Assessment of HBV infection and inter‐host cellular responses using intrahepatic cholangiocyte organoids

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 & C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT‐PCR (RNA, 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 development of personalized drug testing platform for antivirals.


| INTRODUCTION
Chronic hepatitis B infection is the leading cause of liver cirrhosis and hepatocellular carcinoma 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 hepatitis B virus (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. 3 Ectopic expression of NTCP, Na-taurocholate cotransporting polypeptide (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. 4 Importantly, 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. 5 They 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 stemcell 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. 5 This quality is particularly important for potential clinical applications in the area of autologous transplantation. More recently, Crignis et al. described a detailed protocol to achieve HBV infection and demonstrate HBV full lifecycle in this ex vivo ICO model. 6,7 In 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 NTCP 8 and structural changes essential for productive HBV trafficking and export. 9 More 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 ICOs 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. 5

| 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). 10 Primer sequences are provided in the Supporting Information: 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), HepAD38 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 000xg 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.

| Quantification of HBV RNA & DNA
Extracted cellular RNA was quantified using Nanodrop 2000 (Thermo Fisher Scientific) and diluted with nuclease free water to 10 ng/µL. 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 11 and ngsReports, 12,13 and then aligned to the GRCh37.p13 version of the human genome 14 using the transcriptome algorithm STAR. 15 After 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 16 and edgeR, 17 using mean−variance relationship estimates of the log-counts from the limma voom method 18 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. were performed using Imaris software (Bitplane). Immunoblotting was performed as previously described. 19 All primary and secondary antibodies used are provided in Supporting Information:

| Statistical analysis
For qRT-PCR analysis, differential expression of genes between two or more groups were compared using two-way 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: 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

| 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 post differentiation 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 Days 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). Myrcludex B did not as replication had been established ( Figure 6A).

| ICOs demonstrate donor dependent variation in permissiveness to HBV infection
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 ( Figure 6C).

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. The data that supports the findings of this study are available in the supplementary material of this article.