Purpose: To compare the effect of epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor-BB (PDGF-BB), bovine pituitary extract, and fetal bovine serum (FBS), alone or in combination, on proliferation of human corneal endothelial cells (HCEC) cultured from young (<30 years old) and older donors (>50 years old).
Methods: Corneas from donors 2 to 79 years old were obtained from the National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. The HCEC marker, antibody 9.3.E, tested for pure endothelial populations. Antibody Ki67 and ZO-1 tested either before or after cultured cells reached confluence to indicate cell proliferation and cell-cell contact formation. Cell morphology was documented by inverted phase-contrast microscopy. Passages I through VII were used to test the effect of various factors on cell proliferation. For each study, equal numbers of cells were seeded, maintained overnight in 4% FBS to permit cell attachment, washed, and incubated for up to 3 weeks in one of the following: modified Eagle's Minimum Essential Medium (Opti-MEM-I) alone; Opti-MEM-I plus EGF, NGF, PDGF-BB, bovine pituitary extract, or FBS; or a combination of factors. At various times after seeding, cell numbers were determined by electronic cell counter. For each condition, three separate wells were tested and each sample was counted three times. Studies were repeated at least twice using cells from different donors and age groups. Within each study, a one-way ANOVA test was performed to analyze statistical significance.
Results: Cells stained positively with antibody 9.3.E, indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. Positive staining of Ki67, indicating cycling cells, was found in subconfluent cultures. Plasma membrane-associated ZO-1 staining and lack of Ki67 staining indicated that cultured cells formed a contact-inhibited monolayer. Cultured cells decreased in density, increased in size, and became more heterogeneous depending on donor age and on the number of passages. Incubation in OptiMEM-I promoted attachment and induced a moderate proliferative response above that of MEM (P < 0.001). In general, proliferative responses to growth stimuli were relatively slow, with cell counts generally plateauing 10 to 14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 5-50 ng/mL, peak cell counts were significantly higher (P < 0.001) than basal levels. EGF consistently stimulated proliferation in cells from younger donors, but was less effective in stimulating growth of cells from older donors. NGF did not show a consistent significant stimulatory effect at any concentration tested. PDGF-BB (25 ng/mL) tended to stimulate growth to a greater extent than EGF (P < 0.05) in cultures from the same donor. Pituitary extract significantly increased counts at 1.0 (P < 0.05) to 100 ug/mL (P < 0.001). PDGF-BB plus pituitary extract demonstrated greater stimulation than pituitary extract (P < 0.01) or PDGF-BB alone (P < 0.01). FBS (1%-8%) increased cell numbers in a dose-dependent manner, and, at 4%-8%, yielded counts significantly higher (P < 0.001) than that of any single growth-promoting agent tested.
Conclusions: HCEC from both young and older donors can proliferate in vitro in response to growth-promoting agents. Proliferation in the presence of multiple mitogens ceased when confluence was reached, indicating the formation of a contact-inhibited monolayer. In general, cells cultured from young donors were more responsive to the agents tested, but the relative response of HCEC to these agents was similar, regardless of donor age. The relative difference in the extent of the response of the same cell population to different mitogens suggests that these mitogens induce different downstream signals. The relatively robust proliferative response of HCEC to FBS may involve stimulation of multiple downstream signaling pathways may involve stimulation of multiple downstream signaling pathways and/or induce more sustained downstream signaling than the other growth-promoting agents tested.