Acute Wound Healing in the Human Central Corneal Epithelium Appears to Be Independent of Limbal Stem Cell Influence

Abstract

In the adult cornea, epithelial cells are maintained by limbal stem cells (LSCs) that cycle slowly and give rise to transient amplifying (TA) cells. These migrate centripetally, differentiate outward to the surface, and are then lost by desquamation. This study was conducted to analyze the contribution of human central corneal epithelial cells toward corneal epithelial regeneration. A human corneal organotypic culture model was used to assess corneal healing in vitro in 12 matched cornea pairs. Two types of ablation were studied: (1) A ring-shaped, transepithelial, excimer laser (193 nm) ablation, of 7 mm outer diameter and 3 mm inner diameter, to a depth of 80 mum-sparing the central and peripheral corneal epithelium; and (2) an ablation pattern identical to that in (1) with ablation of the limbal epithelium in addition. Corneal healing was followed using time-lapse dark-field microscopy for up to 12 hours, and the corneas were analyzed by using immunohistochemical markers for cell proliferation and stem cells. In the donut model, corneal epithelial repair originated from both the limbus and the central corneal epithelium with the average rate of epithelial recovery from the center being similar to the rate from the periphery (0.06 +/- 0.01 mm/h vs. 0.07 +/- 0.03 mm/h, P = 0.44). When the area of recovery was calculated relative to original edge circumferences, the central epithelial rate tended to be faster than the peripheral (0.06 +/- 0.02 mm(2)/mm/h vs. 0.04 +/- 0.01 mm(2)/mm/h, P = 0.04). Similar rates in epithelial recovery were identified in centripetal and centrifugal directions in both the donut and donut+limbus ablation models. Central epithelial cell density increased 36% over the control cornea within 12 hours after surgery, but there was no change at the periphery. Cell proliferation, assessed using Ki67 and BrdU labeling, was observed across the entire cornea. Expression of the putative stem cell markers p63 and ABCG2 was clearly evident in the basal layer of the limbus. However, weaker labeling was also observed in the central epithelium. Connexin 43 (Cx43), a differentiation marker, was mainly absent in the normal untreated limbal basal cells, but more Cx43-positive cells were labeled in the basal layer of the limbus after wounding. After wounding, the capacity for epithelial cell proliferative and migration appears to be as active in the central cornea as in the periphery/limbus. Central and peripheral epithelial recovery remains equal even after ablation of the limbus. Central human corneal epithelial cells are therefore capable of corneal epithelial regeneration, at least in the first 12 hours after wounding.