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Tanja Ilmarinen, Taina Viheriala, Heidi Hongisto, Teemu Ihalainen, Soile Nymark, Heli Skottman; Maturation status influences the ability of polarized hESC-RPE to tolerate cellular stress. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1226. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of human embryonic stem cell -derived retinal pigment epithelium (hESC-RPE) is a potential treatment strategy for age-related macular degeneration. For effective treatment, hESC-RPE must exhibit the physiological characteristics of native RPE and endure high level of cellular stress caused by e.g. long time periods in cell culture, cryopreservation for cell banking, transplantation surgery and the microenvironment in the diseased eye. Previously, polarized hESC-RPE monolayers have been shown to have higher resistance to oxidative stress-induced cell death than nonpolarized cultures. However, it is not known if the maturation level of polarized hESC-RPE affects their ability to tolerate cellular stress. In order to find the optimal maturation status for hESC-RPE sheet transplantation and criteria to evaluate that prior transplantation, we have here investigated the consequences of cellular stress to hESC-RPE functionality during maturation in vitro.
Human ESC-RPE monolayers were cultured on porous polyester substrate for 3 and 12 weeks to obtain young and mature cultures, respectively. Cellular stress was induced with 1 µM MG132 proteasome inhibitor treatment for 24 h. Maturation level and functionality of 3 week and 12 week cultures without and with MG132 treatment were assessed with transepithelial resistance (TER), phagocytosis assay, immunocytochemistry (ZO-1, claudin-19, Na+/K+-ATPase), calcium imaging, mitochondrial membrane potential assay, and cytokine secretion assay.
The ability of hESC-RPE to tolerate cellular stress was greatly influenced by their maturation status. Young cultures were more susceptible to proteasome inhibition as indicated by greater disruption of functionality compared to mature cultures, including mislocalization of Na+/K+-ATPase, impaired barrier function (a shift of claudin-19 away from tight junctions and lower TER), lower phagocytosis activity, and lower amplitude of ATP-induced calcium response after MG132 treatment. The effects on mitochondrial functionality and cytokine secretion are currently under investigation.
Overall, hESC-RPE can be used to generate sheet transplants which tolerate acute cellular stress in vitro. However, our results suggest that it could be beneficial to use more mature hESC-RPE monolayers to achieve better RPE functionality post-transplantation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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