Characterization of a potential marker of corneal epithelial stem cells

Corneal epithelial stem cells are thought to be localized in the basal cell layer of the limbus. We developed a monoclonal antibody designated 4G10.3 that immunolocalized to limbal basal cells in rat corneas. Western blot analysis demonstrated that 4G10.3 reacted with a single band of 50,000 molecular weight in rat and rabbit corneal epithelium and 48,000-49,000 in human epithelium. Following extraction of corneal epithelium in 20 mM Tris-HCl (pH 6.8) and ultracentrifugation at 100,000 x g, the 50-kD protein was detected in the soluble fraction. 4G10.3 also was used to examine the response of the limbal basal cells to epithelial debridement and thermal burn wounds in the rat. In unwounded control corneas, 40.8 +/- 12.0 (mean +/- standard deviation) cell per limbal area bound 4G10.3. Following a 3 mm debridement wound, the number of cells binding 4G10.3 increased to 77.0 +/- 16.9 two days post-injury and returned to control levels by three days. Following thermal burn, 36.2 +/- 11.2, 68.8 +/- 15.8, 85.4 +/- 15.4, 104.6 +/- 13.8, and 88.0 +/- 40.2 cells per limbal area bound 4G10.3 18 hours, and 1, 2, 3, and 7 days post-injury, respectively. The 50 kD protein and 4G10.3 antibody provided a biochemical and immunological marker of limbal basal cells, hypothesized to be corneal epithelial stem cells.