Fig. 1: Moderate lysosomal expansion and oxidative stress in MLD iPSCs.

a Dot-plot showing the number of lysosomes/cell in ND, MLD, and MLD-ARSA iPSC clones. n = 6 independent experiments, 2–4 clones/group with 2–4 replicates/clone. Clones used: ND 1.1 (O); ND 1.3 (□); ND 2.2 (Δ); ND 2.3 (∇); MLD 1.1 (O); MLD 1.2 (□); MLD 1.3 (Δ); MLD-ARSA 1.1 (O); MLD-ARSA 1.2 (□). Data were normalized to the average ND value of each experiment. Data were analyzed by one-Way ANOVA followed by Bonferroni’s multiple comparisons test. b Representative transmission electron microscopy (EM) images of ND, MLD, and MLD-ARSA iPSCs and quantification of the lysosomal area performed on EM images. Asterisks identify lysosomal vesicles. Scale bars: 2 µm. We counted 60–70 lysosomes/clone; 2–4 clones/group with 1–3 replicates/clone. Clones used: ND 1.1 (O); ND 1.3 (□); ND 2.2 (Δ); ND 2.3 (∇); MLD 1.1 (O); MLD 1.2 (□); MLD 1.3 (Δ); MLD-ARSA 1.1 (O); MLD-ARSA 1.2 (□). Bars indicate the mean ± SEM. Data were normalized to the average ND value of each experiment. Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. *p < 0.05. c Representative immunofluorescence pictures and quantification of the LAMP1-immunopositive area (green) in ND, MLD, and MLD-ARSA iPSCs. Nuclei are counterstained with To-Pro III (blue). Scale bar: 20 µm. Data are expressed as px²/number of cells with a defined nucleus and are normalized to the average ND value of each experiment. Bars indicate the mean ± SEM. n = 5 experiments, 2–3 clones/group with 1–3 replicates/clone. Clones used: ND 1.3 (□); ND 2.1(Δ); MLD 1.1 (O); MLD 1.2 (□); MLD 1.3 (Δ); MLD2.1 (∇); MLD-ARSA 1.1 (O); MLD-ARSA 1.2 (□); MLD-ARSA 1.3 (Δ). d Western blot showing the expression of early endosomal antigen protein 1 (EEA1), GM130 (Golgi marker) and LAMP1 in representative ND, MLD, and MLD-ARSA iPSC clones. β-actin was used as loading control (10 µg of total proteins per lane). The bar graph shows the quantification of WB bands. Data are normalized on β-actin and expressed as the percentage of ND in each experiment; n = 3–8 independent experiments, 4 clones/group with 1–6 replicates/clone. Clones used: ND 1.1 (O); ND 1.3 (□); ND 2.2 (Δ); ND 2.3 (∇); MLD 1.1 (O); MLD 1.2 (□); MLD 1.3 (Δ); MLD 2.1 (∇); MLD-ARSA 1.1 (O), MLD-ARSA 1.2 (□); MLD-ARSA 1.3 (Δ); MLD-ARSA 2.1(∇). Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. **p < 0.01, ***p < 0.001. e Representative confocal fluorescence pictures and quantification of fluorescence intensity of ND, MLD, and MLD-ARSA iPSCs stained with Deep Red CellROX^® to detect reactive oxygen species (ROS; blue). Nuclei counterstained with Hoechst (white). Scale bar: 10 µm. We analyzed n = 10 fields/coverslip/clone in five independent experiments; 2–3 clones/group with 3–6 replicates/clone. Clones used: ND 1.1 (O); ND 2.2 (Δ); ND 2.3 (∇); MLD 1.1 (O); MLD 1.2 (□); MLD-ARSA 1.1 (O), MLD-ARSA 1.2 (□); MLD-ARSA 1.3 (Δ). Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. **p < 0.01; ***p < 0.001. f Representative immunofluorescence picture of ND iPSCs expressing Ki67 (green) and cleaved caspase-3 (CC3, red; arrows). Nuclei are counterstained with DAPI (blue). Single channels and the merged image are shown. Scale bar: 10 µm. g Graphs showing percentages of cleaved caspase-3- and Ki67-positive cells (on total nuclei) in ND, MLD, and MLD-ARSA iPSCs. Data are expressed as the mean ± SEM; n = 3 independent experiments, 3–4 clones/group with 1–2 replicates/clone. Clones used: ND 1.1 (O); ND 1.3 (□); ND 2.2 (Δ); MLD 1.1 (O); MLD 1.2 (□); MLD 1.3 (Δ); MLD 2.1 (∇); MLD-ARSA 1.1 (O), MLD-ARSA 1.2 (□); MLD-ARSA 1.3 (Δ)