Oncostatin

HLCs were harvested after 20 days of hepatic differentiation cells with 2× TrypLE (Life Technologies), and plated onto Matrigel (Corning)-coated 96 well plate at the density of 3 × 10⁴ cells per well for overnight incubation. Cells were then treated with serially diluted concentrations of acetaminophen (APAP, Sigma) and pazopanib hydrochloride (Selleck Chemicals) in the basal differentiation medium supplemented with 20 ng/ml HGF (R&D systems), 100 ng/ml Follistatin-288 (R&D systems) and 20 ng/ml Oncostatin (R&D systems) for 24 hours. Drug dilutions were prepared in DMSO. Cell viability was determined by the MTS assay using CellTiter 96^® AQueous One Solution Cell Proliferation Assay kit (Promega) and the ATP assay was performed with CellTiter-Glo^® Luminescent Cell Viability Assay kit (Promega).
For viability experiments with N-acetyl cysteine (NAC), a similar procedure was adopted except cells were co-incubated with 0.5 mM NAC (Hidonac^®, Zambon S.p.A.) and PZ for 24 hours. Vehicle controls were appropriately modified to include 0.05% EDTA in PBS (in which NAC was dissolved). Cell viability was measured using CellTiter-Glo reagent.

The differentiation of iPSCs into hepatocytes was carried out as described previously35 (link) with slight modification. Briefly, iPSCs were cultured to 70% confluence before switching to a basal differentiation medium containing 100 ng/ml activin A (R&D systems) and 50 ng/ml WNT3a (R&D systems) for 6 days (step 1), followed by 10 ng/ml FGF2 (R&D systems) and 50 ng/ml BMP4 (R&D systems) in differentiation medium (half basal differentiation medium with half STEMdiff™ APEL™ medium (Stemcell Technologies)) for 4 days (step 2). On the following 4 days, 50 ng/ml FGF1 (R&D systems), 10 ng/ml FGF4 (R&D systems) and 25 ng/ml FGF8 (R&D systems) was applied in the differentiation medium (step 3). Finally, the cells were incubated with 20 ng/ml HGF (R&D systems) and 100 ng/ml Follistatin-288 (R&D systems) for 4 days with additional 20 ng/ml Oncostatin (R&D systems) for another 2 days (step 4). The medium was changed every other day during the 20-days differentiation period.

Hepatocytes were generated from hPSCs by a growth factor‐based differentiation protocol described in our previous protocol [15]. After 20 days of differentiation, the cells were harvested by using a serial 2× TrypLE Express treatment and further dissociated into single cells by passing them through a 40‐μm cell strainer. These single cells were then seeded at 2.5 × 10⁵ cells per well in a collagen I (50 μg/ml, Bio Laboratories, Singapore, http://www.biolab.com.sg, catalog no. 354236)‐coated dishes. Attachment and recovery were promoted by seeding them in step IV differentiation medium with hepatocyte growth factor (R&D Systems, catalog no. 294‐HGN‐005), Follistatin (R&D Systems, catalog no. FS‐288), Oncostatin (R&D Systems, catalog no. 295‐OM‐010), and Y‐27632 (Rock Inhibitor) to prevent anoikis in the freshly harvested hPSC‐HEPs. The next day, medium was changed to Williams E medium (Sigma‐Aldrich, catalog no. W1878) without serum, and cells were serum‐starved overnight before nutraceutical treatments. Nutraceuticals quercetin (Sigma‐Aldrich, catalog no. Q4951) and genistein (Sigma‐Aldrich, catalog no. G6649) were administered at a single dose of 10 μM. Hepatocytes in this work were derived from H9‐ESCs. In hepatic characterization, primary human hepatocytes (PHHs) and HUH7 cells were used as positive controls, and HeLa cells were used as negative controls.