Purpose: To determine the relative expression of cell cycle-association proteins in human corneal and limbal epithelium and corneal endothelium in situ, to correlate staining patterns of cell cycle-associated proteins with known proliferative status of corneal and limbal epithelial cells, and to determine the relative proliferative status of corneal endothelial cells in situ by comparing their staining patterns with those of corneal and limbal epithelial cells.
Methods: Corneas from donors 6 weeks and 17, 27, 37, 53, 66, and 67 years of age were preserved in Optisol, received on ice within 24 to 36 hours of death, and immediately fresh frozen. Transverse 6-micron corneal sections were prepared for indirect immunofluorescence localization using commercial antibodies that specifically recognize the following cell cycle-associated proteins: cyclins D, E, A, and B1; protein kinases p33cdk2 and p34cdc2; and Ki67, a marker of actively cycling cells.
Results: All cells of the corneal and limbal epithelium and corneal endothelium stained positively for protein kinases, p33cdk2 and p34cdc2, and for cyclin B1. Staining patterns for cyclins D, E, and A and for Ki67 differed depending on the relative proliferative status of the cells. Terminally differentiated, noncycling corneal epithelial subrabasal cells did not stain significantly for cyclins D, E, or A, or for Ki67. Some corneal epithelial basal cells showed nuclear staining, particularly for cyclin D and Ki67, indicating the presence of actively cycling cells in this regenerative cell layer. In peripheral corneal epithelium, the relative number of basal cells with positive cytoplasmic staining for cyclins D, E, and A increased with proximity to the limbus. Within this region, an occasional nucleus stained positively for Ki67. Limbal basal cells, which contain a slow-cycling stem cell population, stained positively for cyclins D, E, and A within the cytoplasm. Nuclear staining for cyclin D and Ki67 was observed in a few basal cells. Occasional cells within the suprabasal layers of the limbus stained positively for Ki67. The corneal endothelium, considered a nonrenewing population, exhibited staining patterns similar to those of limbal basal cells, except that in no specimen was Ki67 staining observed.
Conclusions: All corneal and limbal epithelial and corneal endothelial cells express protein kinases, p33cdk2 and p34cdc2, and cyclin B1. Relative staining patterns of the cell cycle-dependent proteins, cyclins D, E, and A, and of Ki67 acted as markers to distinguish terminally differentiated epithelial suprabasal cells that have exited the cell cycle, actively cycling epithelial basal cells, and slowly-cycling limbal basal (stem) cells. Staining patterns of the corneal endothelium most closely corresponded to those of limbal basal cells, suggesting that endothelial cells are arrested in G1-phase and have not exited the cell cycle.