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

Background: Wound 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. Methods: The 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 7days 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. Conclusions: This 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.