Notch-1 Immunoexpression in Human and Mouse Cornea

Abstract

Purpose:: To determine whether Notch-1 could be used as a marker to identify stem cells in the limbal basal region. Notch-1 is known to maintain the cells in an undifferentiated manner in several systems. We also wanted to know whether Notch-1 and ABCG2 co-express as ABCG2 is considered as a useful corneal stem cell marker

Methods:: Mouse eyes were embedded in OCT and was prepared for cross- section. Human corneoscleral tissues were obtained from the eye bank and was prepared for cross- section and whole mount analysis. The tissue for whole mount was incubated in Dispase (18 hr at 4 ºC) and the epithelial cell sheet was gently removed and fixed immediately in 4% paraformaldehyde. The sections and whole mounts were stained with antibodies against Notch-1 and ABCG2.

Results:: Immunofluorescence data using Notch-1 monoclonal antibody revealed that Notch-1 was expressed in the limbal basal cells both in mouse and in human cornea. The Notch-1 antigenicity was more pronounced in clusters of cells, mainly in the palisades of Vogt in the human cornea. The mouse and human central cornea was almost devoid of Notch-1 . ABCG2 staining in human tissue showed expression in the limbal region and was not restricted to the few cells in the basal region. However, no staining was detectable in the central cornea. Compared to the human cornea, the ABCG2 expression was not site specific in the mouse cornea. The double staining for Notch-1 and ABCG2 in human cornea showed that all Notch-1 stained cells also expressed ABCG2, but a larger proportion of ABCG2 positive cells did not express Notch-1. The human corneal whole mount stained with Notch-1 and ABCG2 showed only a few cells in the limbal region which was double positive.

Conclusions:: Our findings suggests that Notch-1 is a better marker for corneal stem cells as it marks a unique subpopulation of limbal basal cells that coexpress ABCG2.

Keywords: cornea: epithelium • cornea: basic science • development