key: cord-1056251-k28130h7
authors: Lui, V. C.-H.; Hui, K. P.-Y.; Babu, R. O.; Yue, H.; Chung, P. H.-Y.; Tam, P. K.-H.; Chan, M. C.-W.; Wong, K. K.-Y.
title: Human liver organoid derived intra-hepatic bile duct cells support SARS-CoV-2 infection and replication and its comparison with SARS-CoV
date: 2021-02-15
journal: nan
DOI: 10.1101/2021.02.10.21251458
sha: 16dcaaa8e5fdff31d1b76222f22ef1c019fd3f10
doc_id: 1056251
cord_uid: k28130h7

BackgroundAlthough the main route of infection for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the respiratory tract, liver injury is also commonly seen in many patients, as evidenced by deranged parenchymal liver enzymes. Furthermore, patients with severe liver disease have been shown to have higher mortality. Overall, the mechanism behind the liver injury remains unclear. Approach and resultsWe showed that intra-hepatic bile duct cells could be grown using a human liver organoid platform. The cholangiocytes were not only susceptible to SARS-CoV-2 infection, they also supported efficient viral replication. We also showed that SARS-CoV-2 replication was much higher than SARS-CoV. ConclusionOur findings suggested direct cytopathic viral damage being a mechanism for SARS-CoV-2 liver injury.

Coronavirus disease-2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has rapidly become a worldwide pandemic. As of January 2021, there have been 83,322,449 confirmed cases of COVID-19 and 1,831,412 deaths worldwide [1] .

SARS-CoV-2 is a single-strand positive-sense RNA virus, belongs to the beta coronavirus family, which enters cells through the Angiotensin Converting Enzyme 2 (ACE2) receptor [2] . SARS-CoV-2 interacts with the host cells by first attaching its spike protein to ACE2 on host cells, and then gains entry through hemagglutinin cleavage by host cell protease Transmembrane protease, serine 2 (TMPRSS2) [3, 4] . Human-to-human transmission for SARS-CoV-2 is efficient. While the respiratory tract is a common route of disease transmission [5, 6] , the gastrointestinal tract has also been shown as another possible route of viral transmission [7] [8] [9] . Understanding the cellular/tissue tropism and the route of infection of SARS-CoV-2 virus is essential in overall patient management and infection control.

Liver damage is often identified as a typical occurrence in COVID-19 patients, and 58%-78% of COVID-19 patients were shown to exhibit various degrees of liver injury [10] . Some COVID-19 patients have elevated levels of liver enzymes such as aspartate amino-transferase (AST), alanine aminotransferase (ALT) levels, and gamma-glutamyl transferase (GGT), while some patients have higher overall bilirubin levels and lower serum albumin [11] [12] [13] . Indeed, elevated AST, ALT, and total bilirubin levels but lower serum albumin levels are correlated with higher death rate [6] , and have been found in the severe group of COVID-19 patients [14] . Moreover, activation of coagulation and fibrinolysis accompanied by thrombocytopenia was observed in severe COVID-19 cases [15] . Autopsy examinations of a small number of COVID-19 patients have provided conclusive evidence of secondary liver injury [16, 17] . Liver damage can be aggravated by the increase of COVID-19 infection severity, which indicates that the degree of liver damage may serve as an indicator of COVID-19 progression. Nonetheless, the mechanism of liver injury is poorly understood and may be due to direct viral hepatitis, bystander systemic inflammatory response or complications of drug treatment.

Angiotensin converting enzyme 2 (ACE2), the protein through which SARS-CoV-2 gains entry, is abundantly expressed on many cells, including liver cells, bile duct cells and liver endothelial cells. ACE2 expression levels in bile duct cells is slightly higher than those in hepatocytes and is All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.10.21251458 doi: medRxiv preprint comparable with alveolar epithelial type II cells [18] . Given that bile duct cells play an important role in immune defense and liver regeneration, their impairment may serve as a major cause of virus-induced liver injury in COVID-19 patients [19] . Although typical coronavirus particles characterized by spiked structures in the cytoplasm of hepatocytes have been identified [20] , the susceptibility of bile duct cells to SARS-CoV-2 infection is yet to be confirmed.

