Oncostatin m

Differentiation of hPSCs into hepatocyte-like cells was induced by a modified four-step induction method [4 (link)]. Briefly, suspended cells were seeded onto polystyrene plates coated with Matrigel, human recombinant vitronectin, R-Fc, or NC-Fc. Before confluency, the cells were treated with RPMI-1640 medium containing 100 ng/ml Activin A and B27 supplement (without insulin; Life Technologies) for 5 days in a normoxic atmosphere (20% O₂). The cells were then specified into the hepatic lineage by treatment with RPMI-1640/B27 (with insulin) medium containing 10 ng/ml bFGF and 20 ng/ml BMP4 for 5 days in a hypoxic atmosphere (4% O₂). After specification, the cells were cultured in RPMI-1640/B27 medium with 20 ng/ml hepatocyte growth factor for 5 days (4% O₂) and then in HCM (without epidermal growth factor; Lonza, Basel, Switzerland) supplemented with 20 ng/ml oncostatin M for 5 days (20% O₂).

In all, 2×10⁵ cells/cm² of ESC or iPSC were differentiated to DE by harvesting them with gentle cell dissociation reagent (Stemcell Technologies) and plating onto Matrigel (Corning, Catalog number 354230)-coated culture dishes (Corning) in mTeSR1 medium (Stemcell Technologies). The next day, DE differentiation was initiated by using the STEMdiff Definitive Endoderm Kit (Stemcell Technologies). To induce hepatic differentiation, DE cells were harvested using Accutase (Innovative Cell Technologies), re-seeded in Matrigel-coated transwells (Transwell, Corning, Catalog number 3460) as described in Results and cultured in the presence of medium A (basolateral medium (BM): CTS KnockOut DMEM/F12, 10% KnockOut Serum Replacement, 0.5% GlutaMAX supplement, and 0.5% non-essential amino acids all from ThermoFisher Scientific, supplemented with 100 ng/ml HGF), 1% DMSO) for 8 days followed by incubation in medium B (BM, 100 ng/ml HGF, 1% DMSO, 40 ng/ml dexamethasone) for three 3 days. Cells were further matured in HCM (Lonza, omitting the EGF) supplemented with 20 ng/ml oncostatin-M for 5–7 days. For nonpol-HLCs, DE cells were seeded on Matrigel-coated culture plates and step-wise matured in the media described above. HGF was purchased from Peprotech, dexamethasone from Sigma, and oncostatin-M from R & D Systems.