184 Chapter 6 Conclusion and Outlook In conclusion, the Mat-EHO organoids derived from iPSCs of severe MCADD patients show a phenotype that is characteristic for these patients and provide a good basis for future studies into patient-to-patient variability. For instance, they can be used to assess the phenotype of genetic variants of the ACADM gene that give rise to different residual activities. More interestingly, they can be used to study putative compensatory pathways in asymptomatic patients with a classical c.985A>G and zero residual MCAD activity. Furthermore, nutritional interventions and different stressors can be modulated in vitro, to study liver-specific aspects of MCADD pathophysiology and potential therapeutic interventions. METHODS Human fibroblasts Fibroblasts from patients without documented heritable metabolic diseases (n=2) and also from symptomatic patients with MCADD carrying the classical c.985A>G missense mutation (n=2) were obtained from the Department of Genetics of the University Medical Center Groningen. Patients 5 and 8, previously described in the literature28, were chosen for this study because of their classical mutation, symptomatology and normal growth of fibroblasts. Control fibroblasts from C104 and C105 were also described in the literature 28. All patients were born prior to the implementation of the neonatal MCADD screening in the Netherlands (2007). Both MCADD patients were symptomatic, suffered at least one recorded metabolic crisis, resulting in hospitalization with hypoglycaemia (<2.6 mmol/L), coma, and/or seizures. Fibroblasts were cultured in Ham’s F-10 Nutrient Mix (Thermo Fisher Scientific 11550043), supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco). Generation and culture of iPSCs from patient fibroblasts Human fibroblasts were reprogrammed into induced pluripotent stem cells following the previously published protocol33. iPSCs were cultured in Matrigel (Corning)-coated plates in mTeSRTM Plus medium (STEMCELL Technologies). Cells were passaged every 4-6 days and medium was changed every other day.
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