Extended Data Fig. 10: Generation of gene knockout hepatocyte organoids.
From: Generation of human adult hepatocyte organoids with metabolic functions
a. Confirmation of TP53-knockout in HHOs with Sanger sequencing. Two knockout clones derived from two different donors (PY9 and PY30) were generated. b. Selection of TP53-knockout organoids by a treatment with the MDM2 inhibitor, nutlin-3. Scale bar, 500 μm. Similar results were obtained from at least three technical replications. c. Representative morphology of TP53-knockout HHOs showing their duct-like cystic structures. Scale bar, 100 μm. Identical results were obtained from observation of at least 50 organoids. d. The percentage of EdU+ cells in TP53-knockout HHOs. Culture duration from PHHs are shown on the bottom. n = 47 (218 days) and 73 (345 days) cells were analyzed. e. Albumin secretion, glucose production and urea synthesis from TP53-knockout HHOs. Culture duration from PHHs are shown on the bottom. Data from two TP53-knockout clones. Each dot shows one well. Data and error bars show mean ± SD of three technical replicates. f,g. Ornithine (f) and Urea (g) production from wild type (WT) and OTC-knockout HHOs. Each dot shows one well. Data and error bars show mean ± SD of three technical replicates. h–j. Generation of additional OTC-knockout HHOs. Confirmation of additional OTC-knockout in HHOs with Sanger sequencing (h). Three knockout clones derived from PY9 were generated. A GFP-expressing vector was introduces as a control, and three GFP-expressing clones were also generated. Protein-level confirmation of OTC knockout in OTC-WT and KO clones (i). Morphology of GFP-expressing WT clones and OTC-knockout HHOs (j). Identical results were obtained from at least 50 organoids for each clone. k. Urea synthesis from OTC-knockout HHOs compared with WT HHO clones. HHOs were incubated in gluconeogenesis assay medium without arginine and with 1 mM ornithine. Each dot shows one well. Data and error bars show mean ± SD of four technical replicates. l. The percentage of EdU+ cells in WT GFP clones and OTC-knockout HHOs. Culture duration from PHHs are shown on the bottom. n refers to the number of cells analyzed in each clonal HHO lines.
