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C Zhu, NC Joyce; Induction of Proliferation in Cultured Human Corneal Endothelial Cells (HCEC) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3184.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To compare the effect of EGF, NGF, pituitary extract and FBS, alone or in combination, on proliferation of cultured HCEC. Methods: Donor human corneas (17-71yo) were obtained from National Disease Research Interchange. Descemet's membrane with intact endothelium was dissected. Cells were isolated by EDTA treatment and cultured to confluence. A Live-Dead assay checked for cell viability. The HCEC marker, antibody 9.3E, tested for pure endothelial populations. Passages I-III 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 two weeks in one of the following: MEM or OptiMEM alone; OptiMEM plus EGF, NGF, pituitary extract or FBS; or a combination of all factors. At various times after seeding, cells were counted by Coulter Counter. Within each study, a one-way ANOVA test was performed to analyze statistical significance. Morphology was documented by inverted phase-contrast microscopy. Results: Cells stained positively with antibody 9.3E indicating isolation of HCEC and lack of contamination with epithelial cells or keratocytes. The Live-Dead assay confirmed HCEC viability. Incubation in MEM did not support long-term cell attachment or growth. OptiMEM promoted attachment and induced a moderate proliferative response above that of MEM (p<0.001). Proliferative responses were relatively slow with cell counts plateauing 10-14 days after exposure to growth-promoting agents. EGF yielded a broad, dose-dependent effect and, at 0.5-50ng/ml, peak cell counts were significantly higher (p<0.001) than basal levels. NGF had a minor stimulatory effect at 1-200ng/ml, but only 200ng/ml consistently yielded significant counts (p<0.001). Pituitary extract significantly increased counts at 1.0 (p<0.05) to 100ug/ml (p<0.001). FBS (1-8%) increased cell numbers in a dose-dependent manner. FBS at 8% yielded counts significantly higher (p<0.001) than that of EGF, NGF, or extract alone. Addition of 8% FBS to EGF, NGF or extract, or a combination of all factors yielded counts similar to those of FBS alone and produced a compact monolayer of polygonal cells. Conclusions: EGF, NGF, and pituitary extract alone can induce proliferation in HCEC above basal levels, but 8% FBS alone or a combination of all factors more strongly stimulates proliferation than any single growth factor. These results indicate that a robust proliferative response in HCEC must require activation of multiple signaling pathways.
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