Stemness characteristics of human corneal endothelial cells cultured in various media.

Abstract

To investigate stemness characteristics of human corneal endothelial cells (HCECs) cultured in various media. Human corneal endothelial cells were isolated using a sphere-forming assay. Cells were allowed to attach to the bottom of culture plates and were cultured in different media designated as medium A (Opti-MEM I with 8% fetal bovine serum), medium B (DMEM/F12 with B27 supplement), medium E (DMEM/F12 with epidermal growth factor [EGF]), and medium BE (DMEM/F12 with B27 supplement and EGF), respectively. Cell morphology was evaluated with an phase-contrast inverted microscope. Immunofluorescence staining and western blotting of nestin, octamer-binding transcription factor (OCT3/4), glial fibrillary acidic protein (GFAP), zonula occludens-1 (ZO-1), collagen VIII alpha2, and Na-K ATPase was performed. Cell proliferation was assessed with a cell counting kit-8 assay. A few cultured cells stained with nestin. The cells cultured in medium A expressed high levels of GFAP, OCT3/4, and nestin, and higher levels of ZO-1 were expressed in the cells cultured in medium A and medium B compared with cells cultured in the other media. The cells cultured in medium A assumed a fibroblast-like shape, whereas the cells cultured in medium B and medium BE appeared as mosaics. Cell proliferation was highest in medium A compared with those cultured in the other media. Cultured HCECs expressed stem cell markers, including nestin, OCT3/4, and GFAP. The expression of stem cell markers differed according to the culture media and associated proliferation rate.