Fibroblast growth factor 4

With the approval of the Institutional Review Board (IRB) of CHA General Hospital (IRB07-18; Seoul, Korea), human PD-MSCs were isolated from the chorionic plate membrane of placental tissues, which is a fetal side of the placenta, that were collected at term (38 ± 2 gestational weeks) after obtaining patient consent for the use of their stem cells for research, as previously described by Lee et al. Briefly, PD-MSCs were cultured in alpha-minimum essential medium (α-MEM; HyClone Laboratories Inc., South Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL, Waltham, MA, USA), 1% penicillin–streptomycin (GIBCO-BRL), 25 ng/mL fibroblast growth factor 4 (FGF4; Peprotech, Rocky Hill, NJ, USA), and 1 μg/mL heparin (Sigma-Aldrich, Munich, Germany) at 37 °C in a humidified atmosphere containing 5% CO₂ and 95% air. Before transplantation, PD-MSCs were stained with PKH67 dye (PKH fluorescent cell linker kit; Sigma-Aldrich) to monitor engraftment.

The derivation of hepatic spheres from hPSC aggregates was performed as previously described [19 (link)]. In brief, after 5 days of culture, aggregates cultured with or without DS were collected, washed with PBS and then transferred to differentiation medium directly in an ultra-low 6-well plate. For the induction of endodermal cells, the basal medium consisted of RPMI 1640 (Thermo Fisher, 61870036), 1 × B27 (Gibco, 17504044) and 0.1% bovine serum albumin (BSA, Sigma, SRE0098), 6 μM CHIR99021 (Selleckchem, S1263) was added on day 1, and then 10 ng/ml Activin A (Peprotech, 120-14) was added for 2–3 days. For hepatic differentiation, aggregates were treated in DMEM/F12 (Gibco, 11330032) supplemented with 2% KSR, 10 ng/ml fibroblast growth factor 4 (FGF-4) (Peprotech, 100–31), and 10 ng/ml hepatocyte growth factor (HGF) (Peprotech, 100–39) for 6 days. Finally, for hepatocyte maturation, they were treated in the same medium plus 50% hepatocyte culture media without EGF (HCM, Lonza, CC-3198), 10 ng/ml oncostatin M (Peprotech, 300–10), and 10^–7 M dexamethasone (Sigma, D4902) for another 12 days. On day 21, the hepatic spheres were collected and analyzed.

Mouse primary hepatocytes were isolated from the liver tissue of 8-week-old C57/B6 mice by the traditional collagenase perfusion protocol [34 (link)]. Primary hepatocytes, e-iHeps, and r-iHeps were maintained in hepatocyte culture medium (HCM), consisting of DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Seradigm), 0.1 μM dexamethasone (Sigma), 10 mM nicotinamide (Sigma), 1% insulin-transferrin-selenium (ITS) premix (Gibco), 1% penicillin/streptomycin (PS) (Gibco), GlutaMAX™ (Gibco), 10 ng/ml fibroblast growth factor 4 (FGF4) (Peprotech), 10 ng/ml hepatocyte growth factor (HGF) (Peprotech), and 10 ng/ml epidermal growth factor (EGF) (Peprotech). Freshly isolated mouse primary hepatocytes were cultured onto gelatin-coated dish and collected after 48 hrs for RNA extraction. Mouse embryonic fibroblasts (MEFs) were isolated through single-cell dissociation of E13.5 C57/B6 mouse embryos after removing the head and all the internal organs, including the liver and intestine. Isolated MEFs were cultured in MEF medium (MEFM), composed of DMEM high glucose (Biowest), 10% FBS (Seradigm), 1% MEM/NEAA (Gibco), 1% penicillin/streptomycin (PS) (Gibco), and GlutaMAX™ (Gibco).