Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma

  1. Elisa De Crignis
  2. Tanvir Hossain
  3. Shahla Romal
  4. Fabrizia Carofiglio
  5. Panagiotis Moulos
  6. Mir Mubashir Khalid
  7. Shringar Rao
  8. Ameneh Bazrafshan
  9. Monique MA Verstegen
  10. Farzin Pourfarzad
  11. Christina Koutsothanassis
  12. Helmuth Gehart
  13. Tsung Wai Kan
  14. Robert-Jan Palstra
  15. Charles Boucher
  16. Jan NM IJzermans
  17. Meritxell Huch
  18. Sylvia F Boj
  19. Robert Vries
  20. Hans Clevers
  21. Luc JW van der Laan
  22. Pantelis Hatzis
  23. Tokameh Mahmoudi  Is a corresponding author
  1. Department of Biochemistry, Erasmus University Medical Center, Netherlands
  2. Biomedical Sciences Research Center ‘Alexander Fleming’, Greece
  3. Department of Surgery, Erasmus University Medical Center, Netherlands
  4. Foundation Hubrecht Organoid Technology (HUB), Netherlands
  5. HybridStat Predictive Analytics, Greece
  6. Hubrecht Institute-KNAW, University Medical Centre Utrecht, Netherlands
  7. Department of Viroscience, Erasmus Medical Centre, Netherlands
  8. Max Plank Institute of Molecular Cell Biology and Genetics, Germany
  9. Department of Urology, Erasmus University Medical Center, Netherlands
  10. Department of Pathology, Erasmus University Medical Center, Netherlands
8 figures, 3 tables and 3 additional files

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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 …

