Induction and analysis of oxidative stress in Sleeping Beauty transposon-transfected human retinal pigment epithelial cells

Item Type:	Article
Title:	Induction and analysis of oxidative stress in Sleeping Beauty transposon-transfected human retinal pigment epithelial cells
Creators Name:	Bascuas, T. and Kropp, M. and Harmening, N. and Asrih, M. and Izsvák, Z. and Thumann, G.
Abstract:	Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H(2)O(2)-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H(2)O(2)-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.
Keywords:	Antioxidants, Biomarkers, Cell Count, Cell Death, Cell Line, Cell Survival, Conditioned Culture Media, Cultured Cells, DNA Transposable Elements, Epithelial Cells, Eye Proteins, Gene Expression Regulation, Glutathione, Granulocyte-Macrophage Colony-Stimulating Factor, Hydrogen Peroxide, Nerve Growth Factors, Neuroprotection, Oxidative Stress, Phosphorylation, Proto-Oncogene Proteins c-akt, Retinal Pigment Epithelium, Serpins, Tissue Donors, Transfection, Uncoupling Protein 2
Source:	Journal of Visualized Experiments
ISSN:	1940-087X
Publisher:	JoVE
Volume:	2020
Number:	166
Page Range:	e61957
Date:	11 December 2020
Official Publication:	https://doi.org/10.3791/61957
PubMed:	View item in PubMed

Repository Staff Only: item control page