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

Johannes Krumm, Keisuke Sekine , Patroklos Samaras, Agnieska Brazovskaja , Markus Breunig , Ryota Yasui , Alexander Kleger , Hideki Taniguchi , Mathias Wilhelm , Barbara Treutlein , J Gray Camp , Bernhard Kuster 
Source: Cell Rep
Publication Date: ()
Issue: 38: 110604
Research Area:
Drug Discovery
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Culture Media:

Lastly, cells were cultured in hepatocyte culture medium (Lonza) without EGF and supplemented with 20 ng/mL hepatocyte growth factor and 20 ng/mL oncostatinM (both R&D Systems) until day 21. The medium was exchanged daily during the differentiation process and the cell morphology was monitored by microscopy. At day 0 (iPSC), day 6 (DE), day 8 (HE), day 13 (IH), and day 21 (MH) cells were harvested and subsequently used for RNA extraction and MS sample preparation. For the higher resolution experiment, samples were taken every 12 h between HE and IH. As shown by Camp et al., close to 100% of the iPSCs developed into DE (Camp et al., 2017)


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.