185 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc Generation of EHOs from iPSCs iPSCs were differentiated into expandable hepatic organoids (EHOs) following the previously published protocols for hESCs51,52 with some adaptions. iPSC cells were cultured in Matrigel-coated plates and placed in RPMI1640 medium supplemented with 1X B27-Supplement (Invitrogen), 100ng/ml Activin A (Peprotech), and 30% Wnt3a-conditioned medium (kindly provided by Hans Clevers). Wnt3aCM was removed after 24 hours and cells were kept in the same medium supplemented with 100ng/ml Activin A and 2X B27-supplement for 48 hours. The cells were then transferred to Hepatocyte Culture Medium (HCM) (Lonza, CC-3198) without EGF and supplemented with 20ng/ml BMP4 and 10ng/ml FGF2 (both Peprotech) for the next 5 days. After 5 days, the cells were dissociated using 0.25% Trypsin-EDTA and embedded in BME domes ( 5000-10000 cells per 50µL dome) in Corning 24-well plates. The cells were cultured in an expansion medium (expansion medium) containing Ad. DMEM/ F12 (Gibco) supplemented with 1X Glutamax, 1X HEPES, P/S (all Gibco), 1X B-27 Supplement, 1X N-2 Supplement (all Invitrogen), 10mM Nicotinamide (Sigma Aldrich), 1.25mM N-acetylcysteine (Sigma Adlrich), 50ng/ml EGF (Peprotech), 10uM Forskolin (TOCRIS), 10nM Iso-leu gastrin (Sigma Aldrich), 30% Rspon1-CM (Kindly provided by Calvin J. Kuo) and 10% Wnt-3a CM. Medium was changed every 2-3 days. Organoids were passaged every 7-9 days by manually disrupting the BME domes and split in a ratio 1:8-1:10 depending on the line and supplemented with 10 μM Y 27632 dihydrochloride (Axon Medchem) for 2 days after passaging. Maturation of EHOs into Mat-EHOs Expandable hepatic organoids were matured into hepatocyte-like EHOs (MatEHOs) following the previously published protocol51. Organoids were kept in expansion medium for 4-6 days after passage, and then placed in maturation medium (MM) containing Ad. DMEM/F12 supplemented with 1X Glutamax, 1X HEPES, P/S, 1X B-27 Supplement, 1X N-2 Supplement, 10mM Nicotinamide, 1.25mM N-acetylcysteine, 10nM Iso-leu gastrin, 50 ng/ml HGF, 25 ng/ml BMP7 and 25 ng/ml FGF4 (all growth factors from Peprotech). Organoids were kept in maturation medium for 4 days prior to collection for downstream analysis.
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