Self‐Organizing Human Induced Pluripotent Stem Cell Hepatocyte 3D Organoids Inform the Biology of the Pleiotropic TRIB1 Gene

Establishment of a physiologically relevant human hepatocyte‐like cell system for in vitro translational research has been hampered by the limited availability of cell models that accurately reflect human biology and the pathophysiology of human disease. Here we report a robust, reproducible, and scalable protocol for the generation of hepatic organoids from human induced pluripotent stem cells (hiPSCs) using short exposure to nonengineered matrices. These hepatic organoids follow defined stages of hepatic development and express higher levels of early (hepatocyte nuclear factor 4A [HNF4A], prospero‐related homeobox 1 [PROX1]) and mature hepatic and metabolic markers (albumin, asialoglycoprotein receptor 1 [ASGR1], CCAAT/enhancer binding protein α [C/EBPα]) than two‐dimensional (2D) hepatocyte‐like cells (HLCs) at day 20 of differentiation. We used this model to explore the biology of the pleiotropic TRIB1 (Tribbles‐1) gene associated with a number of metabolic traits, including nonalcoholic fatty liver disease and plasma lipids. We used genome editing to delete the TRIB1 gene in hiPSCs and compared TRIB1‐deleted iPSC‐HLCs to isogenic iPSC‐HLCs under both 2D culture and three‐dimensional (3D) organoid conditions. Under conventional 2D culture conditions, TRIB1‐deficient HLCs showed maturation defects, with decreased expression of late‐stage hepatic and lipogenesis markers. In contrast, when cultured as 3D hepatic organoids, the differentiation defects were rescued, and a clear lipid‐related phenotype was noted in the TRIB1‐deficient induced pluripotent stem cell HLCs. Conclusion: This work supports the potential of genome‐edited hiPSC‐derived hepatic 3D organoids in exploring human hepatocyte biology, including the functional interrogation of genes identified through human genetic investigation.