(A) Representative images of liver organoids in expansion medium (EM) and differentiation medium (DM). (B) Experimental design of infection experiments. Arrows indicate the time points for hepatitis …
Source data for Figure 1F.
Source data for Figure 1G.
(A) Immunofluorescent staining showing the expression of hepatitis B virus core antigen (HBcAg) (green) together with sodium taurocholate co-transporting polypeptide (NTCP) (magenta) or HNF4α (red) …
(A) Bright-field images and levels of hepatitis B virus (HBV) DNA in the supernatant of infected organoids at different days post infection. (B) HBV DNA in the supernatant of infected organoids …
Source data for Figure 1—figure supplement 2C.
Source data for Figure 1—figure supplement 2D.
(A) Levels of hepatitis B virus (HBV) DNA in the supernatant of healthy donor (hD) organoids infected with either the recombinant virus derived from HepG2.2.15 cells or the serum obtained from …
(A) Experimental design of drug treatment of hepatitis B virus (HBV)-infected liver organoids followed by assessment of antiviral activity and toxicity. Arrows indicate time points for HBV detection …
Source data for Figure 2B.
Source data for Figure 2C.
Source data for Figure 2D.
Source data for Figure 2F.
(A) Bar diagrams representing relative cellular viability of differentiated patient-derived liver organoids (iP-BCC2 and iP-BC2) after 2 or 10 days of treatment with fialuridine or tenofovir at the …
(A) mRNA expression levels of the hepatitis B virus (HBV) receptor sodium taurocholate co-transporting polypeptide (NTCP) in undifferentiated (EM) and differentiated (DM) organoids (n = 3); mRNA …
Source data for Figure 3C.
Source data for Figure 3D.
Source data for Figure 3E.
(A) Levels of expression of sodium taurocholate co-transporting polypeptide (NTCP) were evaluated by reverse transcription polymerase chain reaction (RT-PCR) in the untransduced (parental) and the …
(A, B) A fragment encoding 1.3 times the wild-type hepatitis B virus (wt-HBV) genome was amplified from HepG2.2.15 and cloned into the pEntr plasmid. Using the gateway system, HBV was then …
(A) Representative panel showing the procedure to generate organoid cultures or biobanks from liver tissue. (B) Hematoxylin-eosin-stained sections of explanted liver tissue and phase-contrast …
(A) Partial genome map of hepatitis B virus (HBV) with gene names and primer positions used for identifying the integrated regions. (B) Agarose gel electrophoresis of polymerase chain reaction …
Source data for Figure 4—figure supplement 1B.
Source data for Figure 4—figure supplement 1C.
(A) Phase-contrast images (5X and 20X magnification) of organoid cultures seeded from healthy donors (hDs) and infected patients (iPs) show comparable morphological changes in the organization upon …
(A) Hierarchical clustering heatmap of differentially expressed genes derived from the comparison between the group of five healthy donors (hDs) and five infected patients (iPs) presenting hepatitis …
Source data for Figure 5C.
(A) Relative expression of ADAMTS1, CCNA1, CORO1A, STAMN2, and HBV (Core) mRNA in three healthy donor (hD) organoids 6 days post infection with hepatitis B virus (HBV) (each donor represented by …
Source data for Figure 5—figure supplement 1A.
