Semi-automatic Method for Ca²⁺ Imaging Data Analysis of Maturing Human Embryonic Stem Cells-Derived Retinal Pigment Epithelium

Ca²⁺ is a second messenger controlling vital cellular processes, including cell maturation. Changes in Ca²⁺ signaling during maturation of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have not been assessed previously. The aim of this study was to investigate maturation-dependent changes in transient intracellular Ca²⁺ ([Ca²⁺]_i) increases in hESC-RPE. For this, we developed image analysis tools to evaluate cell-specific Ca²⁺ signals from the entire field of view. Spontaneous and mechanically induced transient [Ca²⁺]_i increases (STIs and MITIs) were analyzed in hESC-RPEs cultured for 9 or 28 days, altogether from more than 80,000 cells. Both cultures showed STIs: the longer culture time resulted in twofold increase of amount of cells with STIs. Mechanical stimulation induced intercellular Ca²⁺ waves in cells from both time points, but longer culture time reduced Ca²⁺ wave spreading. Depletion of intracellular Ca²⁺ stores decreased cell fraction with STIs and MITIs at both time points, and absence of extracellular Ca²⁺ had similar effect on cells with STIs. To conclude, hESC-RPE cells undergo significant Ca²⁺ signaling re-arrangements during a short maturation period increasing cell fraction with STIs, while decreasing coordinated cell response to mechanical stimulation. This knowledge and proposed analysis tools can be used for assessment of hESC-RPE maturation in vitro.