Abstract
Purpose: :
To compare replication competence and senescence in human corneal endothelial cells (HCEC) between the central and peripheral areas and between younger and older donors.
Methods: :
Human corneas were separated into two groups: young (<30 years old) and old (>50 years old). Corneas were cut in quarters and a 2–mm scrape wound was created in the endothelium from the periphery to the center. Corneal pieces were incubated for 24, 36, 48, 60, 72, 84, and 96 hrs in growth medium. Tissue was fixed, immunostained for minichromosome maintenance–2 (MCM2)–a marker of replication competence, and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Senescent HCEC in ex vivo corneas were identified by staining for senescence–associated b–galactosidase activity (SA–b–Gal). For all studies, results were compared between central and peripheral cornea and between younger and older donors.
Results: :
In both age groups (n = 4/group), cells repopulated the wound area in a time–dependent manner. In corneas from older donors, significantly fewer HCEC migrated into the wound bed in 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 percent of MCM2–positive cells increased with time until wound healing. In both age–groups, more MCM2–positive cells were present in peripheral cornea than in central cornea. At 36, 48, 60, and 72 hrs after wounding, the percent 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 positive MCM2–positive staining was observed in unwounded areas at any time–point. HCEC in corneas from younger donors (n = 4) showed little–to–no SA–b–Gal activity in either the central or peripheral area. SA–b–Gal activity was easily detectable in corneas from older donors (n = 4) and a significantly higher percentage of central HCEC showed strong SA–b–Gal activity compared with HCEC in the periphery.
Conclusions: :
In ex vivo corneas, HCEC from the peripheral area retain higher replication competence, regardless of donor age. HCEC in the central area of corneas from older donors retain replicative competence, but the relative percent 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 HCEC exhibiting senescence–like characteristics.
Keywords: cornea: endothelium • immunohistochemistry