High temporal resolution proteome and phosphoproteome profiling of stem cell-derived hepatocyte development

Primary human hepatocytes are widely used to evaluate liver toxicity of drugs, but they are scarce and demanding to culture. Stem cell-derived hepatocytes are increasingly discussed as alternatives. To obtain a better appreciation of the molecular processes during the differentiation of induced pluripotent stem cells into hepatocytes, we employ a quantitative proteomic approach to follow the expression of 9,000 proteins, 12,000 phosphorylation sites, and 800 acetylation sites over time. The analysis reveals stage-specific markers, a major molecular switch between hepatic endoderm versus immature hepatocyte-like cells impacting, e.g., metabolism, the cell cycle, kinase activity, and the expression of drug transporters. Comparing the proteomes of two- (2D) and three-dimensional (3D)-derived hepatocytes with fetal and adult liver indicates a fetal-like status of the in vitro models and lower expression of important ADME/Tox proteins. The collective data enable constructing a molecular roadmap of hepatocyte development that serves as a valuable resource for future research. [Display omitted] •High temporal resolution proteome of stem cell-derived hepatocyte differentiation•In vitro hepatocyte differentiation is accompanied by a major molecular switch•3D liver organoids better resemble in vivo hepatocytes than 2D models•Stem cell-derived hepatocytes are fetal-like and not fully ADME/tox compatible Krumm et al. provide an extensive resource of the temporal changes in the proteome, phosphoproteome, and acetylproteome during induced pluripotent stem cell (iPSC)-derived hepatocyte differentiation. They discover a major molecular switch and find that in vitro differentiated hepatocytes are in a fetal-like state with decreased expression of important ADME-tox proteins.