Directed Differentiation of iPSCs into iHeps

Directed differentiation of iPScs into iHeps was performed in vitro using a variation of the 4-step protocol described by Si-Tayeb.^{16 (link)} Briefly, iPScs were single-cell passaged onto growth factor reduced Matrigel™ (BD Biosciences, San Jose, CA) and induced to differentiate into definitive endoderm cells by treatment with RPMI (Invitrogen, Carlsbad, CA), 1X B27 w/o insulin (Invitrogen, Carlsbad, CA) and 0.5X NEAA (Invitrogen, Carlsbad, CA) containing 100 ng/mL activin A (R&D Systems, Minneapolis, MN), 10 ng/mL BMP4 (R&D Systems, Minneapolis, MN) and 20 ng/mL FGF2 (Biosciences, San Jose, CA) for 2 days followed by treatment with RPMI, 1X B27 w/o insulin and 0.5X NEAA with 100 ng/mL activin A for 3 days at ambient O₂/5% CO₂. For hepatic specification, the cells were cultured in RPMI, 1X B27 w/insulin (Invitrogen, Carlsbad, CA) and 0.5X NEAA containing 20 ng/mL BMP4 and 10 ng/mL FGF2 for 5 days at 4% O₂/5% CO₂. For hepatic induction, the cells were treated with RPMI, 1X B27 w/insulin and 0.5X NEAA containing 20 ng/mL HGF (R&D Systems, Minneapolis, MN) for 5 days at 4% O₂/5% CO₂. For hepatic maturation, the cells were cultured for 5 days in HCM™ (Lonza, Walkersville, MD) without EGF and with 20 ng/mL oncostatin M (R&D Systems, Minneapolis, MN) at ambient O₂/5% CO₂.