Vitamin k1

Cell lines (imHC, HepG2 and HEK293T) were transduced with lentivirus at multiplicity of infection (MOI) of 10 in culture media containing 8 μg/mL polybrene. Spinoculation was used for increasing transduction efficiency by centrifugation at 800 g, 32°C for 90 min. At 72 h later, transduced cells were selected with 2 μg/mL puromycin for 2 weeks. Puromycin-resistant cells were then expanded in growth medium containing 10 μg/mL vitamin K1 (Roche, Basel, Switzerland).
Transduced cells were seeded at density of 3x10⁶ cells and maintained in growth medium containing 10% FBS and 10 μg/mL vitamin K1 for 3 days. Next, cells were serum-starved for 2 days in growth medium (without FBS) supplemented with 1x insulin-transferrin-selenium (EMD Millipore) and 10 μg/mL vitamin K1. Cultured media were collected and centrifuged at 3,000 x g for 10 min at 4°C to remove cell debris. Collected media were combined with benzamidine and PEG-8000 at final concentration of 10 nM and 0.1%, respectively, and then concentrated using 30 kDa molecular weight cut-off membranes (Amicon Ultra Centrifugal Filter Devices, Millipore, Carrigtwohill, Country Cork, Ireland) [24 (link), 25 (link)]. Not more than 5 passages of transduced cells were used in all experiments after puromycin selection process was completely done.

siHBs were harvested using cell dissociation buffer (0.1 mg/mL EDTA, 0.5 mg/mL BSA) and seeded onto collagen I-coated plates in RPMI/B27, 10% Fetal Bovine Serum (FBS, Gibco, Waltham, MA, USA), 1% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA), and 50 ng/mL HGF for four hours to enable cell attachment. The medium was then replaced by RPMI/ITS with 50 ng/mL HGF, 3 nM CHIR-99021 (Stemcell Technologies, Vancouver, WA, Canada) and 0.1 ng/mL TGF-β1 (Peprotech, Cranbury, NJ, USA), for 24 h. The medium was changed every day until the end of the differentiation with 20 ng/mL HGF, 0.05 nM dexamethasone (Sigma, St. Louis, MO, USA), 10 ng/mL oncostatin M (Peprotech, Cranbury, NJ, USA), 0.25 nM Compound E (Santa Cruz, Dallas, TX, USA), 2.5 nM SB431452 (Tocris, Bristol, UK) and 10 ng/mL vitamin K1 (Roche, Basel, Switzerland).