Assessment of hepatitis B virus infection and interhost cellular responses using intrahepatic cholangiocyte organoids

The intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma‐derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT‐PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon‐stimulated genes [ISG]) to interferon‐α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well‐differentiated ICOs using spinoculation, with time and donor‐dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor‐dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon‐α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor‐dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals.


| INTRODUCTION
Chronic hepatitis B infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) with an estimated 250 million people affected worldwide and more than 800,000 deaths. 1 At present, there is no effective curative therapy for chronic hepatitis B, although there is a major global initiative to identify strategies for a HBV cure.
However, attempts to develop curative therapy and a greater understanding of the response to infection have been hampered by a lack of satisfactory in vitro model systems that faithfully mimics individual host physiology. 2 Primary human hepatocytes (PHH), often regarded as the "gold-standard" model for in vitro HBV infection, can rapidly dedifferentiate in culture and have a limited lifespan that hampers their use in modeling for chronic HBV infection. 3Ectopic expression of NTCP, the HBV cellular receptor in tumor-derived cell lines such as HepG2 cells has been used extensively in studying HBV infection in vitro but suffer from altered physiology and cellular response. 4Importantly, this approach is nonphysiological and represents an over-simplification of HBV infection for use in translational research.Furthermore, it overlooks genetic variation of hepatocytes, thus limiting its utility in predicting clinical response and the development of a personalized medicine approach to HBV infection management.
Human intrahepatic cholangiocyte organoids (ICO) developed by Huch et al. consist of self-organizing hepatic LGR5+ progenitor cells which can be expanded long-term with genetic stability. 5ey exhibit both mature hepatocyte and ductal metabolic phenotypes following differentiation.Most importantly, they retain host genetics for the liver from which they were derived.
Unlike many other stem-cell or primary hepatocyte-derived model systems, the LGR5+ progenitor cell-derived ICO culture does not involve the process of parental cell reprogramming to achieve multipotency for long-term expansion, thus limiting the introduction of mutations in culture. 5This quality is particularly important for potential clinical applications in the area of autologous transplantation.More recently, De Crignis and Hossain and Hossain et al. described a detailed protocol to achieve HBV infection and demonstrate HBV full lifecycle in this ex vivo ICO model. 6,7In the study by Crignis, ICOs derived from HBV-infected patients exhibit early cancer transcriptomic signatures, thus showing potentials in cancer biomarker research.
In this study, we developed human ICOs from core liver biopsies and liver resections from HBV-naive individuals.In addition to validating the susceptibility of ICOs to HBV as shown by Crignis et al., we provide an extended characterization of key elements in ICOs that support HBV infections, in particular functional expression of Na-taurocholate co-transporting polypeptide (NTCP) 8 and structural changes essential for productive HBV trafficking and export. 9More importantly, using ICOs derived from different donors, we explored the inter-donor differences in HBV replication dynamics and innate immune response.This study is the first to characterize the innate immune competency of this ex vivo culture system and provides further insight into the applications in HBV antiviral assessment.

| Patient selection and ICO isolation
Patients who underwent liver resections for metastatic carcinoma or core biopsies were recruited through the Gastroenterology and Liver Tissue Repository at the Royal Adelaide Hospital with ethics approval from the hospital research committee.Background healthy tissue distant from resected tumor (>10 cm away) or fragments of core biopsied tissues (weight 20-50 mg) were used for ICO isolation as previously described. 5Resected or biopsied tissue was stored at 4°C in basal medium (Advanced DMEM/F-12 supplemented with 1% Pen/Strep, 1% GlutaMAX, 10 mM HEPES), and processed within 48 h.Tissue was minced using scissors under aseptic conditions, and resuspended in 10 mL of cold wash medium (DMEM [high glucose, GlutaMAX, pyruvate] + 1% FBS + 1% Pen/Strep).Once tissue settled, supernatant was removed by suctioning and the washing step repeated 1-2 times.Five milliliters of prewarmed digestion solution (0.125 mg/mL of collagenase and dispase II, 0.1 mg/mL dispase in wash medium) was added to the minced tissue and allow to incubate in waterbath at 37°C.A small aliquot of digesting tissue was examined under the light microscope regularly until at least 80% of material is digested to single cell.Eight milliliters of cold wash medium was added to arrest the digestion process and the digested material was centrifuged at 300 g for 5 min at 8°C.This process was repeated for another 2-3 times until the supernatant was clear.

