Culturing and Differentiating Hepatic Progenitor Cells

AML12 cells were cultured in basic medium containing DMEM/F12, 10% FBS, 1% insulin-transferrin-selenium (ITS) (Sigma, UAS), 40 ng/mL dexamethasone, 1% penicillin, and 1% streptomycin according to ATCC recommendations. For short-term culture, AML12 cells were cultured in basic medium containing LPS (100 ng/mL) for the indicated amounts of time and then collected for RNA and protein analysis. For 3D culture, cells were plated on collagen-coated 12-well plates in reprogramming medium containing DMEM/F12, 1% FBS (Gibco), 10 mM nicotinamide (Sigma, UAS), 0.1 μM dexamethasone, 1% ITS, 20 ng/mL EGF (Peprotech, USA), 20 ng/mL HGF (Peprotech, USA), 10 μM Y27632 (APExBIO, USA), 1 μM A83-01 (APExBIO, USA), and 3 μM CHIR99021 (APExBIO, USA) supplemented with or without LPS (100 ng/mL) [23 (link)]. The medium was replaced every 2 days. After 2 weeks, the cells were harvested for the following assays. Mouse hepatic progenitor cells (mHPCs) were isolated and cultured as previously described [21 (link)].