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.
Highlights
Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease
The presented data are combined from Regea08/017 and Regea08/023 human embryonic stem cells (hESCs)-RPEs
The presented data are combined from Regea08/017 and Regea08/023 hESC-RPEs the wound healing of epithelial cells but less is known about cellular C a2+ dynamics implicated in the response to fresh wounds and wound healing of RPE
Summary
Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The retinal pigment epithelial (RPE) cells form a monolayer of tightly packed cells which lay between neurosensory retina and the choroid [1]. The RPEs absorb stray light, transport the nutrients from the choroidal side to neural retina, regulate visual cycle and secrete various trophic factors maintaining retinal homeostasis [2]. In pathological conditions RPE dysfunction may lead to the cell layer disruption and choroidal neovascularization as observed in wet age-related macular degeneration (AMD) [3]. Choroidal neovascularization evokes hemorrhage, retinal edema, and increased damage to retinal cells, fibrosis and permanent visual loss due to the undergoing wound healing process. The intravitreal injection itself may be traumatic and lead to RPE tear and wound healing
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