Rock inhibitor y 27632

The iPSC lines were generated from different anonymous adult donors by peripheral blood (PB) reprogramming using episomal vectors that express OCT4, SOX2, MYC, KLF4, and BCL-XL (37 (link),38 ,45 (link)). Blood samples were obtained from the Tianjin Blood Center with the approval of the local research ethics committee. All feeder-free iPSCs (passages 6–15) were routinely maintained on Matrigel (Corning)-coated tissue-culture plates (BD) in mTeSR1 (Stemcell Technologies) (46 (link),47 ). On the first day after passaging with Accutase (Stemcell Technologies), 10 μM ROCK inhibitor Y-27632 (Millipore) was added to the culture medium. Human iPSCs were cultured at 37°C with 5% CO₂ and were fed with fresh medium every day.

Cell culture inserts for 24-well plates (pore size of 0.4 µm; Corning, Corning, NY, USA) were coated with 50 µL of growth factor–reduced Matrigel (Corning) before cell seeding. After sorting, cells were isolated by centrifugation and suspended in lung cell medium (LCM) consisting of Dulbecco’s modified Eagle’s medium (DMEM)–nutrient mixture F-12 (Wako, Osaka, Japan) supplemented with B-27 (Thermo Fisher Scientific, Waltham, MA, USA), 15 mM HEPES (Thermo Fisher Scientific), as well as recombinant murine EGF (40 ng/mL, Peprotech, Cranbury, NJ, USA), recombinant murine KGF (20 ng/mL, Peprotech), and 10 µM of the ROCK inhibitor Y-27632 (Millipore, Burlington, MA, USA). The cells were then seeded at a density of 1 × 10⁵ cells/mL in 200 µL on each insert of a 24-well plate, 400 µL of LCM were added to the bottom of each well, and the medium in both chambers was replaced every 2 to 3 days. Colonies were observed and were counted after 10 days with the use of a BZ-9000 microscope (Keyence, Osaka, Japan).

Induced pluripotent stem cells (iPSCs) were differentiated into hepatocytes according to the previously published protocol^{35 (link)}. In brief, cells were seeded into growth factor-reduced Matrigel-coated 6-well plates (2 million cells per well) in STEMdiff definitive endoderm basal medium (StemCell Technologies) containing Supplements A and B and cultivated for one day. From day 2 to 4, medium was exchanged daily and only contained Supplement B. At day 5, cells were detached and re-seeded into 96-well plates with a density of 12,000 cells per well. Medium was changed to Differentiation medium for hepatic specification: High-glucose DMEM/F12 (Gibco); 10% KOSR (Gibco); 1% Glutamin (Gibco); 1% Non-essential amino acids (Gibco); 1% Pen/Strep (Gibco); 100 ng/ml Human Growth Factor (peprotech); 1% DMSO; 10 µM Rock inhibitor Y27632 (Millipore).
From day 7 to 13, medium was changed to Differentiation medium without Rock inhibitor and refreshed daily. At day 14, medium was changed to Cultivation medium, which contained 10⁻⁷ M Dexamethasone (Sigma Aldrich) instead of Human Growth Factor and DMSO. This medium was also changed daily and used during treatments and further analysis.
To assess the level of iPSC differentiation into hepatocytes, expression levels of markers were analyzed on mRNA level by qRT-PCR: ALB, ABCC2, CYP1A1, HNF4A and RXRA.

We knocked down Kat2b in hESCs by TALEN. CAG-GFP fragment was replaced by U6 promoter and shRNA sequence from AAV-CAGGS-EGFP (Addgene, Cambridge, MA, USA). The shRNA targeting sequences were: shKat2b#1 (5′-CGAACTCTAATCCTCACTCAT-3′), shKat2b#2 (5′-CCAGCCAGCTAGGCATCCAAA-3′), shKat2b#3 (5′-GGAAGCTGGATTAATTGACAA-3′). Before electroporation, hESCs were cultured in ROCK-inhibitor Y-27632 (Millipore, Hayward, CA, USA, 688002) for 3 h. About 1 × 10⁷ cells were mixed with 5 μg of each TALEN targeting AAVS1 locus.^{32 (link)} And 40 μg of AAVS1-SA-PURO-PA-U6 promoter-shRNA-PA donor after trypsin/EDTA solution treatment and then electroporated (Gene Pulser Xcell System, Bio-Rad: 250 V, 500 μ F, 0.4 cm cuvettes). Transfected cells were plated on MEF with ROCK-inhibitor for the first 24 h. Then we used 1 μg/ml puromycin (Sigma, St. Louis, MO, USA, P8833) to select cells cultured with MEF-conditioned medium. Individual colonies were picked up after about 15 days and identified by PCR using primers for homologous recombination forward: 5′-CTTCCGCATTGGAGTCGCTTTA-3′ and reverse: 5′-ACAGGAGGTGGGGGTTAGAC-3′ or wild type forward: 5′-CAGCCGGTCCTGGACTTTGTC-3′ and reverse: 5′-AGCCGGGAACCGCTCAACTC-3′.