title: Semi-automatic Method for Ca2+Imaging Data Analysis of Maturing Human Embryonic Stem Cells-Derived Retinal Pigment Epithelium.
abstract: 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+]iincreases (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.
authors.affiliation: Department of Electronics and Communications Engineering, BioMediTech, Tampere University of Technology, Biokatu 6, FM-1, 33520, Tampere, Finland. amna.abukhamidakh@tut.fi.
authors.forename: Amna E
authors.initials: AE
authors.lastname: Abu Khamidakh
authors.affiliation: Department of Electronics and Communications Engineering, BioMediTech, Tampere University of Technology, Biokatu 6, FM-1, 33520, Tampere, Finland.
authors.forename: Florentino Caetano
authors.initials: FC
authors.lastname: Dos Santos
authors.affiliation: BioMediTech, University of Tampere, Biokatu 12, FM-5, 33520, Tampere, Finland.
authors.forename: Heli
authors.initials: H
authors.lastname: Skottman
authors.affiliation: BioMediTech, University of Tampere, Biokatu 12, FM-5, 33520, Tampere, Finland.
authors.forename: Kati
authors.initials: K
authors.lastname: Juuti-Uusitalo
authors.affiliation: Department of Electronics and Communications Engineering, BioMediTech, Tampere University of Technology, Biokatu 6, FM-1, 33520, Tampere, Finland. jari.hyttinen@tut.fi.
authors.forename: Jari
authors.initials: J
authors.lastname: Hyttinen
mesh_terms: D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*
keywords: Intercellular calcium waves; Mechanical stimulation; Mechanically induced transient [Ca2+] i increase; Spontaneous transient [Ca2+] i increase; hESC-RPE