| HBV infection in ICOs and antiviral response
All organoids were pooled together in one tube and resuspended in 10 mL of cold Advanced DMEM/F12.For cell counting, 3 × 10 μL aliquots of resuspended organoids were transferred to Eppendorf tubes, each incubated with 10 mL of TrypLE Express enzymes (ThermoFisher 12604013) for 10 min at 37°C.Once organoids were dissociated into single cells, cell counting was performed using a hemocytometer.Cell count estimate was derived from an average of cell counts from the three aliquots.Before incubation of HBV inoculum, the resuspended organoids were centrifuged at 300×g at 4°C for 5 min, and supernatant removed.Appropriate volume of quantified HBV stock was added to the organoids to achieve the required inoculum (GE/cell).A mixture consists of DM and 4% PEG8000 at 1:1 ratio supplemented with 10 μM Y-27632 was added to the organoids to a final volume of 10 mL, and distributed evenly (1 mL/well) onto a 12-well nonadherent plate.Plate spinoculation was performed at 600×g for 1 h at 35°C, before the addition of R-Spondin (10% vol/vol) and incubation for a further 24 h.Organoids were washed nine times before being re-seeded in fresh nonadherent plate and cultured in DM supplemented with 10 μM Y-27632 (Peprotech).
To test for antiviral response, Myrcludex B (Auspep) and Baricitinib (1187594-09-7, Focus Bioscience) were used to test for the antiviral response on differentiated ICOs at concentration 1 μM and 100 nM, respectively.To evaluate the innate immune response of undifferentiated and differentiated ICOs, pegylated interferon-α2b (98530-12-2, Sigma Aldrich) (at various concentration) and Polyl:C (Polyinosinic:polycytidylic acid, InvivoGen) (at 2 μg/mL concentration) were used to treat the ICOs.Treated ICOs were then harvested at selected timepoints for qPCR analysis of expression of target genes.

| Total cellular RNA isolation and qPCR
Organoids were washed with cold medium to remove extracellular matrix before RNA isolation using TRIsure (Bioline) according to manufacturer's instructions.cDNA first strand synthesis and amplification were performed using Luna Universal qPCR mastermix (NEB) with previously described qPCR conditions on StepOne Plus Real-time PCR System (Applied Biosystems). 10Primer sequences are provided in Supporting Information S1: Table S1.

| HBV stock generation
HBV viral stock was generated from two sources (1) HepAD38 cells and (2) HBV-infected patients.For cell culture-derived HBV (ccHBV), Hep AD38 cells were cultured in T175 flasks until confluent (between 7 and 10 days) whereas for patient-derived HBV, 50-100 mL of plasma samples were used.For virion precipitation, 18 mL of PEG8000 (Sigma, P2139) was added to every 100 mL of cell culture supernatant or plasma.The mixture was allowed to precipitate at 4°C overnight, followed by centrifugation at 10,000 × g at 4°C for an hour.Supernatant was discarded and pellet was resuspended with 10% fetal calf serum in PBS, using a volume 1/100th times of the starting volume.Ten microliters of viral stock is extracted using the Nucleospin Tissue extraction kit (Macherey-Nagel) and quantified for HBV DNA as described below.

