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Tadashi Senoo, Nancy C. Joyce; Cell Cycle Kinetics in Corneal Endothelium from Old and Young Donors. Invest. Ophthalmol. Vis. Sci. 2000;41(3):660-667.
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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 × 100 μm2 areas randomly
selected from each wound, and the mean was used for subsequent
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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