Cell lines and culture
The hiPSC line TkDN4-M was obtained from the Institute of Medical Science, the University of Tokyo20. hiPSCs were maintained in feeder-free culture using Essential 8 Medium or StemFlex medium (Thermo Fisher Sciences). hiPSC-derived LPCs, LSECs, and HSCs were prepared according to previous protocols7,9. HepG2-NTCP cells10 were cultured in Dulbecco’s Modified Eagle Medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS, MEM non-essential amino acids solution, 100 U/ml penicillin, and 100 µg/ml streptomycin. Primary human hepatocytes (PXB-cells) were purchased from PhoenixBio, maintained in its commercial medium; during HBV infection, FBS and EGF were removed and 4% PEG8000 and 2% DMSO were added to the medium.
Isolation and culture of fetal mouse liver cells
LSEC and HSC progenitors were isolated from livers of E14.5 fetal mice. The fetal livers (each sample contained 25–35 embryos) were minced and dissociated in Liver Digest Medium (Life Technologies, California, US). Cells were treated with FcR blocking reagent and incubated with FITC-conjugated anti-stab2 antibody and APC-conjugated anti-Ngfr antibody (Miltenyi biotec). Stab2+ cells and Ngfr+ cells were isolated by a MoFlo XDP cell sorter (Beckman Coulter, Inc, California, US). After sorting, cells were seeded onto a collagen I-coated dish at the density of 40,000 cells/cm2 in Endothelial Cell Growth Basal Medium plus (LONZA) supplemented with VEGF (50 ng/ml) and Y27632 (10 μM) under hypoxic condition. Quantitative RT-PCR was performed using SYBR Premix EX TaqII (Takera bio). Primers and antibodies are listed in Supplementary information.
Endocytosis of EGFR
Cells were seeded at 25,000 cells/cm2 and incubated with FBS-starved medium on the next day for 24 h and then stimulated with EGF for 30 min at 37 °C, followed by HBV infection. To inhibit endocytosis, cells were incubated with 2 µM of ikarugamycin (SIGMA) or 1 µg/ml of filipin (SIGMA) for 30 min before EGF (PeproTech) stimulation. For lysosome inhibition, cells were incubated with 25 µM of chloroquine (SIGMA) for 2.5 h before EGF stimulation. To block EGF stimulation, cells were incubated with 10 µM gefitinib (SIGMA) when EGF was added. The negative control for EGF stimulation, EGF neutralizing antibody (Sino biological, 2.5 µg/ml) was used.
Hepatocytes derived from iPSCs were in the lower chamber of trans-well. To induce hepatic maturation, cells were incubated in Hepatocyte Basal Medium (LONZA) supplemented with HCM SingleQuots (excluding EGF) and Oncostatin M (20 ng/ml) (PeproTech). NPCs were suspended in Endothelial Cell Growth Basal Medium plus (LONZA) supplemented with VEGF (50 ng/ml) and Y27632 (10 μM) and seeded into the upper chamber of trans-well. Co-culture started at HGF stage. After 10 days of co-culture, cells were infected with HBV for 16 h. HepG2-NTCP cells were suspended in HepG2-NTCP maintenance medium and seeded on a collagen I-coated plate at the density of 25,000 cells/cm2. iPSC-derived NPCs were suspended in NPC maintenance medium and seeded into the upper chamber of trans-well. Co-culture started at day 1, and after 6 days of co-culture, cells were infected with HBV for 16 h.
HBV/NL virus was prepared as previously described10. Cells were infected with HBV/NL at 100 GEq/cell in the presence of 4% PEG8000 and 2% DMSO for 16 h. After washing to remove the virus, cells were maintained for 5 days. Activity of NL was then measured using the NL Luciferase Assay Kit (Promega) according to the manufacturers’ protocols.
HBV infection assay
HBV in the culture supernatant of HepAD38 cells was used as previously described16. Medium was harvested and the virus fraction prepared by precipitation with 13% PEG8000 (SIGMA) containing 0.75 M NaCl. Virus was then purified by precipitation at 1,750×g for 30 min, suspended in Opti‐MEM (Life Technologies) and stored at − 80 °C until use. Cells were infected with the virus at 10,000 GEq/cell in the presence of 4% PEG8000 and 2% DMSO for 16 h. After washing to remove virus, cells were cultured for 14 days in the medium containing 4% PEG8000 and 2% DMSO. HBsAg was detected by ELISA assay; HBV DNA and cccDNA were detected by qPCR and southern blot21,22. Primers and probes are listed in Supplementary information.
HBV attachment assay
HBV derived from the culture supernatant of HepAD38 cells. Cells were incubated with the virus at 10,000 GEq/cell in the presence of 4% PEG8000 and 2% DMSO at 4 °C for 3 h. After washing out the free virus, HBV DNA was detected by qPCR. Primers are listed in Supplementary information.
HBV internalization assay
Following HBV attachment assay, the cells were washed to remove free virus and incubated at 37 °C for 24 h. The attached virus was removed by 0.25% trypsin/EDTA and HBV DNA was detected by qPCR.
Cytokine array analysis
Human Cytokine Antibody Array Membrane (Abcam) was used to detect cytokines according to the manufacturer’s protocol. iPSC-derived LSECs were suspended in NPC maintenance medium and seeded at the density of 40,000 cells/cm2 in the upper chamber of trans-well. Control wells contained NPC maintenance medium only. HepG2-NTCP maintenance medium was added to the lower chamber and changed to fresh medium every day. The samples were prepared from the lower chamber at day 5, and the data were analyzed by ImageJ quantitatively. The pictures were taken in ImageQuant LAS 4000 version1.2.
In cross-linking assay, the cells were incubated with the cross-linker BS3 at 4 °C for 30 min, and the cross-linking reaction was terminated using 20 mM of glycine (final concentration) as previously described11. Collected and isolated the cell surface lysates using Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Sciences). Samples were then separated on SDS-PAGE, performed western blotting. Antibodies are listed in Supplementary information.
Knockdown of EGFR
Sequences of siRNAs used for knockdown EGFR are: si-EGFR: 5′- GGAACUGGAUAUUCUGAAA TT-3′ and 5′-GAUCUUUCCUUCUUAAAGA TT-3′.
Cells were transfected with siRNAs at a final concentration of 30 nM using Lipofectamine RNAiMAX Reagent (Life Technologies). The Mission siRNA Universal Negative control were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were used for experiments 48 h post-transfection.
Cultured cells were fixed in 4% PFA and permeabilized with 0.25% tritonX-100. The cells were blocked with SuperBlock T20 Blocking Buffer (Thermo). Then, they were incubated with primary and secondary antibodies. Antibodies are listed in Supplementary information. The images were taken by confocal microscope and merged by OLYMPUS FV1000 Viewer.
Data are expressed as mean ± SEM and analyzed by Student’s t-test, one-way ANOVA and two-way ANOVA. The statistical significance was determined at P < 0.05.
Animal experiments and ethics statement
C57BL/6J mice were purchased from CLEA Japan, Inc (Tokyo, Japan). All mouse experiments were approved by the Animal Experiment Ethics Committees at the Institute for Quantitative Biosciences, University of Tokyo (Approval number: 3004, 3105, 0209). Experiments were performed according to the guidelines of the institutional Animal Care and Use Committee of the University of Tokyo.