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

Abstract

Ca is a second messenger controlling vital cellular processes, including cell maturation. Changes in Ca 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 Ca ([Ca] ) increases in hESC-RPE. For this, we developed image analysis tools to evaluate cell-specific Ca signals from the entire field of view. Spontaneous and mechanically induced transient [Ca] 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 Ca waves in cells from both time points, but longer culture time reduced Ca wave spreading. Depletion of intracellular Ca stores decreased cell fraction with STIs and MITIs at both time points, and absence of extracellular Ca had similar effect on cells with STIs. To conclude, hESC-RPE cells undergo significant Ca 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.

Wound healing of human embryonic stem cell-derived retinal pigment epithelial cells is affected by maturation stage

“…The cells on fluorescence time-series images were identified as described in [ 22 ]. At first cell centers were detected from averaged fluorescence images in Matlab with in-house algorithm developed for this purpose.…”

“…The amplitude of peak threshold was set to 1.1 (10% higher than the baseline). The cell identification and fluorescence curves analysis algorithms together with Matlab codes were similar to the ones used in Abu Khamidakh et al [ 22 ]. The fluorescence kinetics from single cells was corrected for background fluorescence and smoothed with a moving average algorithm with the 5 points span.…”

“…We have recently shown that human RPE cells have distinct spontaneous Ca 2+ activity at different maturation stages. The immature RPEs, where the majority of the cells have fusiform morphology and low level of pigmentation, have lower amount of cells with spontaneous [Ca 2+ ] i increases compared with more mature, cobblestone-shaped, highly-pigmented cells [ 22 ].…”

“…Previously, we found that, similarly to spontaneous [Ca 2+ ] i increases, the properties of these Ca 2+ waves strongly depend on RPE maturation stage. In immature cells, the mechanically induced Ca 2+ waves spread far away from the site of stimulation, while in more mature RPE these waves propagate to minute number of cells surrounding the site of stimulation [ 22 ].…”

Functional Voltage-Gated Calcium Channels Are Present in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium

“…However, much is still not understood about the genetic characteristics of stem cell‐derived RPE or its behavior after transplantation . Furthermore, there is only limited information about the functionality of ion channels and Ca 2+ signaling in stem cell‐derived RPE. In particular, studies about the voltage‐gated Ca 2+ (Ca V ) channels in these cells are lacking.…”