Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Summary

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H₂O₂, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

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 H₂O₂-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 H₂O₂-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.

Introduction

The model described here, offers a useful approach to evaluate the efficiency ofbiopharmaceutical agents for reducing oxidative stress in cells. We have used the model to investigate the protective effects of PEDF and GM-CSF on the H₂O₂-mediated oxidative stress on retinal pigment epithelial cells, which are exposed to high levels of O₂, and visible light, and the phagocytosis of photoreceptor outer segment membranes, generating significant levels of reactive oxygen species (ROS)^1,2. They are considered a major contributor to the pathogenesis of avascular age-related macular de....

Protocol

Procedures for the collection and use of human eyes were approved by the Cantonal Ethical Commission for Research (no. 2016-01726).

1. Cell isolation and culture conditions

1. Human ARPE-19 cell line
   1. Culture 5 x 10⁵ ARPE-19 cells, a human RPE cell line, in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/Ham´s F-12) supplemented with 10% fetal bovine serum (FBS), 80 U/mL penicillin, 80 µg/mL streptomycin, and 2.5 µg/mL amphotericin B (complete medium) at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air in a T75 flask (for other cell densities see Table 1).
   2. Change ....

Results

Induction of oxidative stress in human Retinal Pigment Epithelial cells
ARPE-19 and primary hRPE cells were treated with varying concentrations of H₂O₂ for 24 h and the intracellular level of the antioxidant glutathione was quantified (Figure 2A,B). H₂O₂ at 50 µM and 100 µM did not affect glutathione production, whereas at 350 µM there was a significant decrease of glutathione i.......

Discussion

The protocol presented here offers an approach to analyze the anti-oxidative and protective function of PEDF and GM-CSF produced by transfected cells, which can be applied to cells transfected with any putative beneficial gene. In gene therapeutic strategies that have the objective to deliver proteins to tissue by transplanting genetically modified cells, it is critical to obtain information as to the level of protein expression, the longevity of expression, and the effectiveness of the expressed protein in a model of th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	Corning	353047
6-well plates	Greiner	7657160
96-well culture plate white with clear flat bottom	Costar	3610	Allows to check the cells before measuring the luminescence (GSH-Glo Assay)
96-well plates	Corning	353072
Acrylamid 40%	Biorad	161-0144
Amphotericin B	AMIMED	4-05F00-H
Antibody anti-GMCSF	ThermoFisher Scientific	PA5-24184
Antibody anti-mouse IgG/IgA/IgM	Agilent	P0260
Antibody anti-PEDF	Santa Cruz Biotechnology Inc	sc-390172
Antibody anti-penta-His	Qiagen	34660
Antibody anti-phospho-Akt	Cell Signaling Technology	9271
Antibody anti-rabbit IgG H&L-HRP	Abcam	ab6721
Antibody donkey anti-rabbit Alexa Fluor 594	ThermoFisher Scientific	A11034
Antibody goat anti-mouse Alexa 488	ThermoFisher Scientific	A-11029
ARPE-19 cell line	ATCC	CRL-2302
BSA	Sigma-Aldrich	A9418-500G
chamber culture glass slides	Corning	354118
CytoTox-Glo Cytotoxicity Assay	Promega	G9291
DAPI	Sigma-Aldrich	D9542-5MG
DMEM/Ham`s F12	Sigma-Aldrich	D8062
Duo Set ELISA kit	R&D Systems	DY215-05
EDTA	ThermoFisher Scientific	78440
ELISAquant kit	BioProducts MD	PED613-10-Human
Eyes (human)	Lions Gift of Sight Eye Bank (Saint Paul, MN)
FBS	Brunschwig	P40-37500
Fluoromount Aqueous Mounting Medium	Sigma-Aldrich	F4680-25ML
FLUOstar Omega plate reader	BMG Labtech
GraphPad Prism software (version 8.0)	GraphPad Software, Inc.
GSH-Glo Glutathione Assay	Promega	V6912
hydrogen peroxide (H2O2)	Merck	107209
ImageJ software (image processing program)	W.S. Rasband, NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/; 1997–2014
Imidazol	Axonlab	A1378.0010
Leica DMI4000B microscope	Leica Microsystems
LightCycler 480 Instrument II	Roche Molecular Systems
LightCycler 480 SW1.5.1 software	Roche Molecular Systems
NaCl	Sigma-Aldrich	71376-1000
NaH2PO4	Axonlab	3468.1000
Neon Transfection System	ThermoFisher Scientific	MPK5000
Neon Transfection System 10 µL Kit	ThermoFisher Scientific	MPK1096
Neubauer chamber	Marienfeld-superior	640010
Ni-NTA superflow	Qiagen	30410
Nitrocellulose	VWR	732-3197
Omega Lum G Gel Imaging System	Aplegen Life Science
PBS 1X	Sigma-Aldrich	D8537
Penicillin/Streptomycin	Sigma-Aldrich	P0781-100
PerfeCTa SYBR Green FastMix	Quantabio	95072-012
PFA	Sigma-Aldrich	158127-100G
Pierce BCA Protein Assay Kit	ThermoFisher Scientific	23227
Primers	Invitrogen		See Table 1 in Supplementary Materials
pSB100X (250 ng/µL)			Mátés et al., 2009. Provide by Prof. Zsuzsanna Izsvak
pT2-CMV-GMCSF-His plasmid DNA (250 ng/µL)			Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.
pT2-CMV-PEDF-His plasmid DNA (250 ng/µL)			Constructed using the existing pT2-CMV-PEDF-EGFP plasmid reported in Johnen, S. et al. (2012) IOVS, 53 (8), 4787-4796.
QIAamp DNA Mini Kit	QIAGEN	51304
recombinant hGM-CSF	Peprotech	100-11
recombinant hPEDF	BioProductsMD	004-096
ReliaPrep RNA Cell Miniprep System	Promega	Z6011
RIPA buffer	ThermoFisher Scientific	89901
RNase-free DNase Set	QIAGEN	79254
RNeasy Mini Kit	QIAGEN	74204
SDS	Applichem	A2572
Semi-dry transfer system for WB	Bio-Rad
SuperMix qScript	Quantabio	95048-025
Tris-buffered saline (TBS)	ThermoFisher Scientific	15504020
Triton X-100	AppliChem	A4975
Trypsin/EDTA	Sigma-Aldrich	T4174
Tween	AppliChem	A1390
Urea	ThermoFisher Scientific	29700
WesternBright ECL HRP substrate	Advansta	K-12045-D50
Whatman nitrocellulose membrane	Chemie Brunschwig	MNSC04530301