Replication Competence and Senescence in Central and Peripheral Human Corneal Endothelium

Abstract

To compare replication competence and senescence in human corneal endothelial cells (HCECs) between the central and peripheral areas and between younger and older donors. Human corneas were obtained from the eye bank and separated into two groups: young (younger than 30 years) and old (older than 50 years). Corneas were cut in quarters and a 2-mm scrape wound was created in the endothelium from the periphery to the center. Unwounded endothelium acted as a negative control. Corneal pieces were incubated for 24, 36, 48, 60, 72, 84, and 96 hours in medium containing 8% fetal bovine serum (FBS) plus additional growth factors. Tissue was fixed, immunostained for minichromosome maintenance (MCM)-2, a marker of replication competence, and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Fluorescence microscope images were used to count PI-stained and MCM2-positive HCECs in three 100-microm2 areas within the central and peripheral wound area. Results are expressed as mean number of cells/100 microm2. Senescent HCECs in ex vivo corneas were identified by staining for senescence-associated beta-galactosidase activity (SA-beta-Gal). Whole corneas were cut in quarters and incubated in staining solution containing SA-beta-Gal at pH 6.0. The number of cells stained for SA-beta-Gal and the grade of SA-beta-Gal intensity in three 100-microm2 areas were averaged for the central and peripheral areas from each donor. For all studies, results were compared between central and peripheral cornea and between younger and older donors. In both age groups (n = 4/group), cells repopulated the wound area in a time-dependent manner. In corneas from older donors, significantly fewer HCECs migrated into the wound bed in the central cornea than in the periphery. At each time point, the density of cells in the central wound area was lower in corneas from older donors than from younger donors. In both age groups, the mean percentage of MCM2-positive cells increased with time until wound healing. In both age groups, more MCM2-positive cells were present in the wounded area of the peripheral than of the central cornea. At 36, 48, 60, and 72 hours after wounding, the percentage of MCM2-positive cells in the central or peripheral area of older corneas was significantly less than in the corresponding region in younger corneas. No MCM2-positive staining was observed in unwounded areas at any time point. HCECs in corneas from younger donors (n = 4) showed little to no SA-beta-Gal activity in either the central or peripheral area. SA-beta-Gal activity was easily detectable in corneas from older donors (n = 4) and a significantly higher percentage of central HCECs showed strong SA-beta-Gal activity compared with HCECs in the periphery. In ex vivo corneas, HCECs from the peripheral area retain higher replication competence, regardless of donor age. HCECs in the central area of corneas from older donors retain replicative competence, but the relative percentage of cells that are competent to replicate is significantly lower than in the periphery or in the central area of corneas from younger donors. This reduction in replicative competence negatively correlates with the observed increase in the population of central HCECs exhibiting senescence-like characteristics.