PHHs were purchased from Thermo Scientific Inc. (Waltham, MA, USA), and cultured using the Hepatocyte Medium (Thermo Scientific Inc.) according to the manufacturer’s protocol. PHHs were used for experiments within 3 days after seeding.
STEMPRO human ADSCs were purchased from Life Technologies (Tokyo, Japan). Our HLC generation protocol has been reported1. ADSCs were used in experiments at passages 2–6. Briefly, ADSCs (2 × 106 per well) were seeded in 6-well flat-bottomed collagen-coated plates (Nunclon Sphera Microplates, Thermo Scientific Inc.) and incubated with serum-free Dulbecco’s modified Eagle’s medium with F-12 Supplement (DMEM/F-12) for 24 h. Thereafter, our three-step differentiation protocol was performed. In the first step, to induce definitive endoderm differentiation, cells were incubated with DMEM/F-12 containing 0.5 mg/m bovine serum albumin fraction V (BSA; Sigma- Aldrich, St. Louis, MO, USA) and 2 µmol/L Chir99021 (a glycogen synthase kinase 3 inhibitor; Selleckchem, Tokyo, Japan) for 24 h. The following day, 1% insulin-transferrin-selenium (ITS, Sigma-Aldrich) was added to the medium. In the second step, for hepatoblast differentiation, the medium was changed to minimum essential medium with non-essential amino acids (Thermo Scientific Inc.) containing 0.5 mg/mL BSA, 1% ITS, 20 ng/mL bone morphogenic protein 2 (PeproTech, Inc. NJ, USA), and 30 ng/mL fibroblast growth factor 4 (PeproTech, Inc.) and incubation continued for 5 days. In the third step, for induction of hepatocyte differentiation, the cells were treated with 20 ng/mL hepatocyte growth factor (PeproTech, Inc.) for 5 days, followed by treatment with 20 ng/mL hepatocyte growth factor, 10 ng/mL oncostatin M (PeproTech, Inc.) and 1 × 10−6 M dexamethasone (Sigma-Aldrich) for another 5 days.
An LED irradiation device (3LH-256/3LH-75DPS, Nippon Medical & Chemical Instruments Co., Ltd., Osaka Japan) was used to produce GLED with a wavelength of 520 nm at maximum light intensity. A photoradiometer (Light analyzer LA-105, Nippon Medical & Chemical Instruments Co., Ltd.) was used to measure the light intensity. The cell culture plates were placed on the LED irradiation device and energy density generation was set at 21 W/m2. Cells were exposed to GLED in the third step (hepatocyte differentiation phase) at room temperature for 5 min which gave a radiant exposure of 0.63 J/cm2 for each day. The GLED irradiation protocol is shown in Fig. 6. Control group cells were treated in the same manner except for no GLED exposure. Sufficient ventilation was provided to ensure that the temperature of the culture medium did not change during treatment. The experiments were replicated at least 3 times under the same conditions with three purchased ADSCs.
The LIVE/DEAD Cell Imaging Kit (Thermo Fisher Scientific, Inc.) was used to determine the live/dead nucleated cells among GLED-treated and non-treated HLCs at day 21, following the manufacturer’s protocol. The fluorescence signals were measured using a fluorescence microscope (Keyence Corporation). Cells were counted using Image J software (ver. 1.53, National Institutes of Health, Bethesda, MD, USA).
Apoptosis detection by flow cytometry
Flow cytometry was used to detect apoptosis. Both irradiated and non-irradiated cells were labeled with Annexin V-conjugated fluorescein isothiocyanate and propidium iodide (PI) using a MEBCYTO Apoptosis Kit according to the manufacturer’s instructions (MBL international, Woburn, MA, USA). Quantitative analysis was performed using a FACSVerse cytometer and FACSiute software (Becton Dickinson, Franklin Lakes, NJ, USA).
RT-PCR of HLCs
Total RNA was prepared from each HLC sample using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The following TaqMan assays (with assay identification number and primers) were used: alpha-1 antitrypsin (AAT) (Hs00165475_m1), albumin (ALB) (Hs00910225_m1), OTC (Hs00166892_m1), carbamyl phosphate synthase 1 (CPS1) (Hs00157048_m1), OPN3 (Hs00173892_m1) and PGC-1A (Hs00173304_m1). GAPDH (Hs02786624_g1) was used as an internal control with stable level of expression for normalization (Supplemental Table 1). All primers were purchased from Thermo Fisher Scientific, Inc. The data were analyzed using the 2−ΔΔCt method. Relative expression levels were calculated as ratios to GAPDH expression. The results are presented as the fold change of the relative mRNA expression for a group compared with that in the control group.
