Spontaneous and mechanically induced Ca²⁺ activity changes in hESC‐RPE cells during maturation

Abstract

PurposeThe aim of this study was to investigate changes in spontaneous Ca2+ activity and mechanically induced intercellular Ca2+ communication in human embryonic stem cells‐derived retinal pigment epithelium (hESC‐RPE) during maturation.MethodsIn this study, we assessed Ca2+ activity in hESC‐RPE cells cultured for 9 or 28 days. Ca2+ imaging was done using a Ca2+‐sensitive fluorescence dye fluo‐4‐AM. Spontaneous Ca2+ activity and mechanically induced intercellular Ca2+ communication were recorded in control conditions, in the absence of extracellular Ca2+, after depletion of intracellular Ca2+ stores with thapsigargin, in presence of a gap junction blocker α‐glycyrrhetinic acid and a P2‐receptor blocker suramin.Results9 days cells exhibited twice lower spontaneous Ca2+ activity and 4‐fold higher mechanically induced intercellular Ca2+ communication compared to 28 days cells. Absence of extracellular Ca2+ reduced spontaneous Ca2+ activity in 9 days cells and almost completely inhibited it in 28 days cells, while having no effect on mechanically induced intercellular Ca2+ communication. Depletion of intracellular Ca2+ stores abolished spontaneous Ca2+ activity in 9 days and 28 days cells, as well as mechanically induced intercellular Ca2+ communication in 28 days cells, while not affecting the latter in 9 days cells. Blockade of gap junctions and P2‐receptors had no effect on spontaneous Ca2+ activity or mechanically induced intercellular Ca2+ communication in cells from both time points.ConclusionsOur results show that hESC‐RPE cells undergo significant Ca2+ signaling re‐arrangements during maturation: the cells increase spontaneous Ca2+ activity, while decreasing their intercellular Ca2+ communication.