(A) Hierarchical clustering heatmap of six infected patient (iP), six healthy donor (hD), and four infected donor (iDonor) samples depicting the grouping and expression levels of protein-coding …
Patient | Age | Sex | Diagnosis | Treatment (Y/N) | HBsAg (P/N) | HBcAg (P/N) | HBeAg (P/N) | Anti-HBsAg | Anti-HBcAg | Anti-HBeAg | DNA | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
iDonor 1 | 44 | F | DONOR, cleared HBV | N | N | NA | NA | P | P | NA | NA | |
iDonor 2 | 48 | F | DONOR, cleared HBV | N | N | NA | NA | P | P | NA | NA | |
iDonor 3 | 62 | M | DONOR, cleared HBV | N | N | NA | NA | N | P | NA | NA | |
iDonor 4 | 56 | M | DONOR, cleared HBV | N | N | NA | NA | P | P | NA | NA | |
iP-BC | 49 | M | Liver cirrhosis based on chronic HBV infection | Y | P | NA | NA | P | P | NA | <20 | |
iP-BCC1 | 65 | M | HBV liver cirrhosis with HCC | Y | P | NA | NA | N | P | NA | NA | |
iP-BCC2 | 54 | M | Chronic liver cirrhosis along with HCC | Y | P | NA | NA | N | P | NA | <20 | |
iP-BCC3 | 68 | M | Chronic HBV liver cirrhosis with HCC | Y | P | NA | NA | N | P | NA | <20 | |
iP-BCC4 | 52 | M | HBV liver cirrhosis with multiple HCC | Y | P | NA | N | N | P | P | <20 | |
iP-BCC5 | 51 | M | HBV liver cirrhosis with HCC | Y | P | NA | NA | N | P | NA | <20 | |
iP-BCC6 | 63 | M | HBV liver cirrhosis with HCC | Y | P | N | N | N | P | P | <20 | |
iP-BCC7 | 61 | M | HBV liver cirrhosis with HCC | Y | P | NA | NA | N | P | NA | 28 | |
iP-BCC8 | 53 | M | HBV liver cirrhosis with HCC | Y | P | NA | N | N | P | P | <20 | |
iP-BFA | 54 | F | Acute liver failure based on HBV with Crohn’s disease | N | P | NA | N | N | P | P | <20 | |
iP-BFA2 | 62 | M | Acute liver failure based on HBV | N | P | NA | NA | P | P | NA | NA | |
iP-BDFA | 28 | M | Acute liver failure based on HBV and HDV co-infection | N | P | NA | N | NA | P | P | 1180 | |
iP-BDFA2 | 60 | F | Acute liver failure based on HBV and HDV co-infection | Y | P | NA | NA | N | P | NA | 1080 | |
iP-BDCC | 59 | M | HCC, HBV co-infected with HDV | Y | P | NA | NA | N | P | P | <20 | |
Patient | Age | Sex | Diagnosis | Treatment (Y/N) | HBsAg (P/N) | HBcAg (P/N) | HBeAg (P/N) | Anti-HBsAg | Anti-HBcAg | Anti-HBeAg | DNA | Nash (Y/N) |
CN1 | 69 | F | Nash-HCC | N | N | N | N | N | N | N | NA | Y |
CN2 | 71 | M | Nash-HCC | N | N | N | N | N | N | N | NA | Y |
CN3 | 66 | F | Nash-HCC | N | N | N | N | N | N | N | NA | Y |
CN4 | 60 | F | Nash-HCC | N | N | N | N | N | N | N | NA | Y |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Homo sapiens) | HepG2 | ATCC | HB-8065 RRID:CVCL_0027 | |
Cell line (Homo sapiens) | HepG2.2.15 | CCTCC | CCTCC-GDC0141 RRID:CVCL_L855 | |
Antibody | Hepatitis B Virus Core Antigen Antibody (1-5) (mouse monoclonal) | ThermoFisher | RRID:AB_1075044 | IF: (1:100) |
Antibody | Anti-Hepatitis B Virus Core Antigen antibody (rabbit polyclonal) | Abcam | RRID:AB_10903422 | IF: (1:400) |
Antibody | Hepatitis B Virus Surface Antibody (S 26) (mouse monoclonal) | ThermoFisher | RRID:AB_1075056 | IF: (1:200) |
Antibody | Anti-ALB/Albumin Antibody (F-10) (mouse monoclonal) | Santa Cruz | sc-271605 RRID:AB_10647230 | IF: (1:50) |
Antibody | HNF-4alpha (H-171) antibody (rabbit polyclonal) | Santa Cruz | sc-8987 RRID:AB_2116913 | IF: (1:50) |
Antibody | Anti-SLC10A1 antibody produced in rabbit | Sigma-Aldrich | SAB2104761 RRID:AB_10668685 | IF: (1:200) |
Antibody | Purified Anti-β-Catenin (mouse monoclonal) | BD Transduction Laboratories | 610154 RRID:AB_397555 | IF: (1:200) |
Antibody | ANTI-FLAG M2 antibody (mouse monoclonal) | Sigma-Aldrich | F3165 RRID:AB_259529 | IF: (1:200) |
