Sfrp5

For hepatic differentiation, aggregates on day 3 of DE differentiation were adapted to hepatic differentiation media [20 (link)]. In short, the medium was changed to hepatocyte culture medium (Lonza #CC-3198) with 30 ng/mL of fibroblast growth factor 4 (FGF-4, Peprotech #100-31), 20 ng/mL of bone morphogenetic protein 2 (BMP-2, Peprotech #120-02), and 10 µM SB431542 (Sigma Aldrich #S4317), 0.5 µg/mL of secreted frizzled-related protein 5 (sFRP-5, R&D Systems #6266-SF) for 24 h in Erlenmeyer flasks rotating at 70 rpm. Aggregates were then dissociated into single cells using TrypLE (Thermo Fisher #12604013) and plated on Matrigel^® (Corning #356231) coated plates with a density of 45,000 cells/cm² in hepatic differentiation media containing 10 µM Y-27632 (Tocris #1254). The cells were cultured for three more days with daily medium changes. On day 5 of differentiation, the medium was changed to hepatocyte culture medium supplemented with 20 ng/mL hepatocyte growth factor (HGF, Peprotech #100-39) for a further four days with daily medium change. On day 9 of differentiation, the medium was changed to hepatocyte culture medium (Lonza) with 20 ng/mL HGF (Peprotech #100-39), 10 ng/mL Oncostatin M (OSM; Peprotech #300-10) and 10 ng/mL dexamethasone (Sigma Aldrich #D4902) for an additional four days. Cells were analyzed on day 14 of differentiation.