| RNA sequencing of liver tissue and ICOs
Total RNA was converted to strand-specific Illumina compatible sequencing libraries using the Nugen Universal Plus mRNA-Seq library kit from Tecan (Mannedorf) as per the manufacturer's instructions (MO1442 v2).Briefly, 500 ng of total RNA was polyA selected and the mRNA fragmented before reverse transcription and second-strand cDNA synthesis using dUTP.The resultant cDNA is end-repaired before the ligation of Illumina-compatible barcoded sequencing adapters.The cDNA libraries were strand selected and PCR amplified for 12 cycles before assessment by Agilent Tapestation for quality and Qubit fluorescence assay for amount.Sequencing pools were generated by mixing equimolar amounts of compatible sample libraries based on the Qubit measurements.Sequencing of the library pool was performed using Illumina Nextseq.500 using single read 75 bp (v2.5) sequencing chemistry.
Illumina high-throughput sequencing data was processed using a standard RNA-Seq analysis workflow.Raw single-end FASTQ reads were assessed for quality using FastQC 12 and ngsReports, 13,14 and then aligned to the GRCh37.p13version of the human genome 15 using the transcriptome algorithm STAR. 16After alignment, mapped sequence reads were summarized to GRCh37 gene annotation using featureCounts, available through the package RSubread (https:// bioconductor.org/packages/release/bioc/html/Rsubread.html).Differential gene expression was carried out using R/Bioconductor packages limma 17 and edgeR, 18 using mean-variance relationship estimates of the log-counts from the limma voom method 19 and contrasts defined between each sample group.KEGG pathway and Gene Ontology enrichment were also carried out using R/Bioconductor packages, and heatmaps and upset produced using pheatmap (https://cran.r-project.org/web/packages/pheatmap/index.html) and upSetR (https://cran.r-project.org/web/packages/UpSetR/index.html).
All code carried out in the study is available in the RMarkdown document.

| Immunofluorescence and immunoblotting
Organoids cultured in µ-Slide 8 well (Ibidi) were washed repeatedly with cold PBS to remove BME2 before fixation with 4% PFA for 20 min, followed by a 20-min incubation with 50 mM NH 4 Cl to minimize autofluorescence, and permeabilization with 0.1% Triton-X 100 for 10 min.After washing with cold PBS, organoids were blocked with 5% BSA/PBS for 1 h.Next, organoids were incubated with primary antibodies diluted in 1% BSA, at 4°C for 16 h on a gentle shaker, followed by incubation with secondary antibodies at 1:200 dilutions for a further 2 h.Nuclear staining was performed by incubating with DAPI at 1:1000 dilutions for 15 min.Images were taken using the Olympus FV3000 confocal laser scanning microscope and analyzed with CellSens.3D reconstruction and deconvolution were performed using Imaris software (Bitplane).Immunoblotting was performed as previously described. 20All primary and secondary antibodies used are provided in Supporting Information S1: Table S2.
All microscopy was performed at The University of Adelaide Bio-Imaging facility.

| Detection of secreted HBeAg and HBsAg
The supernatant (200 μL) was collected from infected organoids for HBeAg and HBsAg quantification using the Elecsys HBeAg and HBsAg II kit chemiluminescent microparticle immunoassay (CMIA) on the Cobas e602 instrument (Roche).

| Transmission electron microscopy (TEM)
Organoids were washed with cold medium to remove BME2 and fixed with 4%PFA/1.25%glutaraldehyde/4% sucrose for 1 h.After fixation, organoids were treated with 2% OsO 4 for 1 h, washed with 4% sucrose, followed by gradual dehydration with 70%, 90%, and 100% EtOH and gradual infiltration from 100% propylene oxide to 100% resin before polymerization at 70°C for 24-48 h.Blocks were sectioned at 40 nm slices and imaged using the FEI Tecnai G2 Spirit TEM at the Electron Optic Centre at The University of Adelaide.

| Statistical analysis
For qRT-PCR analysis, differential expression of genes between two or more groups were compared using two-way analysis of variance (ANOVA) analysis in Prism 8. Charts were presented as mean ± SEM with level of significance according to standard GraphPad format for p-values.

| Differentiated ICOs exhibit liver-like structural changes and cellular polarity
Human ICO cultures were generated from normal liver following liver resection (n = 9) and core biopsy (n = 3) samples (Figure 1A).Patient and sample characteristics are provided in Supporting Information S1: Table S3.Despite the small amount of starting material from core liver biopsies, organoids grew to confluency within 2-3 weeks with 100% success rate.Following differentiation, the ICOs changed from cystic structure to a branching "tree in buds" appearance over the course of 20 days (Figure 1B).Histological analysis (H&E staining) indicated a change from a ductal-like phenotype with single-layered epithelium to a multilayered phenotype following differentiation (Figure 1C).This differentiation process is accompanied by cellular polarization as evident following visualization of Zona Occludens-1 (ZO-1: tight junction marker).There is a clear redistribution of ZO-1, in which ZO-1 is present on the complete outer membrane in undifferentiated ICOs to a more targeted localization presumably to tight junctions and interconnecting structures similar to bile canaliculi in the differentiated state (Figure 1D).The ultrastructure of ICOs examined under transmission electron microscopy showed evidence of hepatocyte phenotypes with liver microarchitecture such as hepatocyte microvilli and tight junctions bordering the bile canaliculi and glycogen granules that suggests the development of a hepatocyte-like phenotype (Figure 1E).

