Comparative analyses of the differentiation and stemness status of corneal epithelial cell lines in varying culture conditions.

Cell lines of human corneal epithelial cells (CEC) are widely used as alternatives to primary CEC cultures to understand corneal biology. This study analyzes the expression of differentiation, junctional, limbal and pluripotent stem cell markers of two immortalized cell lines, HCEC (human corneal epithelial cells) and HCLE (human corneal limbal epithelial cells), cultured under different conditions, by flow cytometry, qPCR or immunofluorescence, to identify the culture condition most akin to the native corneal epithelium. Such cells are useful in studies relating to de-differentiation and reprogramming. HCEC and HCLE cells were cultured under the standard culture conditions for each of these cell lines. Accordingly, HCEC were cultured under proliferation (P) and differentiation (D) conditions; HCLE were cultured under proliferation (P), growth (G) and stratification (S) conditions. Native corneal epithelium was included in the comparison. HCEC cultured in proliferation or differentiation growth conditions show better differentiation characteristics as compared to the HCLE cultures, with respect to expression of epithelial differentiation and limbal markers. Interestingly, the adherens junction protein (E-cadherin) and the cytoplasmic expression of gap junction protein (ZO-1) are significantly increased in HCLES, with reduced Ki67 expression, features crucial for ensuring the differentiation and structural integrity of the epithelium. Further, among the HCLE cultures, HCLES showed decreased expression of pluripotency markers, as compared to HCECD; the localization of expression of these markers was similar to that observed in the corneal epithelial tissue. Therefore, HCLES is a superior culture model for studying corneal epithelial differentiation, reprogramming and regulatory mechanisms.