June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Human Embyronic Stem Cell Derived Polarized Retinal Pigment Epithelial Cells have Higher Resistance to Oxidative Stress-Induced Cell Death than Non-Polarized Cultures
Author Affiliations & Notes
  • Jamie Hsiung
    Ophthalmology, Keck School of Medicine of University of Southern California, Los Angeles, CA
    Pathology, Keck School of Medicine of University of Southern California, Los Angeles, CA
  • Danhong Zhu
    Ophthalmology, Keck School of Medicine of University of Southern California, Los Angeles, CA
    Pathology, Keck School of Medicine of University of Southern California, Los Angeles, CA
  • David Hinton
    Doheny Eye Institute, Los Angeles, CA
    Pathology, Keck School of Medicine of University of Southern California, Los Angeles, CA
  • Footnotes
    Commercial Relationships Jamie Hsiung, None; Danhong Zhu, None; David Hinton, RPT (I), RPT (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2227. doi:
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    • Get Citation

      Jamie Hsiung, Danhong Zhu, David Hinton; Human Embyronic Stem Cell Derived Polarized Retinal Pigment Epithelial Cells have Higher Resistance to Oxidative Stress-Induced Cell Death than Non-Polarized Cultures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2227.

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

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Abstract
 
Purpose
 

Oxidative stress mediated injury to the retinal pigment epithelium (RPE) has been attributed as a major factors in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. We hypothesized that polarized monolayers of hESC-RPE cells are less sensitive to oxidative stress-induced cell death relative to non-polarized RPE cells, possibly due to their increased growth factor secretion as well as their intercellular tight junctions. This work has clinical relevance since both cell suspensions and polarized monolayers of hESC-RPE are being evaluated for their potential for cellular therapy in patients with AMD.

 
Methods
 

Polarized, non-polarized/confluent, non-polarized/sub-confluent fetal human RPE cells and hESC H9-RPE cells were treated for 24 hours with varying dosages of H2O2 (0 uM - 500 uM) and tBH (0 uM - 200 uM) and then analyzed for cell death. Polarized RPE cells were grown on transwell plates and treated when transepithelial resistance (TER) reached > 600 Ω●cm2. Non-polarized/confluent cells were plated the day before oxidative treatment at 1x105 cells/cm2 and non-polarized/sub-confluent cells were plated at 5x104 cells/cm2. The cells were treated with H2O2 or tBH the following day.

 
Results
 

Cell viability assays and TUNEL stains reveal highest amount of cell death in non-polarized/sub-confluent fetal and H9 RPE, a decreased amount of cell death in non-polarized/confluent RPE, and little cell death in polarized fetal and H9-RPE cells when all samples were treated with same dosages of tBH and H2O2. Q-RT PCR results indicate higher levels of expression of the anti-apoptotic gene Bcl-2 in non-polarized/confluent cells relative to non-polarized/sub-confluent cells.

 
Conclusions
 

These results indicate that non-confluent/non-polarized RPE cells are most sensitive to oxidative stress-induced apoptosis. Also, both polarized fetal and polarized hESC-RPE have higher tolerance to oxidative stress relative to non-polarized cells. These results suggest that implantation of polarized hESC-RPE for treatment of geographic atrophy in patients with AMD should have better survival than injections of cell suspensions of hESC-RPE.

 
Keywords: 701 retinal pigment epithelium • 721 stem cells • 634 oxidation/oxidative or free radical damage  
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