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

Abstract

BackgroundWound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca2+ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time.MethodsThe 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca2+ activity was measured with live-cell Ca2+ imaging either immediately or 7 days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca2+ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy.Results The Ca2+ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca2+ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca2+ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca2+ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs.ConclusionsThis acquired knowledge about Ca2+ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca2+ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.Electronic supplementary materialThe online version of this article (10.1186/s12938-018-0535-z) contains supplementary material, which is available to authorized users.

Dynamic full-field optical coherence tomography allows live imaging of retinal pigment epithelium stress model

“…The different methods presented to calculate the evolution of scratch assays present consistent results on the speed of either closing or expansion. Differences were observed depending on the origin and maturation stage of the sample, in accordance with the literature 16 . The experiments were performed multiple times ( n = 5 ppRPE; n = 9 hiRPE) giving the same results each time on speed and wound closure.…”

“…Unfortunately, we were not able to determine if the cell movements were due to the scratch assay or breaks in the cell layer caused by lack of membrane adhesion. This problem was also observed by Abu Khamidakh et al 16 . Future developments of this study would be to automatise the realisation of scratch assays with a new setup combining a laser to cut the cell layer with better repeatability.…”

“…Depending on the origin of the cells (ppRPE or hiRPE) and the width of the scratch, cell behaviours differed. Two main features are usually used to quantify wound healing: the speed of closure, i.e., the evolution of the width of the scratch, and the wound closure, measuring the evolution of the area of the scratch 16 . Semi-automatic segmentation based software, named scratch assay velocity evolution (SAVE) profiler (see the “Methods” section), was developed to segment the wound and calculate the width and area of the scratch in order to quantify wound closure.…”

Denuded Descemet’s membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture

“…hESC-RPE cells deposit on the new matrix as cell patches and then proliferate, ultimately organising in a tight monolayer of epithelial-like cells after 7 days of culture. After this time, cells stop proliferating, consistent with the in vivo situation where RPE cells are quiescent and division appears only as a result of disease condition [ 31 ]. Additional coating on the top of the dDM is unnecessary, since the only dDM is capable of supporting the growth of the cells.…”