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1 From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; 2 Dokkyo University School of Medicine, Department of Ophthalmology, Tochigi, Japan.
PURPOSE. To compare cell cycle kinetics in corneal endothelial cells from young and old donors.
METHODS. Human corneas were obtained from the eye bank and separated into two groups: young (19 corneas, <30 years of age) and old (40 corneas, >50 years of age). Corneas were cut in quarters, and the endothelium was released from contact inhibition by producing a 2-mm scrape wound. Unwounded endothelium acted as a negative control. Corneal pieces were exposed for 24, 36, 48, 60, 72, and 84 hours to medium containing 10% fetal bovine serum, 20 ng/ml fibroblast growth factor, and 50 mg/ml gentamicin or the same medium supplemented with 10 ng/ml epidermal growth factor (EGF). Tissue was fixed, immunostained for Ki67 (a marker for the late G1- through M-phase) or for 5-bromo-2'-deoxyuridine (BrdU; a marker for the S-phase), and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Confocal images were evaluated using an image analysis program to count Ki67-positive and PI-stained cells and to evaluate cell cycle position. Cells were counted in 15 x 100 µm2 areas randomly selected from each wound, and the mean was used for subsequent calculations.
RESULTS. Human corneal endothelial cells could be reliably scored for their position within the cell cycle using Ki67 staining patterns. In both age groups, cells repopulating the wound area stained positively for Ki67, whereas no Ki67 staining was observed in unwounded areas under any condition tested. Cells from old donors treated with fetal bovine serum and FGF stained positively for Ki67, indicating that these cells were actively cycling. Compared with cells from young donors, old cells entered the cell cycle more slowly (48 versus 36 hours), the peak of Ki67 staining occurred later (72 versus 60 hours), and fewer cells proliferated (23% versus 47%) or exhibited mitotic figures (4% versus 7%). Addition of EGF to the culture medium increased Ki67 staining in both groups, but the effect on old cells was more dramatic. More cells from old donors entered the cell cycle by 36 hours after wounding, the number of proliferating cells increased 1.6-fold, and the relative number of mitotic figures increased 2.5-fold over cells treated in the absence of EGF.
CONCLUSIONS. Regardless of donor age, corneal endothelial cells can enter and complete the cell cycle. In the presence of fetal bovine serum and FGF, cells from old donors can proliferate but respond more slowly and to a lesser extent than cells from young donors. EGF added to the medium stimulates cells from old donors to enter the cell cycle faster, increases the relative number of actively cycling cells, and increases the number of cells exhibiting mitotic figures. The resultant hypothesis is that it is possible to stimulate a significant proliferative response in corneal endothelial cells from old individuals. Administration of an optimal combination of stimulatory growth factors is required under conditions in which cells have been transiently released from contact inhibition.
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