Identification of Undifferentiated cells in the Limbal Basal Region Using Notch–1

Abstract

Purpose: : To determine whether Notch–1 could be used as a marker to identify stem cells in the limbal basal region since Notch–1 is known to maintain the cells in an undifferentiated manner in other systems.

Methods: : Human corneoscleral tissues were obtained from the eye bank and prepared for cross section and whole mount analysis. The tissue for whole mount was incubated in Dispase (18 hr at 4º) and the epithelial sheet was gently removed and fixed immediately in 4% paraformaldehyde. The sections and whole mount were stained with antibodies against Notch–1, Notch–2, Jagged–1, beta–1 and alpha–6. Epithelial sheet obtained from the central corneal and limbal region was used for Western blot analysis of Notch–1 and Jagged–1. Explant culture was performed and only primary cultures were used in this experiment.

Results: : Immunofluorescence data using Notch–1 monoclonal antibody revealed that Notch–1 is expressed in the limbal basal cells. The expression was not continuous throughout the limbal basal region. The Notch–1 antigenicity was more pronounced in clusters of cells, mainly in the palisades of Vogt, and the corneal zone was almost devoid of Notch–1. The staining of Jagged–1 was more expanded in the limbal region and not restricted to the few cells in the basal region. Staining was almost undetectable in the cornea. The intensity of Notch–1 staining in cultured cells from the limbal explants was high in only a few cells. The dividing cells showed a diminution of the Notch–1 signal. The expression in cultured cells was more cytoplasmic with only a few cells showing additional nuclear staining. Jagged–1 is expressed mostly in the proliferating cells in the explant culture; the staining pattern was more like that of the tissue section. The whole mount stained with Notch–1 showed only a few cells in the limbal region. Western blot analysis showed that Notch–1 and Jagged–1 are expressed in cornea, limbus, and stroma. Cos–7 transfected with Notch–1 showed a 300–kDa and 110–kDa band. A complex pattern of processed forms or degraded products was also detected.

Conclusions: : Immunofluorescence data clearly shows that Notch–1 could be used as a marker to identify stem cells in the limbal zone.

Keywords: cornea: epithelium • cell-cell communication • cornea: basic science