Purpose  The location of stem cells is of great interest. Generally accepted idea is that the corneal stem cells locate in the basal layer of limbus. But still others think that they exist in the central cornea. In this experiment, we observed the wound healing of corneal epithelium after limbal removal to testify the importance of central, non-limbal epithelial cells in the maintenance of epithelial homeostasis.
Methods  Limbal epithelial cells were removed completely or partly  by mechanical scraping in ubiquitous GFP mice. Right eyes were used for injury and left eyes as untouched control. Corneal health was monitored for the life of the animals, up to two years, by observing transparency, vascularization, and centripetal epithelial cell movement (time-lapse GFP fluorescence microscopy). In some mice, a central epithelial scrape injury was made and healing rates were determined in both limbus-removed cornea and untouched cornea.
Results  A technique for complete removal of limbal epithelial cells was verified by thorough examination of DAPI-stained histological specimens that were prepared several hours after the injury. When the limbal epithelium was removed along  half of the circumference, all corneas healed and remained clear and avascular. While the limbal epithelium was removed along half of the circumference together with half of the central corneal epithelium, 80% of the corneas developed central scars and became vascularized. When the limbal epithelium was removed along the entire circumference, 85% healed without incident and remained clear and avascular with normal centripetal epithelial cell movement within a year. One year later, 15% of the eyes showed increased vasculature in the peripheral cornea, which persisted but did not grow into the central cornea and 31% of the eyes developed a central scar and a bundle of blood vessels that grew radially from a small zone of the limbus to the scar. Healing rates of a central epithelial defect, examined more than 50 weeks after the limbal injury, were similar between the limbus-removed right eye and the untouched left eye.
Conclusion  Normal corneal epithelial homeostasis is maintained even after total removal of limbal epithelial cells, provided central corneal epithelial cells are left intact. In such corneas, the restored limbal epithelium appears to function as the site of corneal epithelial stem cells. These results raise possibilities that: non-limbal epithelial cells can support normal epithelial homeostasis in the absence of limbal epithelial cells, or the environment of the limbus can confer stem cell properties to differentiated epithelial cells that migrate into the denuded limbus.