Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation

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

“…In our earlier study, we have shown that mechanical stimulation induces wide-spreading intercellular Ca 2+ waves in immortalized human RPE cell line ARPE-19. 1 In our hands, the morphology of ARPE-19 cells resembles the morphology of 9 days cultured hESC-RPEs, and the waves observed in 9 days cultured hESC-RPEs were similar to those in ARPE-19, spreading far from the site of mechanical stimulation.…”

“…The micropipette was mounted on a Luigs & Neumann SM 5-9 vertical microinjection system (Luigs & Neumann, Ratingen, Germany) and intermittently lowered to touch a target cell membrane. 1 When the pipette touched cell surface, it resulted in temporary deformation of cell shape that was visible in the fluorescence light. If a cell membrane had been damaged during the stimulation, the fluorescence dye leaked out of the stimulated cell by the end of the recordings.…”

“…1 The list of the used primary and secondary antibodies is represented in Table 1. Images were taken with an Olympus IX51 microscope with a 209 air objective.…”

“…20,21 Intercellular Ca 2+ waves induced by mechanical stimulation have been detected in various cell types, for example, neurons, 5 lens epithelial cells, 6 corneal endothelial cells, 7 as well as RPE cells. 1,15 Distinct Ca 2+ dynamics at different differentiation stages has been shown in human osteoblasts, 27 keratinocytes, 9 neurons, 22 chicken RPE 24 and other cell types. 29 Intercellular Ca 2+ waves can propagate via two different routes: intracellularly via gap junctions (GJs), when signaling molecules pass from one cell to another through connexin channels, or via extracellular medium, involving, for example, activation of P2 receptors by ATP.…”

“…Usually, manual cell segmentation is used for analysis of single cell Ca 2+ dynamics. 1,7,21 Automatic cell segmentation on fluorescent images is a challenging developing research area. It has been previously used to assess Ca 2+ signals, for example, in brain cortex slices 19 and neuronal cell cultures.…”