Human Embyronic Stem Cell Derived Polarized Retinal Pigment Epithelial Cells have Higher Resistance to Oxidative Stress-Induced Cell Death than Non-Polarized Cultures

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