hASCs were plated at a density of 40,000 cells/cm2 on collagen I (Invitrogen)-coated dishes (Nunc, Roskilde, Denmark) and cultured in DMEM/F-12 (Invitrogen) supplemented with 10% FBS-MSCS (HyClone), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO2. Once the cells reached 90% confluence, they were washed twice with PBS and incubated with serum-free DMEM/F-12 medium for 48 hours.
To induce definitive endoderm differentiation, the cells were incubated with DMEM/F-12 containing 0.5 mg/mL albumin fraction V(BSA) (Sigma-Aldrich), 50 ng/mL Wnt3a (Peprotech, Rocky Hill, NJ,USA), or GSK3 inhibitors Chir98014 (0.2 μM, Selleckchem, Houston, Texas, USA), or Chir99021 (2 μM, Selleckchem) for 24 hours, or 100 ng/mL activin A (Peprotech) for 72 hours; 1% ITS (Sigma-Aldrich) was added to the medium beginning at the second day.
For subsequent hepatic differentiation, the medium was changed to MEM/NEAA (Invitrogen), supplemented with 0.5 mg/mL BSA, 1% ITS, 20 ng/mL BMP2 (Peprotech) and 30 ng/mL FGF4 (Peprotech) for 5 days. To allow for hepatocytes maturation, the cells were further treated with 20 ng/mL Hepatocyte Growth Factor (HGF) for 5 days, and 20 ng/mL HGF, 10 ng/mL oncostatin M (OSM) (Peprotech) plus 10−6 M Dexamethasone (DEX, Sigma-Aldrich) treatment for another 5 days. The differentiation media were changed every 2 days.
To induce definitive endoderm differentiation, the cells were incubated with DMEM/F-12 containing 0.5 mg/mL albumin fraction V(BSA) (Sigma-Aldrich), 50 ng/mL Wnt3a (Peprotech, Rocky Hill, NJ,USA), or GSK3 inhibitors Chir98014 (0.2 μM, Selleckchem, Houston, Texas, USA), or Chir99021 (2 μM, Selleckchem) for 24 hours, or 100 ng/mL activin A (Peprotech) for 72 hours; 1% ITS (Sigma-Aldrich) was added to the medium beginning at the second day.
For subsequent hepatic differentiation, the medium was changed to MEM/NEAA (Invitrogen), supplemented with 0.5 mg/mL BSA, 1% ITS, 20 ng/mL BMP2 (Peprotech) and 30 ng/mL FGF4 (Peprotech) for 5 days. To allow for hepatocytes maturation, the cells were further treated with 20 ng/mL Hepatocyte Growth Factor (HGF) for 5 days, and 20 ng/mL HGF, 10 ng/mL oncostatin M (OSM) (Peprotech) plus 10−6 M Dexamethasone (DEX, Sigma-Aldrich) treatment for another 5 days. The differentiation media were changed every 2 days.