177 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc Figure 3. Maturation of EHOs into Mat-EHOs. (A) Schematic depiction of the maturation protocol (B) Representative brightfield images of EHOs and Mat-EHOs. Scale bar = 500 µm. (C) Relative gene expression of mature hepatic markers and peroxisomal markers in EHOs and Mat-EHOs. Data represents 8 biological replicates from independent experiments ± SEM. (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 two-tailed unpaired t test). (D) Albumin secreted into the supernatant after 24 hours in EHOs and Mat-EHOs. Data represents 4 biological replicates ± SEM. (*P<0.05, **P<0.01, two-tailed unpaired t test). Peroxisomal adaptation in MCADD Subsequently, we investigated if mitochondrial and peroxisomal fatty-acid oxidation pathways (Figure 4a) were regulated in response to loss of MCAD protein. In order to mimic the fasting conditions, which often trigger symptoms in patients, we exposed the Mat-EHO organoids to glucose-free medium in the presence of BSA-palmitate and carnitine. Again similar to the clinical phenotype, medium-chain acyl-carnitines accumulated in MCADD and the ratio C8/C10 was strongly elevated (Figure 4b-c). Interestingly, and unlike in
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