Spontaneous and mechanically induced Ca2+ activity changes in hESC-RPE cells during maturation
Abstract
Purpose
The 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.
Methods
In 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.
Results
9 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.
Conclusions
Our 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.