| ICOs exhibit a mixed-cell phenotype
Using qRT-PCR, metabolic markers of mature hepatocytes were assessed in undifferentiated and differentiated ICOs isolated from three patients and their respective parental liver tissues.Upregulations of hepatocyte markers (albumin, CYP3A4, and CYP2B6) were seen following at least 10 days of differentiation (Figure 2A).In contrast, LGR5, a progenitor cell marker, was only detected in undifferentiated ICO cultures.The hepatocyte marker (HNF4a) and ductal cell markers (KRT19) were comparably expressed at the mRNA level, before and after differentiation, suggesting retention of bicellular phenotypes.This finding was confirmed with immunofluorescent staining for ductal (SOX9) and hepatocyte (HNF4a) markers in both undifferentiated and differentiated ICOs, while albumin expression was only seen in differentiated ICOs (Figure 2B).
To explore the relationship between ICOs and the tissue of origin, we performed RNASeq analysis of differentiated and undifferentiated ICOs and their cognate liver tissue from four donors.Analysis of transcriptomic abundance revealed clustering of samples based on gene expression of liver metabolic markers with undifferentiated and differentiated ICOs more similar than cognate liver tissue.Interestingly, distinct donor variation in gene expression were seen among the differentiated ICOs despite similar culture and differentiation conditions (Figure 2C).

| Advanced differentiation is required for functional HBV entry receptor expression
The temporal development of differentiation was monitored through detection of Albumin mRNA that revealed a steady increase at Day 10 postdifferentiation process (Figure 3A).NTCP mRNA expression while low in undifferentiated ICOs did not appreciably increase until Day 15 of differentiation (Figure 3A,B), with observed variation between donors.Confocal microscopy revealed that NTCP expression was detected at the cell surface and between adjoining cells, distant from ZO-1 that is localized to apical surface (Figure 3C).Using a fluorescent labeled HBV PreS1 peptide that detects functional NTCP expression, we identified that NTCP is not expressed at the cell surface until Day 10-15 following differentiation, consistent with increased mRNA expression discussed above.In contrast, low-level NTCP expression was observed before Day 10 although it remains intracellular (Figure 3D,E).

| ICOs demonstrate donor-dependent variation in permissiveness to HBV infection
ICOs from different donors were grown in DM for 15 days before infection with cell culture-derived HBV (ccHBV, derived from HepAD38).HBV replication was validated by visualization of cytoplasmic HBcAg expression by Immunofluorescence confocal microscopy (Figure 4A-C). 21An inoculum of at least 200 genome copies/cell of ccHBV is required for infection to occur (Figure 4A).The use of patient-derived plasma HBV as inoculum resulted in infection with as low as 50 GEq/cell (Figure 4D).HBV replication was also confirmed through demonstration of a temporal increase in intracellular HBV pregenomic RNA, HBV pregenomic and subgenomic RNA (total RNA), covalently closed circular DNA (cccDNA) and HBV DNA and HBeAg and HBsAg in the supernatant over a 2-week period (Figure 5).In all cases heat-inactivated (HI) HBV acted as a control for input inoculum.Interestingly differences in donor efficiency of infection were observed that is consistent with variation in HBV permissiveness in PHH and most likely in natural infection. 22