RIPA buffer (Thermo Fisher Scientific, Inc.) containing a protease inhibitor cocktail (Sigma- Aldrich) and a PhosSTOP phosphatase inhibitor cocktail (Roche, Tokyo, Japan) was used for protein extraction. Total protein concentrations were assessed with a BCA Kit (Thermo Fisher Scientific, Inc.) and equal quantities of extracted proteins were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). To evaluate protein abundance, blots were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti‑OPN3 (1:1,000; SAB2700986; Sigma-Aldrich) and anti‑β‑actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc. MA, USA). Blots were then incubated with anti‑rabbit IgG, HRP‑linked (1:2,000; cat. no. 7074; Cell Signaling Technology, Inc.) as the secondary antibody for 1 h at room temperature. The proteins were detected with chemiluminescence (GE, Little Chalfont, Buckinghamshire, UK).
CYP3A4 activity assay
CYP3A4 enzyme activity was assessed using the P450- Glo™ CYP3A4 Assay with Luciferin-IPA (Promega Corporation. WI, USA) according to the manufacturer’s instructions. Luminescence was measured using a microplate reader (SpectraMax i3; Molecular Devices, LLC). Luminescent measurements were normalized to the total amount of viable cells.
Ammonium metabolism assay
Ammonium metabolism was evaluated by the change in ammonium ion concentration in the cell culture supernatant at 24 h after the addition of ammonium chloride (NH4Cl). Briefly, NH4Cl (FUJIFILM Wako Pure Chemical Corporation) diluted with Hanks’ Balanced Salt Solution (HBSS, FUJIFILM Wako Pure Chemical Corporation) to a standard of 300 µmol/L was added to culture plates after the plates were washed twice with HBSS. The plates were placed adjacent to each other in the same incubator and incubated under the same conditions. The supernatants were then collected and applied to an ammonia assay kit (Cell Biolabs) to measure ammonium concentrations at 24-h intervals after NH4Cl addition. Controls were culture plates containing only standard ammonia solution. As a reference value, ammonium metabolism assays were performed using PHHs with Hepatocyte Medium in the same manners described above.
HLCs were immobilized in iPGell (Geno staff, Tokyo, Japan) and fixed with 4% paraformaldehyde according to the manufacturer’s protocol. Frozen jellified cells were then sectioned, mounted on glass slides and incubated with an anti-OPN3 primary antibody (Abcam, ab140901) overnight at 4 °C. Cells were then incubated with a fluorophore-conjugated secondary antibody (Thermo Fisher Scientific Inc., A11008) and then with DAPI (Thermo Fisher Scientific Inc., P306931). Slides were observed under a fluorescence microscope (Keyence Corporation, Itasca, IL, USA).
Ca 2+ and ATP assays
Intracellular Ca2+ concentration in GLED-treated HLCs was measured using a Ca2+ detection assay kit (Abcam, Cambridge, UK). Briefly, after cell homogenization and centrifugation at 20,000 × g for 5 min at 4 °C, supernatants were collected. Samples were mixed with chromogenic reagent and incubated at room temperature for 10 min according to the manufacturer’s protocol. The absorbance values of each sample were then measured at 575 nm with a microplate reader. Measurements for each cell lysate sample were standardized at 1 × 106 HLCs.
To evaluate the amount of ATP in GLED-treated and non-treated HLCs, cell homogenization and centrifugation was performed as for the above Ca2+ assay and an adenosine triphosphate (ATP) assay kit (FUJIFILM Wako Pure Chemical Corporation) was used according to the manufacturer’s protocol. Measurements for each cell lysate sample were standardized at 1 × 106 HLCs.
Detection of reactive oxygen species (ROS)
The ROS Assay Kit (Dojindo Lab., Kumamoto, Japan) was used to measure the formation of intracellular ROS. Briefly, HLCs on differentiation day 21 (10 days after GLED irradiation) were seeded into a 6-well dish and incubated with 2′,7′-dichlorodihydrofluorescin diacetate (DCFDA) for 30 min in the dark at 37 °C. After washing twice with HBSS, the cells were incubated with 100 µmol/L hydrogen peroxide for 30 min according to the manufacture’s protocol. The fluorescence signals were measured and analyzed using a FACSVerse cytometer and FACSiute software (Becton Dickinson). A fluorescence microscope (Keyence Corporation) was used to image cells and the level of cellular fluorescence was measured using the Cell Magic Wand plugin for Image J software.
All data are presented as the mean ± standard difference. Statistical analysis and graph production were conducted using GraphPad Prism v7.0 (GraphPad Software, Inc.) and Image J software. Comparisons between two groups were analyzed using the Mann‑Whitney test. P-values less than 0.05 (two‑sided) were considered statistically significant.