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Plössl et al. J Transl Genet Genom 2022;6:46-62 https://dx.doi.org/10.20517/jtgg.2021.39 Page 48

for developing AMD, we applied a genetic risk score (GRS) based on a minimal set of selected risk variants
[20]
from eight gene loci associated with AMD at genome-wide significance . This allowed us to discriminate
iPSC-RPE cell lines in those with very high and those with very low genetic risk for AMD. Oxidative stress
was induced by treating the cell lines with sodium iodate (SI), a potent stressor of RPE cells [21,22] . By
comparing cell lines of the two extreme ends of the genetic risk spectrum in defined experimental settings,
we aimed to sort out the genetic influence of NRF2-mediated stress response in AMD pathogenesis.

METHODS
Genotyping and GRS calculation
Patients were recruited at the Eye Clinic of the University Hospital Regensburg and clinically examined by
an experienced ophthalmologist (Brandl C). AMD phenotyping was performed in a clinical routine setting
via gold standard multimodal imaging. This included funduscopy and color fundus imaging (FF450 plus
fundus camera, Zeiss, Oberkochen, Germany), macular optical coherence tomography, fundus
autofluorescence, and fluorescein angiography (all Spectralis, Heidelberg Engineering, Heidelberg,
Germany). Patients were assessed for the presence or absence of features of early AMD, such as drusen
and/or hyper/hypopigmentation, or signs of late-stage disease such as geographic atrophy or neovascular
manifestations, the latter characterized by sub/intra-retinal fluid, leakage of choroidal neovascularization,
and bleeding. Using Sanger sequencing or restriction fragment length polymorphisms, all probands were
genotyped for 13 selected AMD-associated SNPs at eight different loci known to be highly correlated with
[20]
AMD risk. Individual GRSs were calculated using the model reported by Grassmann et al. , 2012.

Generation of iPSCs and differentiation into RPE
Based on the established GRS, blood samples or skin biopsies were again collected from high-risk (HR) and
low-risk (LR) probands, fibroblast cultures or peripheral blood mononuclear cells (PBMCs) were
established, reprogrammed to induced pluripotent stem cells (iPSCs), and subsequently differentiated into
iPSC-RPE as described [23-25] . Pigmented cell clusters were picked from six-well plates and sub-cultured
followed by cryopreservation in liquid nitrogen. Prior to experimental analysis, iPSC-RPE cells were
thawed, seeded on six-well plates coated with Matrigel® GFR (Corning Inc., Corning, NY, USA), passaged
once after two weeks, and cultivated for another two weeks before seeding the cells onto Transwell filters
(ThinCert® Cell Culture Inserts by Greiner Bio-One, Kremsmünster, Austria) in a 6- or 12-well format.
Unless stated otherwise, cells were matured on Transwell inserts for sux weeks and used in experiments
during weeks 6-8. The culture medium was replaced every 72 h.

Verification of RPE cell characteristics
Immunocytochemistry
For immunocytochemistry, a section of the Transwell filter was excised and transferred into 1× PBS. After
fixation of the cells with 2% PFA for 10 min followed by three washing steps for 5 min with 1× PBS, the
filters were blocked in 10% normal goat serum, 0.3% Triton X-100 in PBS. After 30 min of incubation at
room temperature (RT), the blocking solution was replaced by the primary antibody solution and incubated
at 4 °C overnight (ON). After three washing steps with PBS, cells were incubated in secondary antibody for
1 h at RT, washed again, and mounted onto microscope slides using Dako Fluorescence Mounting Medium
(Agilent Technologies, Santa Clara, USA). Antibodies used for immunochemistry were rabbit polyclonal
anti-hBEST1-334 (targeting amino acids 572-585 of the human BEST1 protein) (diluted 1:250) and rabbit
[26]
polyclonal anti-ZO-1 (Thermo Fisher Scientific, Waltham, USA) (diluted 1:500). Secondary antibodies for
immunofluorescence were goat Alexa 594-conjugated anti-rabbit and Alexa 488-conjugated anti-mouse
(dilution 1:1000, Thermo Fisher Scientific). All filters were counterstained with DAPI for visualization of
cell nuclei.