131 Metabolic maturation increases susceptibility to hypoxia-induced damage in human iPSC-derived cardiomyocytes 6 experiment was performed with both cell lines. Statistical analysis was performed using Prism 8 (GraphPad) software. Normality was assessed using a Shapiro-Wilk test. To compare two normally distributed groups, a Student’s t-test was performed. For three or more groups and the assessment of one parameter, one-way ANOVA statistical test was used. With multiple parameters and three or more groups, a two-way ANOVA was used. For one-way ANOVA and two-way ANOVA, Dunnett multiple comparisons was used as post-hoc analysis to determine significance at a P<0,05. Results are shown as mean ± SEM. RESULTS Immature iPSC-CMs are not sensitive to hypoxia Human iPSCs were subjected to directed differentiation to cardiomyocytes (iPSC-CMs) using the Wnt pathway inhibition differentiation protocol27,28, resulting in a homogeneous, autonomously contractile cell population robustly expressing CM-defining markers troponin T and ACTN1 (Figure 1a-c) and only low ratios of cells expressing proliferation marker Ki67 (5,9% ± 0,8%; Figure 1d), similar to values reported for neonatal mice and young infant hearts.30,31 To test the sensitivity of these, presumably immature, iPSC-CMs to oxygen and nutrient deprivation, we cultured the cells in four different media of variable nutrient compositions (Supplementary table 1) before exposure to hypoxia: 1) DMEM-Glu/L (25 mM glucose, 10% KOSR), 2) RPMI-Glu/ B27 (11,1 mM glucose, 2% B27), 3) RPMI/B27 (0mM glucose, 2% B27), and 4) RPMI-Lac/L (0 mM glucose, lactate, 10% KOSR). DMEM-Glu/L is highest in glucose and lipids, followed by RPMIGlu/B27 with high glucose and low lipids, where both types are commonly used to maintain iPSC-CMs after differentiation. RPMI/B27 and RPMI-Lac/L are both used during the purification steps of iPSC differentiation into CMs and contain no glucose where RPMI/B27 is low in lipids. RPMI-Lac/L contains additional lactate and higher lipid concentration to inhibit glycolysis and stimulate the use of the respiratory chain32. Incubation in 1% O2 for 24 hours did not lead to increased cell death of iPSC-CMs in high-glucose media (DMEM-Glu/L, RPMI-Glu/B27) and noglucose and low-lipid media (RPMI/B27) compared to incubation in 21% O2 (Figure 1e, f). Only exposure to 1% O2 for 24 hours in no-glucose, high-lipid and high-lactate medium (RPMI-Lac/L) induced minimal, yet significantly increased cell death (ethidium homodimer-1 [EthD1] / calcein AM ratio: 0,02 ± 0,005 [21% O2] vs. 0,11 ± 0,004 [1% O2]; P=0,04). To validate the assessment of cell death, we included a Triton X-100 treatment as a positive control, that resulted in 100% cell death for all media conditions (Figure 1e). To more accurately quantify results, we repeated the respective experiments using RPMI-Lac/L medium and analysed via flow cytometry using an amine-reactive dye to detect membrane disruption. Although in 12 of 30 experiments a small reduction in cell viability was observed, differences were statistically non-significant in flow cytometric analyses (viable cells: 85% ± 1,9% [21% O2] vs. 78,3 ± 3,0 [1% O2]; P=0,1; Figure 1g, h). These findings show immature iPSC-CMs are insensitive to hypoxic damage irrespective of media glucose content. The presence of lipids and lactate in combination with low glucose media led to minimal, yet detectable hypoxic damage in immature iPSC-CMs.
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