Human hepatocyte growth factor hhgf

For hepatoblast culture, control and L1L2_Alb livers were dissected and digested in 1 mg ml⁻¹ collagenase/dispase (Roche) for 30 min at 37 °C with constant shaking. Total cells were passed through a 100-μm cell strainer (BD Bioscience) and centrifuged at 250g for 10 min. Red blood cells were lysed with 1X Ammonium-Chloride-Potassium buffer and cells were counted. The hepatoblasts (1 × 10⁶) were plated on collagen I- (BD Bioscience) or gelatin- (Sigma) coated 60-mm dishes and cultured in Dulbecco's modified Eagle's medium/Nutrient Mixture F12 (DMEM/F12) supplemented with 10% foetal bovine serum (FBS), 25 ng ml⁻¹ human epidermal growth factor (hEGF) (Peprotech), 25 ng ml⁻¹ human hepatocyte growth factor (hHGF), 5 μg ml⁻¹ insulin and 40 ng ml⁻¹ dexamethasone (Sigma). For hepatic differentiation, cells were treated with 12.5 ng ml⁻¹ Oncostatin M (Sigma) for 6 days. For BEC differentiation, hepatoblasts were cultured on Matrigel (BD Bioscience) for 14 days. For Yap overexpression, we infected retroviruses-containing vector, Yap WT, YAP 2SA and YAP 5SA 2 days after plating. The retroviruses were produced in 293 T cells with pMSCV puro, pMSCV-Yap WT, pMSCV-Yap 2SA, pMSCV-Yap 5SA and packaging vectors (Gag-pol and VSV-G).