Differentiation of Human iPSCs to Hepatocytes

Use of human iPSCs was approved by the MUSC Stem Cell Research Oversight Committee. Human K3 iPSCs were generated previously and have been described elsewhere^{40 (link)}. The hepatocyte differentiation protocol has been published elsewhere^{21 ,25 ,40 (link)} with minor modifications. Briefly, human iPSCs were seeded on Matrigel-coated tissue culture plates with a density of 7 × 10⁵ cells/ml to form a monolayer. Cells were induced to form definitive endoderm in RPMI 1640 Medium (Invitrogen, Waltham, MA) supplemented with 2% B-27 Supplement minus insulin (Invitrogen), 10 ng/mL of BMP4 (Invitrogen), 20 ng/mL FGF2 (Invitrogen), and 100 ng/mL Activin A (Invitrogen) for two days, followed by 100 ng/mL Activin A for three days. Definitive endoderm was then converted to hepatocyte progenitor cells by addition of BMP4 (20 ng/mL) and FGF2 (10 ng/mL) for an additional 5 days. Immature hepatocytes were generated by the inclusion of Hepatocyte Growth Factor (20 ng/mL) (Invitrogen) for 5 days. Cells were finally induced to mature to hepatocyte-like cells by the addition of Oncostatin M (20 ng/mL) (Invitrogen) for 5 days.