Following the protocol of Cai et al. (2012), we used feeder-free differentiation conditions entailing the addition of a variety of growth factors and chemicals to the growth media. We incubated cells in (1) RPMI-B27 (RPMI-1640 from Thermo Fisher Scientific; 2% B-27 Supplement Minus Insulin from Thermo Fisher Scientific) medium supplemented with recombinant human/mouse/rat activin A (100 ng/mL; R&D Systems), recombinant human BMP-4 (10 ng/mL; Peprotech), and recombinant human FGF basic (20 ng/mL; R&D Systems) for 2 days, followed by 3 days of incubation in RPMI-B27 (minus insulin) supplemented with activin A alone, in ambient oxygen/5% CO2, yielding definitive endoderm; (2) RPMI-B27 (with insulin, i.e., made with 2% B-27 Supplement) supplemented with BMP-4 (20 ng/mL; PeproTech) and FGF basic (10 ng/mL) for 5 days in 5% oxygen/5% CO2, yielding hepatic progenitor cells; (3) RPMI-B-27 (with insulin) supplemented with recombinant human HGF (20 ng/mL; PeproTech) for 5 days in 5% oxygen/5% CO2, yielding immature HLCs; and (4) HCM Hepatocyte Culture Medium (Lonza) without EGF and supplemented with recombinant human oncostatin M (20 ng/mL; R&D Systems) for 5 days in ambient oxygen/5% CO2,
