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

Amna E. Abu Khamidakh, Alejandra Rodriguez-Martinez, Kai Kaarniranta, Anne Kallioniemi, Heli Skottman, Jari Hyttinen, Kati Juuti-Uusitalo

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    Abstract

    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.

    Original languageEnglish
    Article number102
    JournalBioMedical Engineering Online
    Volume17
    Issue number1
    DOIs
    Publication statusPublished - 2018
    Publication typeA1 Journal article-refereed

    Keywords

    • Ca waves
    • Ca2+ waves
    • Cell maturation
    • HESC-RPE
    • Image analysis
    • Mechanical stimulation
    • Mechanically induced intercellular Ca waves
    • Mechanically induced intercellular Ca2+ waves
    • RPE
    • Spontaneous [Ca2+]i increases
    • Spontaneous [Ca] increases
    • Wound healing
    • hESC-RPE

    Publication forum classification

    • Publication forum level 1

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