Hepatocyte-like Cell Differentiation

Differentiation of hepatocyte-like cells was performed as described previously (Si-Tayeb et al., 2010 (link)). Briefly, hESCs were plated on matrigel-coated plates as single cells and cultured in the presence of ROCK inhibitor for 24 hr. After reaching 80% confluency, differentiation was initiated by adding RPMI medium supplemented with B27 (GIBCO, Life Technologies), 0.5% nonessential amino acids (Life Technologies) and Activin A (100 ng/ml, R&D Systems) for 5 days to induce definitive endoderm stage cells. Cells were further differentiated by exposure to RPMI medium supplemented with B27, 0.5% nonessential amino acids, FGF4 (10 ng/ml, R&D Systems) and BMP4 (20 ng/ml, R&D Systems) for 5 days. After an additional 5 days of cultivation in RPMI/B27 supplemented with 0.5% nonessential amino acids and HGF (20 ng/ml, Peprotech), cultures mainly consisted of immature hepatocytes. Maturation to hepatocyte-like cells was initiated by cultivation in HBM medium (Lonza, UK) supplemented with HCM SingleQuots (Lonza) and Oncostatin M (20 ng/ml, R&D Systems). Hepatocytes were probed for cell-specific markers, such as α-fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α).