| DISCUSSION
International efforts are currently underway to achieve either complete or functional cure for HBV infection.Emerging therapeutics in preclinical and Phase I/II clinical studies range from direct-acting agents that target the HBV life cycle (e.g., anti-HBV RNA, entry inhibitors, RNA translation inhibitors, capsid and HBsAg inhibitors), [23][24][25] enhancement of the innate immune pathways (RIG-I NOD2, TLR7 and TLR8 agonizts), 26,27 modulation of host-factors (apoptosis inducer, ciclofillin inhibitor) 28,29 and gene editing technology (CRISPR/Cas9, ARCUS platform). 30However, assessment of the efficacy of these emerging novel therapeutic strategies will require an in vitro model system that not only supports the full lifecycle of HBV but also accounts for genetic differences among patients.PHH may fulfill some of these roles, but rapid dedifferentiation as a result of major transcriptomic alterations of genes involved in TCA cycle, mitochondrial dysfunction and oxidative phosphorylation can occur as early as 4 h after isolation from liver 31,32 remain the main physiological constraint of this model.That is not to mention their limited proliferative capacity, availability and cost which restricts their use for viral infection and cell response studies.
To overcome the current limitations of in vitro HBV model systems, we developed human ICOs as originally described by Huch et al that allow long-term expansion in the undifferentiated form with genetic stability. 5The process of ICO generation from liver derived stem cells requires stimulation of the Wnt-β-catenin pathway, which recapitulates the regenerative process of liver lobules from progenitor cells that reside in close proximity to the central veins, following liver injury. 33Using a mouse transplantation model, successful engraftment of ICOs can result in development of mature hepatocytes with suppressed ductal phenotypes. 34Previously, Crignis et al.
had demonstrated successful HBV infection using this culture system. 6In this study, we adapted the ICO isolation from both core biopsies and liver resections, validated the findings by Huch and Crignis, and provided a detailed characterization of HBV infection and cellular responses using this model system.NTCP (SCL10A1) is an essential HBV entry receptor expressed at the basolateral surface of the hepatocytes 35 and it has been suggested that PHH lose their permissiveness to HBV infection as a result of NTCP expression following isolation and de-differentiation in culture. 36NTCP is a transmembrane glycoprotein with the C-terminus located extracellularly and N-terminus located intracellularly in hepatocytes. 37In our study, expression of NTCP mRNA correlates with differentiation status, and while low-level expression of NTCP protein was noted in undifferentiated ICOs, this was predominantly intracellular.NTCP was not detected at the cell surface until day 10 postdifferentiation as determined by a HBV PreS1 peptide binding.This suggests (1) posttranslational modification of NTCP may occur following differentiation and (2) that differentiation is essential for functional NTCP expression and productive HBV binding in ICOs.Consistent with this, infection of ICOs was dependent on NTCP as evidenced by attenuation of HBV infection when ICOs were treated with the NTCP competitive HBV entry inhibitor Myrcludex-B.We also noted a significant difference in NTCP expression at the mRNA level between three donors suggesting possible individual variation which may impact susceptibility to HBV infection.However, our infection data from the three donors suggests that this may not be the case and recent reports have suggested that higher expression of NTCP in ICO cultures does not result in more efficient HBV infection. 6Further experiments are required to explore genetic differences in HBV infection susceptibility.
Given that ICOs can be readily isolated from liver biopsy samples and amenable to biobanking, the ICO model can act as a suitable platform for the study of existing and novel inhibitors of HBV replication that may inform genetic susceptibility to HBV antivirals.To this end we assessed the antiviral activity of Myrcludex-B, a licenced HBV entry inhibitor that competitively binds the cellular HBV receptor NTCP, either in a pre or postinfection scenario.As expected, treatment of ICOs before HBV infection resulted in a significant decrease in HBV RNA and complete loss of expression of HBV core antigen expression in infected hepatocytes, while addition of Myrcludex-B following infection had no impact on HBV infection, consistent with its entry mode of action.Baricitinib is an immunosuppressive oral selective Jak1 and Jak2 inhibitor and reactivation of HBV replication is a well-recognized complication in patients receiving Baricitinib especially those with rheumatoid arthritis. 38Baricitinib treatment of HBV-infected ICOs resulted in an increase (approximately 1 log) in HBV RNA suggesting that HBV replication is sensitive to innate activation of antiviral responses and is consistent with studies showing that HBV can be cleared from the liver in a cytokine-mediated nontoxic manner 27,39 and as a result of interferon dependent antiviral responses. 40ven that the JAK-STAT signaling pathway plays a role in signal transduction for a number of key cytokines a number of possible mechanisms are possible.Interleukin-6 plays an important role in hepatocyte homeostasis and its impairment can lead to impaired innate responses to viral infection while disruption of interferon signaling, that is important for an antiviral state through production of antiviral ISGs may explain the increase in HBV replication following inhibition of Jak1 and 2 by Baricitinib.Collectively, the ICO infection model shows potentials as a model system to dissect the molecular mechanisms for new HBV antivirals.
The ability of HBV to induce a robust antiviral response is controversial with inconsistent reports on the ability of HBV to activate and evade the innate antiviral response. 41,42This is primarily due to the limitations of model systems used to investigate HBV replication such as hepatoma-derived cell lines (i.e., HepG2) that have defects in innate immune sensing and associated signal transduction. 43Previous study by Gerold et al, comparing the RNA sensing properties of a range of human hepatoma cell lines (Huh-6, Huh-7, HepG2, Hep-G2-HFL, Hep3B, and HepaRG) with PHH, showed that PHH demonstrates competent RNAsensing mechanism through both RIG-I and TLR3 mediated pathways, with considerably higher IFN-β expression than hepatoma cell lines following stimulation of TLR3 pathway using polyI:C.In their study, many hepatoma cell lines are refractory to stimulation of either or both of these pathways. 43In our study, significant differences in ISG response is seen between RNA-sensing and effector ISGs in ICO (both undifferentiated and differentiated) as compared with Huh7 cells (Supporting Information S1: Figure S1), but would require further validation across multiple hepatoma cell lines and organoids.However, there is evidence that HBV can activate an antiviral innate response in physiological relevant culture systems.Micropatterned cocultures in which PHH are cocultured with stromal fibroblasts are permissive for HBV infection for up to 3 weeks and pluripotent stem-cell derived hepatocytes (iHeps) induce an interferon and antiviral ISG response.Furthermore, interferon can degrade cccDNA from the HBV-infected liver and in a small subset of individuals, pegylated interferon can eliminate HBV. 44Indeed, in infected chimpanzee, HBV replication is controlled by a type I interferon response. 27,45However, HBV infection of PHH neither activates or inhibits pattern recognition receptors and thus evades type-I antiviral responses as a stealth virus. 46Consistent with the report from Lucifora