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.
PatientAgeSexDiagnosisTreatment (Y/N)HBsAg
(P/N)
HBcAg
(P/N)
HBeAg
(P/N)
Anti-HBsAgAnti-HBcAgAnti-HBeAgDNA
iDonor 144FDONOR, cleared HBVNNNANAPPNANA
iDonor 248FDONOR, cleared HBVNNNANAPPNANA
iDonor 362MDONOR, cleared HBVNNNANANPNANA
iDonor 456MDONOR, cleared HBVNNNANAPPNANA
iP-BC49MLiver cirrhosis based on chronic HBV infectionYPNANAPPNA<20
iP-BCC165MHBV liver cirrhosis with HCCYPNANANPNANA
iP-BCC254MChronic liver cirrhosis along with HCCYPNANANPNA<20
iP-BCC368MChronic HBV liver cirrhosis with HCCYPNANANPNA<20
iP-BCC452MHBV liver cirrhosis with multiple HCCYPNANNPP<20
iP-BCC551MHBV liver cirrhosis with HCCYPNANANPNA<20
iP-BCC663MHBV liver cirrhosis with HCCYPNNNPP<20
iP-BCC761MHBV liver cirrhosis with HCCYPNANANPNA28
iP-BCC853MHBV liver cirrhosis with HCCYPNANNPP<20
iP-BFA54FAcute liver failure based on HBV with Crohn’s diseaseNPNANNPP<20
iP-BFA262MAcute liver failure based on HBVNPNANAPPNANA
iP-BDFA28MAcute liver failure based on HBV and HDV co-infectionNPNANNAPP1180
iP-BDFA260FAcute liver failure based on HBV and HDV co-infectionYPNANANPNA1080
iP-BDCC59MHCC, HBV co-infected with HDVYPNANANPP<20
PatientAgeSexDiagnosisTreatment (Y/N)HBsAg
(P/N)
HBcAg
(P/N)
HBeAg
(P/N)
Anti-HBsAgAnti-HBcAgAnti-HBeAgDNANash (Y/N)
CN169FNash-HCCNNNNNNNNAY
CN271MNash-HCCNNNNNNNNAY
CN366FNash-HCCNNNNNNNNAY
CN460FNash-HCCNNNNNNNNAY
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HepG2ATCCHB-8065
RRID:CVCL_0027
Cell line (Homo sapiens)HepG2.2.15CCTCCCCTCC-GDC0141
RRID:CVCL_L855
AntibodyHepatitis B Virus Core Antigen Antibody (1-5) (mouse monoclonal)ThermoFisherRRID:AB_1075044IF: (1:100)
AntibodyAnti-Hepatitis B Virus Core Antigen antibody (rabbit polyclonal)AbcamRRID:AB_10903422IF: (1:400)
AntibodyHepatitis B Virus Surface Antibody (S 26) (mouse monoclonal)ThermoFisherRRID:AB_1075056IF: (1:200)
AntibodyAnti-ALB/Albumin Antibody (F-10) (mouse monoclonal)Santa Cruzsc-271605
RRID:AB_10647230
IF: (1:50)
AntibodyHNF-4alpha (H-171) antibody (rabbit polyclonal)Santa Cruzsc-8987
RRID:AB_2116913
IF: (1:50)
AntibodyAnti-SLC10A1 antibody produced in rabbitSigma-AldrichSAB2104761 RRID:AB_10668685IF: (1:200)
AntibodyPurified Anti-β-Catenin (mouse monoclonal)BD Transduction Laboratories610154
RRID:AB_397555
IF: (1:200)
AntibodyANTI-FLAG M2 antibody (mouse monoclonal)Sigma-AldrichF3165
RRID:AB_259529
IF: (1:200)
Recombinant DNA reagentpENTR/D-TOPO (plasmid backbone)ThermoFisherInvitrogen: K240020
Recombinant DNA reagentpLenti6/V5-DEST Gateway Vector (plasmid backbone)ThermoFisherInvitrogen: V49610
Commercial assay or kitMonolisa
HBsAg ULTRA
BioradBiorad: 72346
Commercial assay or kitHuman Hepatitis B e Antigen (HBeAg) ELISA KitCusabioCSB-E13557h
Commercial assay or kitRealiaPrep RNA Cell Miniprep SystemPromegaPromega: Z6012
Commercial assay or kitDNase IThermoFisherInvitrogen: 18047019
Commercial assay or kitSuperScript II Reverse TranscriptaseThermoFisherInvitrogen: 18064022
Commercial assay or kitGoTaq qPCR Master MixPromegaPromega: A6001
Commercial assay or kitQIAamp MinElute Virus Spin KitQiagenQiagen: 57704
Commercial assay or kitDNeasy Blood and Tissue KitsQiagenQiagen: 69504
Commercial assay or kitT5 ExonucleaseNew England BiolabsNEB: M0363
Commercial assay or kitPlasmid-Safe ATP-Dependent DNaseEpicentreE3101K
Chemical compound, drugTenofovir disoproxil fumarateSigma-AldrichSML1794
Chemical compound, drugFialuridineCayman Chemical15867
Chemical compound, drugMyrcludex-BCreative Peptides10-101-139
Chemical compound, drugBlasticidine S hydrochlorideSigma-AldrichSigma: 15205
Chemical compound, drugCollagenase DSigma-AldrichSigma: 11088858001
chemical compound, drugTrypLE ExpressThermoFisherGibco:12604013
Chemical compound, drugCultrex Reduced Growth Factor Basement Membrane Extract, Type 2, PathclearR and D Systems3533-010-02
Chemical compound, drugB-27 Supplement, minus vitamin AThermoFisherGibco: 12587010
Chemical compound, drugN-2 SupplementThermoFisherGibco: 17502048
Chemical compound, drugN-Acetyl-L-cysteineSigma-AldrichSigma: A7250
Chemical compound, drugNicotinamideSigma-AldrichSigma: N3376
Chemical compound, drugRecombinant [Leu15]-Gastrin I humanSigma-AldrichSigma: G9145
Chemical compound, drugRecombinant Human EGFPeproTechGMP100-15
Chemical compound, drugRecombinant Human FGF-10PeproTech100–26
Chemical compound, drugRecombinant Human HGFPeproTech100–39H
Chemical compound, drugForskolinSigma-AldrichSigma: F3917
Chemical compound, drugRecombinant Human NogginPeproTech120–10C
Chemical compound, drugA 83–01TocrisTocris: 2939
Chemical compound, drugROCK Inhibitor (Y-27632)Sigma-AldrichSigma: SCM075
Chemical compound, drugDAPTSigma-AldrichSigma: D5942
Chemical compound, drugDexamethasoneSigma-AldrichSigma: D4902
Chemical compound, drugRecombinant Human BMP-7PeproTech120–03P
Chemical compound, drugRecombinant Human FGF-19PeproTech100–32
Software, algorithmCode used for RNA-Seq data analysisMoulos and Hatzis, 2015The R package used for the RNA-Seq data analysis is available at https://www.bioconductor.org/packages/release/bioc/html/metaseqR.html
Software, algorithmGraphPad Prismhttps://graphpad.comRRID:SCR_002798Version 5
Table 2
Recommended scale of starting material.
ProcedureNumber of wells/conditionNumber of organoids/well
DNA isolationOne well of a 24-well plate≈100–200 organoids/well
RNA isolationOne well of a 24-well plate
cccDNA isolation20–24 wells of a 24-well plate
Immunofluorescence staining5–10 wells of a 24-well plate

Additional files

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