In Vitro Analysis of Gene and Protein Expression in Primary Limbal Epithelial Cells Exposed to Differentiation-Inducing Medium

Purpose: To study the time course of the differentiation process and its regulatory networks in primary limbal epithelial cells (pLECs) using serum-free, low calcium Keratocyte growth medium 3 (KGM3) and CnT-2D differentiation medium. Methods: pLECs were isolated from corneoscleral rims from healthy donors and cultured in serum-free low calcium (0.06 mM Ca2+) KGM3. Differentiation was induced by supplementation with CnT-2D differentiation medium, while control cells were maintained in low-calcium KGM3 medium. Gene and protein expression analyses were performed using qPCR and Western blotting, respectively, at 72 h and at 5, 7, 10, and 14 days post-supplementation to determine the optimal time course of differentiation induction. Results: CnT-2D differentiation medium supplementation resulted in a significant upregulation of differentiation-associated markers, including desmoglein 1 (DSG1), paired box domain 6 (PAX6), keratin 3 (KRT3), fatty acid binding protein 5 (FABP5), cellular retinoic acid binding protein 2 (CRABP2), alcohol dehydrogenase 7 (ADH7), aldehyde dehydrogenase 1A1 (ALDH1A1), with the most pronounced changes observed at day 10 post-supplementation (p ≤ 0.05). Conclusions: CnT-2D differentiation medium effectively initiates differentiation of limbal epithelial cells in vitro. The gradual increase in the expression of key differentiation markers, including DSG1, KRT3, and PAX6, indicates that CnT-2D medium successfully induces differentiation in 2D cultured primary limbal epithelial cells. However, subcellular localization of these markers, epithelial barrier function, and differentiation in 3D models were not assessed and remain to be investigated.