Influence of pressure on in vitro human corneal endothelial cells derived from human induced pluripotent stem cell (hIPSC)

Purpose Human induced pluripotent stem cells (hIPSC) have infinite self-renewal capacity and can differentiate into somatic cells. We obtained hIPSC-derived corneal endothelial-like cells (CE-likeC) using a sequential supply of growth factors. As, in vivo, the functionality of CEC may depend on various environmental factors, including intraocular pressure, we investigated whether pressure could influence the hIPSC differentiation into EC Methods We developed a specific device for pressurizing cell cultures placed in a CO2 incubator. The device consists of a pump injecting the gas mixture from the incubator into a sealed container equipped with a pressure sensor and an electronic control system. hIPSC-derived CEC were cultured in the differentiation medium for 1 week either in a standard incubator at atmospheric pressure or under a pressure of 20 mmHg. The medium was changed every 2 days. Differentiation was determined by cell morphology analysis and ionic pumps immunostaining (CLCN3, VDAC3, SLC4A and Na+/K+/ATPase) and tight junctions (ZO-1). Results At D8, the 2 cultures showed similar endothelial morphology, ZO-1 and SLC4A, but expression of ionic pumps CLCN3, VDAC3, and Na+/K+/ATPase were increased under pressure. Conclusion The device developed is fully functional. Our results show that, in vivo, a pressure of 20 mmHg did not modify the cell morphology. However, it tends to modulate the expression of some ionic pumps. Pressure level seems to be an important parameter in the differentiation into EC. Considering this, it could improve the EC functionality, i.e. deswelling capacity Grants: ABM2013, IUF2012-2017 and postdoc UJM 2013