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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 56

the SI concentrations consistent over time. Concentrations of 0.125 and 0.25 mM were chosen for these
experiments based on findings in preliminary tests (data not shown). Initially, we confirmed that both SI
concentrations had no negative effect on iPSC-RPE cytotoxicity [Supplementary Figure 2A], stable TEER
values [Supplementary Figure 2B], and monolayer integrity and intact cobblestone morphology, as
indicated by uniform ZO-1 staining [Supplementary Figure 2C]. Relative cytotoxicity of the SI treatments
was not increased in comparison to control, but it was increased when cells were treated with 1.5 mM SI for
72 h [Supplementary Figure 2A]. Treatment with SI resulted in a dose-dependent upregulation of HMOX1
expression, with statistical significance being reached for the 0.25 mM treatment in all cell lines (Kruskal-
Wallis test with post hoc Dunn’s multiple comparison test using Benjamini-Hochberg method, P < 0.01)
[Figure 5A].

When data were analyzed with regard to differences between HR and LR cell lines, the dose-dependent
effect was clearly visible in both groups, although cell lines with differing AMD-associated GRS failed to
show differences in HMOX1 expression after treatment with either of the concentrations (Mann-Whitney U
-test, P > 0.05) [Figure 5A]. In line with the results from the 24 h treatment, NQO1 expression was impacted
more strongly by 72 h SI treatment than HMOX1 expression. NQO1 expression also increased dose
dependently and reached values of 3-10-fold upregulation, showing statistically significant differences
between control and SI treatment for both concentrations in all cell lines (Kruskal-Wallis test with post hoc
Dunn’s multiple comparison test using Benjamini-Hochberg method, P < 0.05 for comparison control vs.
-5
0.125 mM SI, P < 5 × 10 for comparison control vs. 0.25 mM SI). Concordant with data on HMOX1
expression, NOQ1 expression upon oxidative stress treatment was not differentially expressed in HR and LR
cell lines (Mann-Whitney U-test, P > 0.05) [Figure 5B]. The qRT-PCR results were confirmed by Western
blot analysis and a SI dose-dependent increase in HMOX1 and NOQ1 expression was observed at 72 h of SI
treatment compared to control. After densitometric evaluation of Western blot signal intensities, the
observed differences failed to reach statistical significance in all cell lines (Kruskal-Wallis test with post hoc
Dunn’s multiple comparison test using Benjamini-Hochberg method, P < 0.05), even though a clear SI dose-
dependent trend in increased HMOX1 and NQO1 protein was notable in the Western blot images as well as
the quantifications. No differences were found when comparing oxidative stress induced protein amounts
of HMOX1 and NQO1 between HR and LR cell lines (Mann-Whitney U-test, P > 0.05) [Figure 5C and D].

Taken together, oxidative stress was robustly induced in an acute 24 h setting and an extended duration
situation using lower SI concentrations for 72 h. NRF2 downstream response genes HMOX1 and NQO1
were upregulated under the two conditions chosen, although in no situation a genetic influence in HR and
LR AMD iPSC-RPE lines was apparent. This led us to conclude that the genetic risk to develop AMD has no
measurable effect on influencing the NRF2-associated first-line oxidative stress defense.

DISCUSSION
Our aim was to establish a cellular model system which allows us to combine both genetic and
environmental risk factors in vitro crucial to elucidate the individual contributions to AMD pathogenesis.
Consequently, we selected our study participants based on their genetic risk profile for AMD rather than on
their AMD phenotype alone, resulting in four CNV patients with a very high GRS for AMD and four
individuals with a very low GRS who had no AMD phenotype. Patient-derived iPSC-RPE cells were
generated from these individuals and characterized for RPE integrity before they were subjected to analysis
on NRF2-mediated oxidative stress response mechanisms. We established a short term, acute stress model
in which cells were treated with SI for 24 h as well as an intermediate stress duration model of 72 h SI
treatment. While significant upregulation of NRF2 pathway response genes was established under all
experimental conditions, there was no difference in stress reaction between HR and LR cell lines. This led us