Gene expression in calcium triggered healthy primary limabal epithelial cells, in vitro

Abstract

Aims/Purpose: Calcium supplementation can induce differentiation of several cell types. The tear film has an important role in maintenance of the ocular surface homeostasis with an ionic calcium concentration of 0.4 mM‐1.0 mM. The effect of calcium concentration on healthy primary limbal epithelial cells (LECs) has not been analysed, yet. Our purpose was to investigate the effect of calcium supplementation on gene expression of primary limbal epithelial cells (LECs) of healthy donor corneas, in vitro.Methods: Primary LECs were isolated from corneoscleral rims of healthy donors (n = 6) and were cultured in serum free low calcium (0.06 mM Ca+2) Keratocyte growth medium 3 (KGM3). The primary LECs were then supplemented by 0.3 mM‐2.0 mM Ca+2 concentrations for 72 h. Control LEC cultures were supplemented by low calcium (0.06 mM Ca+2) KGM3 medium. After cell harvesting, RNA and protein were extracted from treated and control LECs to perform qPCR and western blot analysis.Results: The calcium supplementation had no significant effect on PAX6 mRNA expression after 72 h. The differentiation marker and adhesion protein DSG1 and the cornea specific keratin K3 expression did not change significantly through Ca+2 supplementation (p ≥ 0.5). The retinoic acid signalling component fatty acid binding protein FABP5 expression (p ≥ 0.5) the wound healing and vascularization marker CAV1 gene expression (p ≥ 0.5) did not change significantly through Ca+2 supplementation, after 72 h.Conclusions: Calcium supplementation for 72 h did not change PAX6 expression and did not trigger differentiation of healthy primary limbal epithelial cells. We plan to analyse the effect of longer incubation time in the future in order to trigger a full differentiated status of limbal epithelial cells, using calcium supplementation.