An organoid is a miniaturized and simplified version of an organ produced in vitro in 3dimension. It shows realistic micro-anatomy and retain the biology of individual tissues. Lung and gut organoids have been successfully used to demonstrate SARS-CoV-2 infection [21, 22] .

Recently, human liver ductal organoids were shown to express ACE2 and TMPRSS2, and were permissive to SARS-CoV-2 infection [23] . The extra-and intra-hepatic bile duct cells are derivatives of different progenitors, in that extra-hepatic ducts arise from a common SOX17+/PDX1+ pancreatobiliary progenitor, while the intra-hepatic ducts arise from CK19+/AFP+ hepatoblasts [24] . Furthermore, the cellular properties and functions of extra-and intra-hepatic cholangiocytes (bile duct cells) are very different. It remains to be investigated if intra-hepatic bile ducts are susceptible to SARS-CoV-2 infection and support viral replication.

Recently, we have shown that the human liver tissue organoids derived from EPCAM+ve cells of liver biopsies are from the hepatoblast progenitor (CK19+) lineage rather than from the pancreatobiliary (extrahepatic ducts) progenitor (SOX17+/PDX1+) lineage, and thus recapitulates the intrahepatic cholangiocyte development [25] .

In this study, we utilized human liver tissue derived organoids from hepatoblast progenitor as an ex-vivo tool to investigate the infection, tropism and replication competence of SARS-CoV-2 on intra-hepatic bile ducts and compared to SARS-CoV. We showed that liver tissue derived organoids consisted of intra-hepatic cholangiocytes and hepatoblasts as major cell types, which expressed ACE2 and TMPRSS2 and could be rapidly infected by both SARS-CoV-2 and SARS-CoV. SARS-CoV-2 was shown to have a much higher replication rate than SARS-CoV.

Pediatric liver tissues were obtained from non-tumour margin of hepatoblastoma (HB). Liver biopsies were obtained during operations with full informed consent from parents or patients, All rights reserved. No reuse allowed without permission.

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Wedge liver biopsies (1-2 cm 3 ) from the non-tumor margin of pediatric patients (n=4) with hepatoblastoma (HB)). Liver tissues were minced in cold wash medium (Advanced DMEM/F12; 1% GlutaMAX; 1% FBS; 1% Penicillin/Streptomycin (P/S)) and digested in digestion medium 

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Single cells from organoids were prepared. The organoid medium along with matrix gel containing organoids was transferred to a 15 ml tube, 1-2 ml cold Advanced DMEM/F12 was added and the mixture was incubated on ice for 10 min to dissolve matrix gel before centrifugation (300g; 5 min). The supernatant was aspirated until the organoid pellet and a layer of matrix gel remained. 1 ml 5X TrypLE Express (12604013; GIBCO) was added, mixed well and incubated at 37°C for 5 min. 1 ml FBS was added to the mixture, which was pipetted up and down for 40-50 times with a small circumference opening glass pipette (diameter 0.3-0.5 mm) to efficiently dissociate organoids into single cells. 5-10 ml cold Advanced DMEM/F12 medium was added, and the suspension was passed through a 30 µm strainer before centrifugation (300 g; 5 min) at 4°C. The supernatant was aspirated until only the pellet remained and the cells were The CellRanger (10X Genomics) analysis pipeline was used to generate digital gene expression matrix (UMI counts per gene per cell) from sequencing data by aligning to the human genome.

The raw gene expression matrix was filtered, normalized and clustered using standard Seurat package procedures 16. The low quality cells were removed from the analysis using the following thresholds: cells with a very small library size or UMI counts per cell (nUMI >1500), genes detected per cell (nGene >1000), UMIs vs. genes detected (log10GenesPerUMI >0.8), mitochondrial counts ratio (mitoRatio <0.1). Cell-cycle phases were predicted using a function included in Seurat that scores each cell based on expression of canonical marker genes for S and G2/M phases, this was used to regress out the cell-cycle effects from the downstream analysis.

Clusters were visualized using uniform manifold approximation and projection (UMAP) as All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.10.21251458 doi: medRxiv preprint implemented in Seurat. First 13 PCs were selected for UMAP based on the points where the principal components cumulatively contribute 90% of variation associated with entire data. The cell-type identities for each cluster in UMAP were determined using presence of known marker genes in each cluster. 

The 3D liver organoids were sheared mechanically using syringe to expose the apical surface to the virus inoculum. