Abstract
Purpose: :
The purpose of this study was to establish a serum-free culture systems for human corneal epithelial cells without compromising their ability to differentiation into a mucous epithelium and to investigate the regulation of membrane associated mucins by retinoic acid (RA).
Methods: :
Human Corneal limbal epithelial (HCLE) cells were dissociated from donor eyes and were grown in growth factor supplemented serum-free media. Passage 3 cells were cultured in air-liquid interface after confluence. HCLE was cultured under RA-deficient and various range of concentrations (10-9 to 10-6 M) of RA. Expression of P63, cytokeratin 3, cytokeratin 19, and MUC 16 were used to investigate the differentiation pattern. Three membrane associated mucins (MUC1, 4, 16) expressions were analyzed by quantitative real time polymerase chain reaction and immunoblot analysis under different culture conditions with various concentrations of RA or with various time of exposure.
Results: :
Cultured HCLE cells showed features of a multi layered squamous epithelium. RA- deficient culture resulted in overproduction of cornified envelope, and low concentration of RA (10-9 to 10-7 M) induced normal appearance of non keratinized squamous epithelium. Under these conditions, P63, CK3, CK 19 stain pattern were similar to the limbal corneal tissue. Higher concentration (10-6 M) of RA resulted in fewer layered cells with abnormal differentiation pattern. Cultured HCLE expressed 3 membrane associated mucins (MUC1, 4, 16) mRNA and their expressions were highly associated with RA concentrations and exposure times.
Conclusions: :
Serum free HCLE culture systems retained physiologic features of fully differentiated corneal epithelium. RA was a key modulator for normal differentiation and had a dose dependent effect on the expression of membrane associated mucins in cultured corneal epithelial cells.
Keywords: cornea: epithelium • differentiation • cornea: surface mucins