Abstract
Purpose :
The implantation of a polarized retinal pigment epithelial (RPE) cell sheet derived from human embryonic stem cells is a promising treatment under investigation for RPE dysfunction related diseases, including age related macular degeneration (AMD). However, long-term culture of RPE cells under high oxygen exposure may cause RPE cell stress and senescence. Mesobiliverdin, an analog of biliverdin, and the peptide Humanin were reported to exhibit cytoprotective effects on pancreatic cells and neurons. This study aimed at investigating whether these molecules have cytoprotective effects on polarized HESC-RPE cells as well.
Methods :
1). RPE cells derived from H9 human embryonic stem cells were cultured on vitronectin coated transwell or 24-well culture plates for 4 weeks to form polarized monolayers. 2). The polarized RPE monolayers were treated with different concentrations of Mesobiliverdin or Humanin. 3). The treated RPE cells were measured for cell viability. 4). Western blot, immunostaining, qPCR, and senescence associated beta-gal (SA beta-gal) staining were used to detect the oxidative stress- and senescence-related gene and protein expression.
Results :
1. All control and mesobiliverdin as well as humanin treated HESC-RPE cells exhibited excellent cell viability but did not showed any further increase in viability after treatment with mesobiliverdin and humanin, as compared to the non-treated control cells.
2. Both mesobiliverdin and humanin treatments have modestly increase HESC-RPE cell viability under oxidant stress.
3. Moreover, mesobiliverdin and humanin decreased the senescence of HESC-RPE cells after long-term culture under normal cell culture conditions: Humanin treated HESC-RPE cells displayed only about 30% SA-beta-gal stained positive cells, mesobiliverdin treated cells showed about 60% SA positive cells, and as comparison, the non-treated control cells had about 90% positive cells.
Conclusions :
Neither mesobiliverdin nor humanin increased HESC-RPE cell viability when grown as a monolayer; however, untreated cells already demonstrated excellent viability. Both mesobiliverdin and humanin exhibited protective effects on HESC-RPE cells against oxidative stress, and humanin had an even stronger ability in preventing the polarized HESC-RPE cells from undergoing senescence in vitro. Future studies will focus on the mechanism of Humanin in the inhibition of HESC-RPE cell senescence.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.