Abstract
Purpose: :
To develop a reliable method to identify human corneal epithelial stem cells in human corneas and track their proliferation and migration within the epithelium.
Methods: :
Fresh, matched pairs of normal human corneas from donors not suitable for transplantation were provided by the Melvin Jones Eye Bank (Genoa) and used for the study. Carboxyfluorescein diacetate succinimidyl esters (CFSE) (Molecular Probes) was used to target corneal epithelial cells, fluorescence was digitally recorded for five consecutive days, and the rate of cell movement was determined following the different position of the CFSE labelled limbal stem cells. The shape and distribution of CFSE positive cell clusters and the cell movements were analyzed in frozen cross- sections by fluorescence microscopy counterstained with 4 ', 6-diamidino-2-phenylindole (DAPI).
Results: :
CFSE and DAPI staining allowed for the identification of stem cells in green fluorescence, while the overall corneal cell components were stained in blue. Clusters of high-CFSE labelled cells were tracked in cornea and an analysis of time sequences revealed that they moved centripetally. Following the corneas for five days, the limbal cells were observed migrating centripetally and superficially.
Conclusions: :
The use of CFSE in human corneas followed by fluorescence microscopic observation allowed us to detect the movement of epithelial cells in normal human cornea. Human corneal epithelium exhibits a fluorescent pattern of CFSE expression that is suitable for studying cell movement in the normal human cornea. This experimental system should be valuable for further studies on epithelial cell migration in human corneas.
Keywords: cornea: epithelium