2. 5 |
Quantification of HBV RNA and DNA Extracted cellular RNA was quantified using Nanodrop 2000 (ThermoFisher) and diluted with nuclease-free water to 10 ng/μL.Fifty nanograms of RNA was used directly as template in each qPCR reaction using LunaScript Mulitplex One-Step RT-qPCR kit (New England Biolabs, NEB #E1555) along with 1 μM of primers, and PCR cycling conditions are: 55°C 10 min 1 cycle, 98°C 1 min 1 cycle, 40 cycles of 98°C 10 s, 60°C 10 s, 72°C 20 s, and 72°C 5 min.The results are compared with a standard curve generated from linear regression analysis of seven 10-fold dilutions of a linearized 1-mer HBV plasmid (concentrations between 1.7 × 10 −18 and 4.46 × 10 −13 g/μL).Control without reverse transcription was used to rule out DNA contamination.Supernatant (100 μL) of infected organoids were collected from the wells (12 well plate) and subjected to two DNaseI treatment (2 U/ μL) with 1 μL MgCl 2 before extraction.Intracellular and supernatant HBV DNAs were isolated using the Nucleospin Tissue extraction kit (Macherey-Nagel) following manufacturer's instructions, eluted to a final volume of 20 μL.Quantification was performed as described earlier.For cccDNA qPCR quantification, infected cells (12 well plate) were first harvested and DNA extracted using Nucleospin Tissue extraction kit (Macherey-Nagel), and eluted to 20 μL for T5 exonuclease digestion (NEB M03653) at 37°C for 30 min and 99°C for 5 min to remove rcDNA, dsl DNA dn PF-rcDNA.(using pre-and posttreatment of rcDNA as digestion control).The qPCR conditions are: 95°C, 10 min 1 cycle, 40 cycles of 95°C 15 s, 60°C 5 s, 72°C 45 s, 88°C 2 s.The cccDNA primers were validated against previously described primers by Singh et al. 11 cccDNA quantification (2.5 μL/ reaction) was determined using the standard curve generated from linearized 1-mer HBV plasmid, and the final determination of the total cccDNA copies in each well was calculated by multiplying the copies/reaction by a factor of 8 (total elution volume of 20 μL = 8 reactions).

