For each human iPSC line for hepatocyte differentiation, all differentiated cells are constantly removed by manually picking up. Before the initiation of hepatocyte differentiation, human iPSCs were dissociated into clumps by using dispase (Roche) and plated onto BD Matrigel Basement Membrane Matrix Growth Factor Reduced (BD Biosciences). These cells were cultured in the MEF-conditioned medium for 3-4 days. The differentiation protocol for the induction of definitive endoderm cells, hepatoblasts, and hepatocytes was based on our previous reports with some modifications. Briefly, in the definitive endoderm differentiation, human iPSCs were cultured for 4 days in the L-Wnt3A-expressing cell (ATCC, CRL2647)-conditioned RPMI1640 medium (Sigma) which contains 100 ng/ml Activin A (R&D Systems), 4 mM L-Glutamine, 0.2% FBS (PAA Laboratories), and 1×B27 Supplement Minus Vitamin A (Life Technologies). For the induction of HBCs, the definitive endoderm cells were cultured for 5 days in RPMI1640 medium (Sigma) which contains 30 ng/ml bone morphogenetic protein 4 (BMP4) (R&D Systems) and 20 ng/ml FGF4 (R&D Systems), 4 mM L-Glutamine, and 1×B27 Supplement Minus Vitamin A. The protocol for HBC proliferation using human recombinant laminin-111 (BioLamina) was based on our previous report with some modifications. Briefly, the hPSC-derived HBCs were first purified from the hPSC-derived cells (day 9) by selecting attached cells on a human recombinant LN111-coated dish (final coating concentration was 1.0 μg/cm2) at 15 min after plating. The hPSC-derived HBCs were cultured on a human LN111-coated dish (2.5 × 104 cells/cm2) in maintenance DMEM/F12 medium (DMEM/F12 medium (Invitrogen) was supplemented with 10% FBS (PAA laboratories), 1×insulin/transferrin/selenium, 10 mM nicotinamide, 10-7 M Dexamethasone (DEX) (Sigma), 20 mM HEPES, 25 mM NaHCO3, 1×GlutaMAX, 40 ng/ml hepatocyte growth factor (HGF) (R&D Systems) and 20 ng/ml epidermal growth factors (EGF) (R&D Systems)). The medium was refreshed every day. The HBCs were dissociated with Accutase (Millipore) into single cells, and subcultured every 6 or 7 days. To perform the hepatocyte differentiation, the HBCs were cultured for 5 days in RPMI1640 medium (Sigma) which contains 20 ng/ml HGF, 4 mM L-Glutamine, and 1×B27 Supplement Minus Vitamin A. And then, the cells were cultured for 11 days in Hepatocyte Culture Medium (HCM, Lonza) without EGF but with 20 ng/ml oncostatin M (OsM). Unlike our previous reports, the current differentiation method could omit the use of adenovirus-mediated overexpression of hepatic transcription factors.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using RNeasy Mini Kit.
Label
Cy3
Label protocol
100 ng total RNA from all samples was used as template to produce Cy3- labeled cRNA. The RNA samples were amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer’s protocol.
Hybridization protocol
Cy3 labeled cRNAs were hybridized overnight (17 hours, 65°C) to an Agilent Whole Human Genome Oligo Microarrays (8x60K v2, Design ID 039494) using Agilent’s recommended hybridization chamber and oven.Finally, microarrays were washed once with wash buffer 1 for 1 min at RT followed by a second wash with wash buffer 2 for 1 min at 37°C.
Scan protocol
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent’s Microarray Scanner System (G2505C, Agilent Technologies, Palo Alto, USA).
Description
HepG2
Data processing
The Agilent Feature Extraction Software (FES 10.7.3.1 ) was used to read out and process the microarray image files.