133 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 ◀ Figure 1. Hypoxia does not induce damage in iPSC-CMs A. Schematic representation of experimental setup. B, C. ACTN1 and cTnT expression of iPSC-CMs assessed by immunofluorescence microscopy (B) and flow cytometry (C) (n=8) D. Ki67 expression of iPSC-CMs assessed by flow cytometry (n=9). E. Ethidium homodimer-1 (EthD1)/Calcein AM staining of iPSC-CMs cultured in four different media (DMEM-Glu/L, RPMI-Glu/B27, RPMI-B27, RPMI-Lac/L) with indicated nutrient compositions, exposed to indicated O2 concentrations and durations. Arrowheads indicate a representative selection of EthD1-positive cells. F. Quantification (n=8) and media compositions of E. G. Flow cytometry analysis of viability of iPSC-CMs cultured for 24 hours in 21% O2 or 1% O2 and quantification (H) (n=30). Data is collected from 2-3 technical replicates (exact amounts specified in separate panels), 3 biological replicates (cardiac differentiations) of 2 cell lines. H: 3 technical replicates from 10 biological replicates (cardiac differentiations). Data were analysed using one-way ANOVA and Dunnett multiple comparison. *P<0,05. Scale bar: 200μm. Data are shown as mean ± SEM. Metabolically matured iPSC-CMs are sensitive to hypoxia To investigate whether maturation of iPSC-CMs would increase susceptibility to hypoxia, we applied the recently published protocol for metabolic maturation (MM)18 (Figure 2a), resulting in increased ratio of ACTN expressing cells (83,3% ± 1,3% [RPMI/Lac/L (non-MM)] vs. 94,4% ± 2,6% [MM]; P<0,01; Figure 2b, c) and decreased ratio of proliferating KI67 expressing cells (5,9% ± 0,77% [non-MM] vs. 1,4% ± 0,32% [MM]; P<0,01; Figure 2d). MM iPSC-CMs displayed a significant increase in cell death after 16 and 24 hours of hypoxia (fold increase EthD1/ calcein AM ratio compared to normoxic: 1,78 ± 0,14 [1% O2, 16 hours]; 1,83 ± 0,15 [1% O2, 24 hours]; Figure 2e, f). Hypoxia induced damage of MM iPSC-CMs was confirmed by an increase in media lactate dehydrogenase concentration (Figure 2g) (25,0 ± 0,01 [U/l; 21% O2] vs 39,4 ± 5,8 [U/l; 1% O2, 24 hours], P<0,05) and a reduction in cell viability assessed via flow cytometry (56,5% ± 2,8% [21% O2] vs 14,0% ± 1,2% [1% O2, 24 hours]; P<0,001; Figure 2h, i). Additionally, analyses of iPSC-CM beating rates after 24 hours exposure to 1% O2 showed significant decrease only for MM (Supplementary figure 1).
RkJQdWJsaXNoZXIy MTk4NDMw