Mtesr1

For induction of hepatocytes, liver progenitor cells were collected and seeded onto collagen type I-coated plates at 2 × 10⁴ cells/cm². The cells were cultured for 2 days in SHM medium and the medium was supplemented with 1 μM Dexamethasone (Dex) (Sigma) and 20 ng/ml human Oncostatin M (PeproTech) for 6 days. For induction of biliary epithelial cells, the liver progenitor cells were isolated and co-cultured with pre-inoculated low density mouse embryonic fibroblasts (passage 2). The mouse embryonic fibroblasts were isolated from E13.5 C57BL/6 J mouse embryos and cultured in DMEM high-glucose media (Gibco) supplemented with 10% FBS (Ausbian). The medium was replaced with mTeSRTM1 (STEMCELL Technologies) containing YAC (mTeSR1+YAC), and on day 6, the medium was replaced with mTeSR1+YAC supplemented with 2% Matrigel (Corning) for another 6 days. All media were changed every other day.

CHA15 human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSC-NT4-S1), established by CHA University, Seoul, South Korea, were cultured under feeder-free conditions. CHA-15 were cultured on Matrigel (cat. no. #356231, Corning Life Sciences, Tewksbury, MA, USA)-coated 35 mm dishes in mTeSR1 (cat. no. #85850, STEMCELL Technologies, Vancouver, BC, Canada), while hiPSCs were cultured on Vitronectin (cat. no. #07180, STEMCELL Technologies, Vancouver, BC, Canada)-coated 35 mm dishes in TeSR-E8 (cat. no. #05990, STEMCELL Technologies, Vancouver, BC, Canada), and subcultured every 4–5 days using Gentle Cell Dissociation Reagent (cat. no. #07174, STEMCELL Technologies, Vancouver, BC, Canada). Cells were seeded onto 35 mm dishes in mTeSR1 or TeSR-E8 supplemented with 10 μM Y-27632 (ROCK inhibitor, cat. no. #1254, Tocris Bioscience, BS, United Kingdom) to enhance cell survival and attachment.
Human pluripotent embryonal carcinoma (NCCIT) cells were cultured in RPMI media (RPMI 1640, GIBCO BRL, Rockville, MD, USA). Human embryonic kidney (HEK293) cells were cultured in DMEM media (GIBCO BRL, Rockville, MD, USA). Both RPMI and DMEM media were supplemented with 10% fetal bovine serum (GIBCO BRL, Rockville, MD, USA) and 1% penicillin and streptomycin (GIBCO BRL, Rockville, MD, USA), and cells were grown at 37 °C in a humidified atmosphere with 5% CO₂.

The iPSC line iPSC-UkB-Ctrl-XX was generated from PBMCs via expansion to a cellular intermediate of EPCs, using Stem Span SFEM II (Stem Cell Technologies) and addition of Erythropoietin (R&D Systems), Stem Cell Factor (PeproTech), Interleukin-3 (PeproTech), Insulin Growth Factor-1 (PeproTech), and dexamethasone (Sigma-Aldrich). Following expansion, EPCs were electroporated with Epi5 Reprogramming (Thermo Fisher Scientific) Lonza 4D-Nucleofector X-Unit (Lonza). Cells were plated on Corning ESC-grade Matrigel (Corning) in ReproTeSR (Stem Cell Technologies) before changing to mTeSR 1 (Stem Cell Technologies) when colonies first appeared. Following iPSC clones were picked and expanded before characterization. iPSC-UkB-Ctrl-XX line is cultured on Corning ESC-grade Matrigel and in mTeSR 1. Cells are passaged when colonies are too large using 0.5 mM EDTA in PBS and cultured in a humidified incubator at 37°C and 5% CO₂.