Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma
- Department of Biochemistry, Erasmus University Medical Center, Netherlands
- Biomedical Sciences Research Center ‘Alexander Fleming’, Greece
- Department of Surgery, Erasmus University Medical Center, Netherlands
- Foundation Hubrecht Organoid Technology (HUB), Netherlands
- HybridStat Predictive Analytics, Greece
- Hubrecht Institute-KNAW, University Medical Centre Utrecht, Netherlands
- Department of Viroscience, Erasmus Medical Centre, Netherlands
- Max Plank Institute of Molecular Cell Biology and Genetics, Germany
- Department of Urology, Erasmus University Medical Center, Netherlands
- Department of Pathology, Erasmus University Medical Center, Netherlands
Figures
Figure 1 with 3 supplements
Modeling HBV infection in vitro using human liver organoids.
(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 …
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Figure 1—source data 1
Source data for Figure 1F.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig1-data1-v2.xlsx
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Figure 1—source data 2
Source data for Figure 1G.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Expression of HBV core Ag in infected human liver organoids.
(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) …
Figure 1—figure supplement 2
Release of HBV DNA declines over time in hD organoid lines infected in vitro.
(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 …
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Figure 1—figure supplement 2—source data 1
Source data for Figure 1—figure supplement 2C.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig1-figsupp2-data1-v2.xlsx
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Figure 1—figure supplement 2—source data 2
Source data for Figure 1—figure supplement 2D.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig1-figsupp2-data2-v2.xlsx
Figure 1—figure supplement 3
HBV-specific DNA, RNA, and proteins are detected in hD-derived organoids upon infection with recombinant HBV virus as well as with HBV-infected patient’s serum.
(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 …
Figure 2 with 1 supplement
HBV-infected liver organoids as a model for HBV antiviral drug screening and toxicity.
(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 …
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Figure 2—source data 1
Source data for Figure 2B.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig2-data1-v2.xlsx
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Figure 2—source data 2
Source data for Figure 2C.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig2-data2-v2.xlsx
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Figure 2—source data 3
Source data for Figure 2D.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig2-data3-v2.xlsx
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Figure 2—source data 4
Source data for Figure 2F.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig2-data4-v2.xlsx
Figure 2—figure supplement 1
Human liver organoids as a model for anti-HBV drug-induced toxicity screening.
(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 …
Figure 3 with 2 supplements
Lentivirally transduced transgenic liver organoids in the study of HBV.
(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 …
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Figure 3—source data 1
Source data for Figure 3C.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig3-data1-v2.xlsx
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Figure 3—source data 2
Source data for Figure 3D.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig3-data2-v2.xlsx
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Figure 3—source data 3
Source data for Figure 3E.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig3-data3-v2.xlsx
Figure 3—figure supplement 1
hD organoid lines expressing high levels of functional NTCP can be generated by lentiviral infection.
(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 …
Figure 3—figure supplement 2
Generation of a lentiviral construct used to establish human liver organoid lines expressing viral RNA and releasing virus in the supernatant.
(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 …
Figure 4 with 2 supplements
Characterization of organoid cultures from liver explants of HBV-infected patients.
(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 …
Figure 4—figure supplement 1
Parts of HBV genome are found integrated in patient-derived liver organoids.
(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 …
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Source data for Figure 4—figure supplement 1C.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig4-figsupp1-data2-v2.xlsx
Figure 4—figure supplement 2
iP-derived and hD-derived organoids have comparable differentiation potential.
(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 …
Figure 5 with 1 supplement
Differential gene expression in patient-derived liver organoids.
(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 …
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Figure 5—source data 1
Source data for Figure 5C.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Upregulation of signature genes in ex vivo HBV infected liver organoids.
(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 …
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Figure 5—figure supplement 1—source data 1
Source data for Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/60747/elife-60747-fig5-figsupp1-data1-v2.xlsx
Figure 6
Characterization of early HCC gene signature.
(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 …
Figure 7
Schematic of applications of human liver organoids in HBV studies.
Author response image 1
Detection limit of Southern Blot analysis of HBV genome.
A. Serial dilution of cloned HBV genome (top row) and non HBV plasmid (bottom row) was spotted on Hybond-XL membrane and detected using a PCR generated biotinylated near full length HBV probe. …
Tables
Table 1
Characteristics of patients included in the study.
| 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 |
Key resources table
| 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 |
Table 2
Recommended scale of starting material.
| 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 |
Additional files
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Supplementary file 1
Gene expression signature.
- https://cdn.elifesciences.org/articles/60747/elife-60747-supp1-v2.xlsx
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Supplementary file 2
GO analysis.
- https://cdn.elifesciences.org/articles/60747/elife-60747-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60747/elife-60747-transrepform-v2.docx
