173 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc Figure 1. Protocol for the generation of human expandable hepatic organoids (EHOs) from iPSCs. (A) Schematic depiction of the differentiation protocol from iPSCs to hepatobiliary organoids. (B) Representative images depicting morphological changes at different stages of the differentiation. From left to right: iPSCs, DE, HB, cystic EHOs and “lobule-like” EHOs. Scale bars, 200 µM for B1-3 and 500 µM for B4-5. (C) Relative gene expression at different stages of the differentiation of endoderm markers, (D) early hepatocyte markers and (E) early biliary specification. Data represents 3 biological replicates from independent control donors. For C-E, human liver samples were included in the analysis. (F) Immunofluorescent staining of EHOs using albumin (green) on top and HNF4 (green) bottom, with nuclei in blue and cell membrane in red. Scale bar = 100 µm. (G) Upper graph: Urea released in supernatant by organoids (blue) and human precision-cut liver slices (PCLS) (yellow) in 24 hours. Lower graph: Albumin released in supernatant by organoids (blue) in 24 hours. Data represents 10 biological replicates for the organoids and 3 biological replicates for the PCLS from independent experiments. All graphs: error bars indicate SEM.
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