Hepatocyte Differentiation from Human iPSCs

Hepatic differentiation from human iPSCs was performed as described previously ^{(5) (link)} with some modifications. Confluent human iPSCs were passaged at a split ratio of 1:3 on iMatrix-511 with Essential 8^® medium and cultured for 2 d. To initiate differentiation, the cells were subsequently treated with RPMI-1640 plus 2% B27 Minus Insulin (RPMI-B27), containing 3-6 μM CHIR-99021 (MCE, NJ, USA #HY-10182) and 1% GlutaMAX^® (Invitrogen) for 24 h, followed by treatment with RPMI-B27 alone for 24 h. To initiate differentiation into hepatic progenitors, the cells were treated with RPMI-B27 containing 1% GlutaMAX^®, and 1% dimethyl sulfoxide (DMSO; Wako) for 5 d. The cells were detached using TrypLE Select (Invitrogen) and re-seeded on culture dishes coated with Matrigel (Growth Factor Reduced; Corning, Inc.) in hepatocyte maturation medium: Leibovitz’s L-15 medium (Wako) containing 8.3% tryptose phosphate broth, 10 μM hydrocortisone 21-hemisuccinate, 50 μg/mL sodium-L-ascorbate, 100 nM dexamethasone (DEX) (all from Sigma-Aldrich), 0.58% insulin-transferrin-selenium (ITS), 2 mM GlutaMAX^® (all from Invitrogen), 8.3% fetal bovine serum (Biowest), and 100 nM Dihexa (TRC, Ontario, Canada #H293745). This culture was continued for 20 d to obtain definitively differentiated hepatocyte-like cells. During the culture, the medium was changed every 2 d.