Abstract
Abstract: :
Purpose: The aim of this study was to determine the differentiation characteristics of a previously established telomerase immortalized human corneal epithelial cell (THC) line compared with normal human corneal epithelium. Methods: THC were grown on cell culture inserts (12mm diameter, 3.0µm pore size, Corning Inc., Corning, NY) submersed in high calcium containing (1.15mmol/L) KGM-2 media (CloneticsTM, BioWhittaker, Inc., Walkersville, MD) for seven days. THC cultures were then air-lifted and evaluated at day 0, 7 and 10 days by TdT-mediated dUTP-biotin nick end labeling (TUNEL, ApopTag®, Serologicals Corporation, Norcross, GA) and immunohistochemistry (IHC) using antibodies specific for Muc1, Bcl-2 and Keratin type 3 (K3). Expression of proteins was confirmed by Western Blotting. TUNEL and IHC image were obtained by either Leica SPII laser confocal or fluorescence microscopy with CoolSNAPfxTM CCD camera (Roper Scientific, Tucson, AZ). Results: After 7 days of submersed culture (day 0), THC formed a confluent layer, one to two cells thick. After air-lifting for 7 days (day 7), there was clear stratification of the epithelial sheet to form a cuboidal basal cell layer, a polygonal wing cell layer (1-2 cells) and a squamous superficial cell layer (1-2 cells). At 10 days occasional intraepithelial cysts appeared in the basal/wing cell layer. Similar to normal cornea, anti-Bcl-2 Mabs to the aa 11-44 epitope stained the nucleus of HTC. Interestingly, occasional superficial epithelial cell nuclei failed to stain with anti-Bcl-2 by day 7 and 10, again similar to differentiated normal human and rabbit corneas. TUNEL positive cells were not detected at day 0, but by day 7 and 10 occasional TUNEL labeling was detected in the superficial epithelial cells. Importantly, superficial TUNEL positive cells were negative for nuclear Bcl-2, again similar to normal cornea. Muc1 localized to cytoplasmic membrane in all cells at day 0; however, by days 7 to 10 Muc1 localized only to superficial epithelial cells and on occasional basal cell. K3 showed staining in the cytoplasm of THC at day 0, but was enhanced in upper layer and reduced in basal layer at days 7 to 10. Conclusions: Overall these data show that cultured-THC exhibit natural human/rabbit epithelial differentiation pattern after 7-days of air-lifting. Importantly, differentiation in culture appears to mimic normal programmed cell death of surface cells with loss of Bcl-2 prior to apoptosis.
Keywords: cornea: epithelium • cell death/apoptosis • cornea: surface mucins