Modeling ocular surface ion and water transport by generation of lipid- and mucin-producing human meibomian gland and conjunctival epithelial cells

Despite the importance of the ocular surface in human physiology and diseases, little is known about ion channel expression, properties, and regulation in ocular epithelial cells. Furthermore, human primary epithelial cells have rarely been studied in favor of rat, mouse, and especially rabbit animal models. Here, we developed primary human meibomian gland (hMGEC) and conjunctival (hConEC) epithelial cells. We show that hConEC and hMGEC produce MUC5AC and lipids, respectively. With cell cultures maintained at the air-liquid interface, we recorded transepithelial short-circuit currents (<i>I</i>_sc) by the Ussing chamber method. We identified in the apical membrane Na⁺, Cl^-, and K⁺ ion channels; amiloride-sensitive epithelial sodium channel (ENaC), cAMP-dependent CFTR, UTP-dependent TMEM16a, and chromanol 293B-sensitive KCNQ1. At the basolateral membrane, we identified bumetanide-sensitive NKCC and barium-sensitive K⁺ channels. We also found that vasoactive intestinal peptide, concentration-dependent (EC₅₀ of 1-8 nM), stimulates the CFTR-dependent <i>I</i>_sc in both cells. Western blot analysis confirms the expression in both cell cultures of βENaC subunit, CFTR, TMEM16a, and KCNQ1 proteins. We recorded water influx by quantitative phase microscopy and identified a cAMP-dependent and mercury-sensitive water flux and identified by Western blot AQP3 and AQP5 proteins in hConEC and hMGEC. Taken together, we propose a model of the ion transports of human conjunctival and meibomian gland epithelial cells that will set the stage for future molecular dissection of the regulation of these transport proteins in the context of tear secretion and related diseases.<b>NEW & NOTEWORTHY</b> We generated human meibomian gland and conjunctival epithelial cells producing lipids and mucins. We identified ion channels including ENaC, CFTR, TMEM16a, and KCNQ1, as well as NKCC. We found that electrolyte and water flux are regulated by signaling pathways mediated by purinergic and VIP receptors. Our findings provide valuable insights into epithelial ion and water transport in the human conjunctiva and meibomian gland, enhancing understanding of these processes in both physiological and disease states.