2.1 Cell maintenance and hepatogenic differentiation in vitro
hESC-MSCs, kindly provided by Eun Ju Lee (Seoul National University Hospital, Republic of Korea), were cultured in endothelial cell basal medium MV2 (C-22221, Promocell, Heidelberg, Germany). hASCs, provided by Anterogen Co., Ltd. (Seoul, South Korea), and hBM-MSCs (PT-2501; Lonza, Basel, Switzerland, http://www.lonza.com) were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM; cat. no.: SH30021.01; Hyclone, Logan, UT, USA) containing 0.1% gentamicin and 10% fetal bovine serum (FBS). hESC-MSCs (1.3 × 106 cells) and hASCs (1 × 106 cells) were seeded on fibronectin (F1141-2MG; Sigma)-coated 100-mm tissue culture plates. The next day, the cells were washed with HBSS twice, and the medium was changed to low-glucose DMEM (in case of hASCs) or endothelial cell basal medium MV2 (in case of hESC-MSCs) containing 0.1% gentamicin, 20 ng/mL epidermal growth factor (EGF; CYT-217; ProSpec, East Brunswick, NJ, USA), and 10 ng/mL bFGF (CYT-218; ProSpec) for 2 days. The medium was then changed to low-glucose DMEM or endothelial cell basal medium MV2 containing 0.1% gentamicin, 1% FBS, 20 ng/mL oncostatin M (OSM; CYT-231; ProSpec), w/ or w/o 20 ng/mL HGF (CYT-244; ProSpec), and 5 × 10− 3 M nicotinamide for 7 days. The medium was then changed to low-glucose DMEM containing 0.1% gentamicin, 1 × insulin-transferrin-selenium (ITS) supplement (100×), 20 ng/mL OSM (CYT-231; ProSpec), w/ or w/o 20 ng/mL HGF (CYT-244; ProSpec), 10 ng/mL bFGF, 1 × 10− 6 M dexamethasone, 5 × 10− 3 M nicotinamide, and 0.1% DMSO for an additional 14 days. The medium was replaced every 2–3 days.
2.2 RNA isolation and quantitative real-time polymerase chain reaction (PCR) analysis
Total RNA was isolated using Easy-Blue reagent (Intron Biotechnology, South Korea) according to the manufacturer’s protocol. Gene-specific human primers were as follows: HGF (forward: 5′-AAGGTGACTCTGAATGAGTC-3′, reverse: 5′-GGCACATCCACGACCAGGAACAATG-3′), ALB (forward: 5′-GTCACCAAATGCTGCACAGA-3′, reverse: 5′-ACGAGCTCAACAAGTGCAGT-3′), TDO2 (forward: 5′-GTGTGCATGGTGCACAGAAT-3′, reverse: 5′-GGGTTCATCTTCGGTATCCA-3′), CYP2E1(forward: 5′-TTCCTCCTGCTGGTGTCCAT-3′, reverse: 5′-CCCACGTACAGCGTGAACA-3′) and β-actin (forward: 5′-GTCCTCTCCCAAGTCCACAC-3′, reverse: 5′-GGGAGACCAAAAGCCTTCAT-3′). All amplifications were conducted in a premixture (20 µL) containing of 5 µM gene-specific primers 4 µL, 2 × SYBR 10 µL, and 6 µL template, under the following conditions: denaturation at 95 °C for 5 min; 40 cycles of 95 °C for 10 s, 59 °C for 34 s; and a final extension at 72 °C for 5 min. The reactions were carried out using a Roche LC96 instrument (Roche Diagnostics, Penzberg, Germany). For validation of real-time PCR, PCR was conducted under the following conditions: denaturation at 95 °C for 5 min; cycles (34 for ALB, 34 for TDO-2, 31 for CYP2E1, and 18 for β-actin) of 95 °C for 30 s, 59 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min using the same cDNA and primers. PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining.
Cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed with an appropriate amount of tissue lysis buffer (RIPA buffer containing protease and phosphatase inhibitor cocktail), incubated on ice for 30 min, and centrifuged at 13,000 rpm for 10 min at 4 °C. Next, 30 µg of total protein was loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T), and incubated with the appropriate primary antibodies in TBS containing 1% bovine serum albumin overnight at 4 °C. Membranes were washed several times with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (0.1 µg/mL; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Immunoreactivity was detected using an enhanced chemiluminescence detection system (WSE 6100 LuminoGraph I; ATTO, Tokyo, Japan).
2.4 Human Hepatocyte Growth Factor (HGF) ELISA
To evaluate levels of HGF secretion, 200 µL of conditioned media were obtained from each group. HGF ELISA was conducted using RayBio Human HGF ELISA kit (RayBiotech, ELH-HGF) according to manufacturer’s instructions. HGF standards and undiluted supernatant from each group were added to the assay microplate and were allowed to incubate for 2.5 hours at room temperature. After washing, biotinylated anti-human HGF antibody and HRP-conjugated streptavidin were then applied for an hour and 45 minutes. Absorbance at 450 nm was read using an Epoch microplate spectrophotometer (Bio Tek Instruments).
2.5 Immunofluorescence staining
Cells grown or differentiated on round glass coverslips in 24-well plates were fixed and permeabilized with 100% cold methanol for 10 min. Fixed cells were incubated for 1 h in PBS containing 3% bovine serum albumin for blocking, followed by 2 h of incubation with specific primary antibodies. Cells were washed three times with TBS-T, then incubated with Cy2-conjugated goat anti-rabbit/mouse IgGs (Jackson Immunoresearch Laboratories) or Alexa 594-conjugated goat anti-rabbit/mouse IgGs (Molecular Probes, Eugene, OR, USA) as required according to the primary antibody. Cellular DNA was counterstained with 4′,6-diamidino-2-phenylindole (0.2 µg/mL in PBS).
2.6 Periodic acid-Schiff (PAS) staining
For PAS staining (1.01646.0001; Merck Millipore, Germany), hASCs, hESC-MSCs and their individual hepatogenic differentiated cells attached to coverslips were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, and washed with water. The cells were incubated with 0.5% periodic acid solution for 5 min. After rinsing with tap water, the cells were incubated with Schiff’s solution for 15 min and counterstained with hematoxylin solution for 2 min. The stained cells were observed and photographed under a microscope.
2.7 Flow cytometry analysis
Cells (5 × 105) were washed with PBS two times and stained with the following secondary antibodies conjugated with fluorophores: PE-CD105 (560839, BD Pharmigen), PE-CD135 (558996, BD Pharmigen) and PE-ASGRP-1 (Asialoglycoprotein receptor-1, 563655, BD Pharmigen) for 1 h. Information on the antibody for the negative control is PE-IgG (555749, BD Pharmigen). The cells were washed with PBS two times and measured by flow cytometry on a FACS Calibur (BD Biosciences). The acquired data were analyzed using BD FACS Calibur software.
Graphical data were presented as means ± standard errors of the means (SEMs). Statistical significance among the three groups and between groups was determined using one-way or two-way analysis of variance (ANOVA) followed by Bonferroni post-tests. Differences with p values of less than 0.05 were considered significant.