July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Generation of Reversible Immortalized Cell Lines to Model Pseudoexfoliation Syndrome
Author Affiliations & Notes
  • Coralia Luna
    Ophthalmology, Duke University, Durham, North Carolina, United States
  • Pratap Challa
    Ophthalmology, Duke University, Durham, North Carolina, United States
  • Ursula Schlotzer-Schrehardt
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Pedro Gonzalez
    Ophthalmology, Duke University, Durham, North Carolina, United States
  • Footnotes
    Commercial Relationships   Coralia Luna, None; Pratap Challa, None; Ursula Schlotzer-Schrehardt, None; Pedro Gonzalez, None
  • Footnotes
    Support  The Glaucoma Foundation, Research to Prevent Blindness, NIH EY029400, NIH EY05722
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 5129. doi:
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    • Get Citation

      Coralia Luna, Pratap Challa, Ursula Schlotzer-Schrehardt, Pedro Gonzalez; Generation of Reversible Immortalized Cell Lines to Model Pseudoexfoliation Syndrome. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5129.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : A major limitation to understand the pathogenesis of pseudoexfoliation syndrome (PEX) is the lack of experimental models. Our objective is to develop a workable in vitro model of PEX syndrome using reversible immortalized cell lines to facilitate generation of reproducible results without the limitations of replicative senescence.

Methods : IPSCs from PEX and control donors were generated from primary cultures of dermal fibroblasts and differentiated into smooth muscle like (SML) cells. Levels of differentiation were evaluated by western blot and immunocytochemistry for alpha SMA and SM22. To model PEX conditions cells were subjected to gamma radiation induced senescence, and further exposed to treatment conditions associated with pseudoexfoliation, including ultraviolet radiation B (UVB), TGFβ1, IL6, and oxidative stress (40%O2). Microfibril morphology was analyzed by electron microscopy after 14 days of the respective treatments. Reversible immortalization was conducted using two lentiviral vectors containing the LoxP sequence in their long terminal repeats, and expressing SV40 TAg+timidine kinase or the catalytic component of human telomerase+timidine kinase. Immortalization was reverted by deletion of the immortalizing genes through adenoviral mediated expression of Cre-recombinase followed by selection of unexcised cells with ganciclovir.

Results : SML cells generated from iPSCs from 3 PEX donors showed consistent alterations in microfibril morphology compared to controls after induction of senescence by gamma radiation and chronic exposure to UVB radiation or to 40% oxygen. Conditional immortalization resulted in cell lines expressing similar levels of smooth muscle cell markers SMA and SM22 as non-immortalized cells after removal of the immortalizing genes. These cell lines also showed the characteristic abnormal microfibril morphology observed in non-immortalized cells from PEX donors after induction of cellular senescence and exposure to either UVB radiation or 40% oxygen.

Conclusions : Reverse immortalization of SML cells generated from iPSCs from PEX donors is a promising approach to provide an unlimited number of cells to model consistently some characteristics of PEX in vitro. Availability of such cell lines should provide a useful tool to investigate the mechanisms responsible for the increase in outflow resistance in PEX glaucoma and identify novel therapies for this disease.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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