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 2D and 3D-derived hepatocytes to fetal and adult liver, indicate 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 serve as a valuable resource for future research.