182 Chapter 6 DISCUSSION In this paper we have established a patient-specific iPSC-derived hepatic organoid system for the study of MCADD. These organoids recapitulated the metabolic profile with high levels of medium-chain acyl-carnitines and an elevated C8/C10 ratio that is typical of severe MCADD patients. Furthermore, the Mat-EHOs expressed pathways that are relevant for future studies of the pathophysiology of the disease. It may not be surprising that we observed only minor adaptations in putative compensatory pathways, since the MCADD organoids were derived from symptomatic patients. Compensatory pathways would rather be expected to be more pronounced in asymptomatic patients. Nevertheless, we observed some conspicuous differences between MCADD organoids and controls in respect of CoA metabolism, evidenced by reduced levels of C14-acyl-carnitine and upregulation of NUDT7 in MCADD. Studying metabolic functions in iPSC-derived hepatobiliary MCADD organoids Discovery of organoids in 200916, revolutionized the field of biomedical research, providing an alternative to traditional primary hepatocytes. Recent work has focused on the development of organoids from iPSCs. iPSC-derived hepatobiliary organoids have often been described as closer to fetal tissue25, and advancing their maturity levels to that of liver tissue remains a challenge. Here we compared different maturation stages of the organoids for the study of MCADD. While the original protocol uses a 3-step procedure which relies on spontaneous aggregation of hepatic single cells20, we found that this step was hard to reproduce and did not yield enough biomass for metabolic readouts. Therefore, for the purpose of our study, EHOs were matured to Mat-EHOs (equivalent of “pre-maturation” stage in the reference paper20). At this stage, Mat-EHOs were found to already exhibit liver-like phenotypes such as typical hepatocyte mRNA markers, albumin and urea secretion, and the presence of functional mitochondria and peroxisomes. Importantly, both MCADD EHOs and Mat-EHOs showed accumulation of medium-chain acyl-carnitines (C6-C10), recapitulating a major clinical phenotype. Interestingly, only MCADD Mat-EHO organoids presented reduced levels of short- and long-chain acyl-carnitine relative to their control counterparts. These changes are in alignment with a recent in silico study from our group, based on a detailed computational model of the fatty-acid oxidation, which predicted reduced levels of short- and long-chain acyl-CoAs and their corresponding acyl-carnitines in MCADD28. Decreased short-chain
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