168 Chapter 6 ABSTRACT Background Medium chain acyl-CoA dehydrogenase deficiency (MCADD) is an inherited metabolic disease, characterized by a mutation in the ACADM gene. Interestingly, even with the same mutation, patients often present with very heterogeneous symptoms, ranging from fully asymptomatic to life-threatening hypoketotic hypoglycemia. The mechanisms underlying this heterogeneity remain unclear. Therefore, there is a need for in vitro models of MCADD that recapitulate the clinical phenotype as a tool to study the pathophysiology of the disease. Methods Fibroblasts of control and symptomatic MCADD patients with the c.985A>G (p.K329E) were reprogrammed into induced pluripotent stem cells (iPSCs). iPSC were then differentiated into hepatic expandable organoids (EHOs), further matured to Mat-EHOs, and functionally characterized. Results EHOs and Mat-EHOs performed typical hepatic metabolic functions, such as albumin and urea production. The organoids metabolized fatty acids, as confirmed by acyl-carnitine profiling and high-resolution respirometry. MCAD protein was fully ablated in MCADD organoids, in agreement with the instability of the mutated MCAD protein. MCADD organoids accumulated mediumchain acyl-carnitines, with a strongly elevated C8/C10 ratio, characteristic clinical phenotype of the disease. Notably, C2 and C14 acyl-carnitines were found decreased in MCADD Mat-EHOs. Finally, MCADD organoids exhibited differential expression of genes involved in mitochondrial β-oxidation and peroxisomal coenzyme A metabolism, particularly upregulation of NUDT7. Discussion iPSC-derived organoids of MCADD patients recapitulated the major clinical phenotype of the disease. Mat-EHOs expressed relevant pathways involved in putative compensatory mechanisms, notably CoA metabolism. The upregulation of NUDT7 expression may play a role in preventing excessive accumulation of dicarboxylic acids in MCADD. This patient-specific-hepatic organoid system is a promising platform to study the phenotypic heterogeneity between MCADD patients.
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