September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
A cell-based model to study wound repair in retinal pigmented epithelial cells
Author Affiliations & Notes
  • YingHsuan Shih
    Neuroscience Research Institute , University of California-Santa Barbara, Santa Barbara, California, United States
    Molecular, Cellular, and Developmental Biology, University of California-Santa Barbara, Santa Barbara, California, United States
  • Monte J Radeke
    Neuroscience Research Institute , University of California-Santa Barbara, Santa Barbara, California, United States
  • Pete Coffey
    Neuroscience Research Institute , University of California-Santa Barbara, Santa Barbara, California, United States
  • Footnotes
    Commercial Relationships   YingHsuan Shih, None; Monte Radeke, None; Pete Coffey , None
  • Footnotes
    Support  LA1-02086; Garland Initiative for Vision Research Grant
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 265. doi:
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      YingHsuan Shih, Monte J Radeke, Pete Coffey; A cell-based model to study wound repair in retinal pigmented epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):265.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : In rodents, RPE can repair laser-induced wounds by migration and proliferation. Whether human RPE employs a similar mechanism is unclear. To gain further understanding on wound healing of differentiated human RPE, we developed a cell-based system to study RPE behavior, morphology, and gene expression in response to chronic wounds.

Methods : Human fetal RPE were cultured on Electric Cell-substrate Impedance Sensing (ECIS) 96-well plates for 30-60 days to differentiate. Each well contains 2 electrodes, which can introduce elevated electrical pulses to kill overlying RPE and create 0.25mm2 wounds. To create chronic wounds, electrical pulses were applied to kill RPE once a day. TGFβ1 was applied to study the roles of TGFβ signaling pathway on RPE repair. Wound healing was monitored by real-time impedance recording. Cell proliferation was detected using EdU labeling and cell size was measured based on ZO-1 staining. Gene expression was analyzed by RNA-Seq.

Results : Immediately after wounding, cell death was detected as an abrupt drop in impedance. After 3 hours, bystander cells began to repopulate the electrodes. After 15 hours, the wounds were enclosed by cells that originated from the peripheral. Both EdU+ and EdU- cells were observed in/outside of the enclosed wounds indicating that monolayer repair resulted from cell proliferation and migration. Cell size and density remained stable following a single wound. Conversely, after 20 treatments, RPE became larger and less dense. After repeated treatments, gene expression of the whole population remained constant despite the substantial changes in cell behavior, morphology and cell numbers. These results suggest that monolayer repair resulted from localized signaling. Moreover, activation of the TGFβ signaling pathway repressed cell proliferation, prolonged repair, and led to larger and less dense RPE.

Conclusions : Here we used a cell-based system to study the repair mechanism of differentiated human RPE in response to chronic wounding. We showed that human RPE employs both cell proliferation and migration to repair disruption of the monolayer. However, the capability to repair is reduced by chronic cell loss and activation of the TGFβ signaling pathway. This cell-based system not only allows us to dissect the detailed mechanisms that regulate RPE repair under different conditions, but may also provide a platform to understand the lack of repair in diseased RPE.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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