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

Abstract

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.

Competing tissue-specific functions for the Tribbles-1 plasma lipid associated locus

“…Additionally, the authors showed that TRIB1 KO 3D organoids had increased triglyceride mass compared to the WT model, but the TRIB1 KO 2D organoids had decreased TG mass. Taken together, the authors proposed that their 3D hiPSC-derived hepatic organoid model recapitulates TRIB1 KO biology [37 ]. This work by Abbey et al is an example of the crucial step of replicating phenotypes from mouse models in a human model, showing the continued functional relevance of TRIB1 in human health beyond mouse biology.…”

The Use of Cell-penetrating Peptide for Delivery of Recombinant Transcription Factor DNA into Primary Human Fibroblast

“…25 CEBPA is a transcription factor that plays a role in hepatocyte cell maturation, hepatogenesis, as well as hepatic metabolism including albumin expression and urea production. 13,26 Another core receptor used in this study, NR1I2, has been reported to have a modulating effect on functional liver genes, such as the CYP family. 13 Increased expression of CYP3A4 in hepatocytelike cells is important, since the production of cytochrome P450 enzymes play a role in drug metabolism.…”

Hepatocyte Differentiation from iPSCs or MSCs in Decellularized Liver Scaffold: Cell–ECM Adhesion, Spatial Distribution, and Hepatocyte Maturation Profile

“… 39 Other approach is to involve these various techniques, including 3D co-culture with non-parenchymal cells (endothelial cells and mesenchymal cells) in the form of organoid. 40 , 41 …”