Following
infection of ICOs with patient-derived HBV genotype B and treatment with Myrcludex B either 1 day pre-or 1 day postinfection, antiviral activity was as assessed by detection of HBV total RNA and confocal immunofluorescence imaging to detect HBcAg at 21 days postinfection (Figure 6A,B).Consistent F I G U R E 2 Human intrahepatic cholangiocyte organoids (ICOs) exhibit Interpatient variation in metabolism.(A) Quantitative real-time polymerase chain reaction (qRT-PCR) of metabolic and cellular markers for three corresponding sets (patients 01, 10, and 12) of undifferentiated ICOs, differentiated ICO and parental liver tissue (fold change, relative to GAPDH, Mean ± SEM).(B) Representative immunofluorescence staining of differentiated and undifferentiated ICOs for ductal marker (SOX9), hepatocyte marker (HNF4α), and albumin (×20 magnification, scale bar = 10 μm).Zoom images are shown as inset.(C) Heat map comparison of metabolic and cell signature transcriptomes from RNA-seq of four undifferentiated and differentiated ICOs cultured under similar conditions with corresponding parental liver tissue (green arrow indicates LGR5 progenitor marker).with the mechanism of action of Myrcludex B as an entry inhibitor, preinfection Myrcludex treatment resulted in significant reduction of HBV RNA and HBcAg whereas postinfection treatment with Myrcludex B did not as replication had been established (Figure6A).Interestingly, postinfection treatment of two donor organoids with the JAK inhibitor resulted in an increase in HBV RNA that was not observed with preinfection treatment (Figure6C).

3. 6 |
ICOs demonstrate a robust ISG response to interferon-α and dsRNA mimics but not HBV infection Before investigating cellular responses to viral infection, we first determined the innate immune competency of ICOs.ICOs from several different donors, either undifferentiated or differentiated were stimulated with interferon-α or PolyI:C and ISG mRNA expression evaluated.Timepoint experiments revealed comparable upregulation of mRNA for the antiviral ISGs IFITM1, ISG15, and Viperin following stimulation with 1000 U/mL of interferon-α for both undifferentiated and differentiated ICOs (Figure 7A).Even at lower concentrations of interferon-α (10 U/mL and 100 U/mL), we noted significant upregulation of mRNA for ISGs and the proinflammatory cytokine, CXCL10 (Figure 7B).While only a small number of donors were investigated, the response to interferon-α and low-dose RNA viral genome mimic PolyI:C (2 μg/mL) was donor dependant (Figure 7C,D).Similar to interferon-α, we also noted a 2-4 log induction of ISG mRNA following stimulation of undifferentiated and differentiated ICOs with the RNA viral genome mimic PolyI:C (2 μg/mL) that activates the innate response through cytosolic (RLRs) and endosomal toll-like receptors (TLR).Collectively these results suggest that human ICOs retain innate immune factors for detection and response to viral infection.Next, we assessed the innate response to HBV-infected ICO cultures.Three individual donor ICOs previously characterized for HBV replication (Figure 5), were infected with HBV and monitored for innate immune activation over a course of 12 days.Using heatinactivated HBV as a control, we noted no induction of interferon-β production or ISG expression across all donors (Figure 7E) even in the face of productive HBV infection.This demonstrates at least in human-derived ICOs that HBV does not activate innate immune responses.
When cultured ex vivo, metabolic characterization of the ICO transcriptome reveals a mixed cellular but predominantly ductal phenotype.Despite this, ICO derived from "normal" liver tissue from three different donors were successfully infected with recombinant HBV derived from HepAD38 cells or HBV derived from patient plasma (genotype B) and over a 12-day time course revealed F I G U R E 3 Human intrahepatic cholangiocyte organoids express functional hepatitis B virus (HBV) entry receptor sodium taurocholate cotransporting polypeptide (NTCP) upon differentiation.(A) Quantification of albumin and NTCP mRNA expression with quantitative real-time polymerase chain reaction (qRT-PCR) throughout differentiation (mean ± SEM, *p < 0.05; **p < 0.01, ***p < 0.001).qRT-PCR of three corresponding undifferentiated and differentiated ICOs (patients 01, 10, and 12) at differentiation Day 15 (fold change, relative to GAPDH, mean ± SEM, two-way analysis of variance).(B) Western blot analysis of NTCP expression at indicated time point of differentiation, vinculin as expression control.NTCP expression is indicated by the intensity of 37 kDa bands (red box) (B, C) Immunofluorescence staining of differentiated ICO at Day 15, cross sectional views show distribution of NTCP (green), ZO1 (red), and DAPI (blue).Scale bar is 15 μm.(D, E) Immunofluorescence staining of ICOs with NTCP antibody and fluorescent tagged PreS1 throughout differentiation at ×20 magnification.(E) Zoom image from the inner square.

