cell state of in vitro hepatogenesis: mature hepatocytes
Treatment protocol
Differentiation of ES into hepatocytes was carried out as described (Fig. 1A) (4). Briefly, ES were cultured to be confluent in conditioned medium on a feeder-free system for 3 days. Then, ES were incubated in RPMI-1640 (HyClone, Logan, UT) containing 0.5 mg/mL albumin fraction V (Sigma-Aldrich, St. Louis, MO) and 50 ng/mL activin A (Peprotech, Rocky Hill, NJ) for 1 day and further cultured in the same RPMI medium supplemented with 1% insulin-transferrin-selenium (ITS; Sigma-Aldrich) for 4 days. After treatment with activin A, EP were harvested. For further differentiation, EP were cultured in hepatocyte culture medium (HCM; Lonza, Baltimore, MD) containing 30 ng/mL FGF4 (Peprotech) and 20 ng/mL BMP2 (Peprotech) for 5 days and further incubated in HCM supplemented with 20 ng/mL hepatocyte growth factor (Peprotech) for 5 days. Maturation of ES-derived hepatocytes was induced by culturing in HCM supplemented with 10 ng/mL oncostatin M (R&D Systems, Minneapolis, MN) and 0.1 μM dexamethasone (Sigma-Aldrich) for 5 days. MH were harvested at this time point. The medium was changed daily.
Growth protocol
Human embryonic stem cells (CHA-hES4) were provided by the College of Medicine, Pochon CHA University, Seongnam, Korea.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with Trizol reagent, followed by clean-up and DNase I treatment with QIAGEN RNeasy mini kit in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.
Label
biotin
Label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
Hybridization protocol
Standard Illumina hybridization protocol
Scan protocol
Standard Illumina scanning protocol
Description
replicate 3-4
Data processing
The data were normalised using quantile normalisation with IlluminaGUI in R
