stem cell markers, integrin ß1, cytokeratin 19 (K19), EGF receptor (EGFR) and nuclear protein p63, and differentiation markers, cytokeratin 3 (K3) and involucrin. Epithelial cells freshly isolated from human corneal limbus were allowed to adhere to collagen IV-coated dishes for 20 and 60 minutes, sequentially. The rapid adhering cells in 20 minute (RAC), later adhering cells in 60 minutes and non-adhering cells after 60 minutes were evaluated for stem cell properties by a) p63 staining, b) RT-PCR for ΔNp63 mRNA, and c) proliferative potential on a mitomycin C-treated 3T3 fibroblast feeder layer. Results: The expression of p63 protein and transcripts was located only in the nuclei of limbal epithelial basal layer. Integrin ß1, K19 and EGFR antibodies stained limbal basal cells much stronger than superficial cells. K3 and involucrin antibodies only stained the superficial cells. Among three populations isolated from limbal epithelial cells by adhesion to collagen IV, the RAC, accounting for 5-10% of whole population, displayed the highest number of p63 positive cells (47 ± 8 %), the strongest expression of ΔNp63 mRNA, and the greatest clonal growth capacity on a 3T3 feeder layer. The colonies generated from the RAC could be serially passaged and preserved. Conclusions: It was demonstrated for the first time that corneal epithelial cells with stem cell features can be isolated at least partially by adhesion to collagen IV. These rapid adhering cells have potential for use in corneal bioengineering and gene therapy.

Keywords: cornea: epithelium • gene/expression • immunohistochemistry