F
I G U R E 6 Human intrahepatic cholangiocyte organoids (ICOs) show drug responsiveness to entry inhibitor and JAK1/2 inhibitor.Drug treatment of ICOs infected with plasma hepatitis B virus (HBV) (genotype B) for 21 days.(A) Schematic timeline of experiment and quantitative real-time polymerase chain reaction (qRT-PCR) of infected ICOs, normalized to total RNA (two-way analysis of variance, mean ± SEM) for Myrcludex B and JAKi (Baricitinib).(B) Corresponding immunofluorescence for HBcAg in the same experiment.(C) Schematics of treatment timeline for two ICOs (patients 07 and 10) in a shorter time frame and qRT-PCR of infected ICOs, normalized to total RNA, expressed as mean ± SEM.F I G U R E 7 (See caption on next page).
LIM ET AL. | 13 of 16 expression of HBV pregenomic RNA, total RNA and cccDNA and extracellular HBV DNA and expression of HBeAg and HBsAg.Moreover, immunofluorescence imaging revealed cytoplasmic HBV core antigen expression in organoid cells.These results collectively indicate that human ICOs are a viable platform for the study of the molecular mechanisms of HBV replication and the cellular response in a primary cell setting.
et al., HBV infection in ICOs does not induce an appreciable innate response as assessed by monitoring ISG mRNA expression.It is possible F I G U R E 7 Human intrahepatic cholangiocyte organoids (ICOs) demonstrate a robust interferon-stimulated genes (ISG) response to interferonα and RNA mimics but not hepatitis B virus (HBV) infection.(A) Temporal fold induction of ISGs in undifferentiated ICOs and differentiated ICO at 6, 16, and 24 h poststimulation with interferon-a at 1000 U/mL (n = 2).(B) Fold induction of ISGs at 24 h poststimulation with two concentrations of interferon-α.(C) Fold induction of ISGs in differentiated ICO derived from three separate donors (patients 01, 10, and 12) at 24 h poststimulation with interferon-a.(D) Fold induction of viral sensing and antiviral effector ISGs in undifferentiated ICO an differentiated ICO at 24 h poststimulation with PolyI:C at 2 μg/mL and (E) following HBV infection among three ICOs at 1000 GEq/cell.mRNA expression relative to HPRT and normalized to uninfected controls at each specific time points (*p < 005; **p < 0.01, two-way analysis of variance, mean ± SEM).
that this observation may be due to low level of HBV infection in the ICO model.However, our assessment of ICO cytosolic RIG-I and endosomal TLR pathways with low dose PolyI:C or JAK-STAT pathway using interferon-α resulted in significant induction of the ISGs, IFITM1, ISG15, Viperin and CXCL10, similar to that of PHH, confirming their ability to respond at the innate immune level.Collectively, our study demonstrated a predominant cholangiocytelike transcriptomic signatures in ICO when cultured ex vivo.Despite this, advanced differentiation in vitro allows selective development of hepatocyte-like phenotypes with structural and metabolic changes essential for HBV infection.The differentiated ICOs are susceptible to infection with both cell culture and plasma derived HBV.Importantly, ICO infection model demonstrates significant donor variation in NTCP expression, HBV susceptibility, HBV replication dynamics, antiviral and ISG response (antiviral effectors), thus extends the application of ICOs in translational virology, particularly in assessing personalized host responses to antivirals.