Recombinant DNA reagent | pENTR/D-TOPO (plasmid backbone) | ThermoFisher | Invitrogen: K240020 | |
Recombinant DNA reagent | pLenti6/V5-DEST Gateway Vector (plasmid backbone) | ThermoFisher | Invitrogen: V49610 | |
Commercial assay or kit | Monolisa HBsAg ULTRA | Biorad | Biorad: 72346 | |
Commercial assay or kit | Human Hepatitis B e Antigen (HBeAg) ELISA Kit | Cusabio | CSB-E13557h | |
Commercial assay or kit | RealiaPrep RNA Cell Miniprep System | Promega | Promega: Z6012 | |
Commercial assay or kit | DNase I | ThermoFisher | Invitrogen: 18047019 | |
Commercial assay or kit | SuperScript II Reverse Transcriptase | ThermoFisher | Invitrogen: 18064022 | |
Commercial assay or kit | GoTaq qPCR Master Mix | Promega | Promega: A6001 | |
Commercial assay or kit | QIAamp MinElute Virus Spin Kit | Qiagen | Qiagen: 57704 | |
Commercial assay or kit | DNeasy Blood and Tissue Kits | Qiagen | Qiagen: 69504 | |
Commercial assay or kit | T5 Exonuclease | New England Biolabs | NEB: M0363 | |
Commercial assay or kit | Plasmid-Safe ATP-Dependent DNase | Epicentre | E3101K | |
Chemical compound, drug | Tenofovir disoproxil fumarate | Sigma-Aldrich | SML1794 | |
Chemical compound, drug | Fialuridine | Cayman Chemical | 15867 | |
Chemical compound, drug | Myrcludex-B | Creative Peptides | 10-101-139 | |
Chemical compound, drug | Blasticidine S hydrochloride | Sigma-Aldrich | Sigma: 15205 | |
Chemical compound, drug | Collagenase D | Sigma-Aldrich | Sigma: 11088858001 | |
chemical compound, drug | TrypLE Express | ThermoFisher | Gibco:12604013 | |
Chemical compound, drug | Cultrex Reduced Growth Factor Basement Membrane Extract, Type 2, Pathclear | R and D Systems | 3533-010-02 | |
Chemical compound, drug | B-27 Supplement, minus vitamin A | ThermoFisher | Gibco: 12587010 | |
Chemical compound, drug | N-2 Supplement | ThermoFisher | Gibco: 17502048 | |
Chemical compound, drug | N-Acetyl-L-cysteine | Sigma-Aldrich | Sigma: A7250 | |
Chemical compound, drug | Nicotinamide | Sigma-Aldrich | Sigma: N3376 | |
Chemical compound, drug | Recombinant [Leu15]-Gastrin I human | Sigma-Aldrich | Sigma: G9145 | |
Chemical compound, drug | Recombinant Human EGF | PeproTech | GMP100-15 | |
Chemical compound, drug | Recombinant Human FGF-10 | PeproTech | 100–26 | |
Chemical compound, drug | Recombinant Human HGF | PeproTech | 100–39H | |
Chemical compound, drug | Forskolin | Sigma-Aldrich | Sigma: F3917 | |
Chemical compound, drug | Recombinant Human Noggin | PeproTech | 120–10C | |
Chemical compound, drug | A 83–01 | Tocris | Tocris: 2939 | |
Chemical compound, drug | ROCK Inhibitor (Y-27632) | Sigma-Aldrich | Sigma: SCM075 | |
Chemical compound, drug | DAPT | Sigma-Aldrich | Sigma: D5942 | |
Chemical compound, drug | Dexamethasone | Sigma-Aldrich | Sigma: D4902 | |
Chemical compound, drug | Recombinant Human BMP-7 | PeproTech | 120–03P | |
Chemical compound, drug | Recombinant Human FGF-19 | PeproTech | 100–32 | |
Software, algorithm | Code used for RNA-Seq data analysis | Moulos and Hatzis, 2015 | The R package used for the RNA-Seq data analysis is available at https://www.bioconductor.org/packages/release/bioc/html/metaseqR.html | |
Software, algorithm | GraphPad Prism | https://graphpad.com | RRID:SCR_002798 | Version 5 |
Procedure | Number of wells/condition | Number of organoids/well |
---|---|---|
DNA isolation | One well of a 24-well plate | ≈100–200 organoids/well |
RNA isolation | One well of a 24-well plate | |
cccDNA isolation | 20–24 wells of a 24-well plate | |
Immunofluorescence staining | 5–10 wells of a 24-well plate |
Gene expression signature.
GO analysis.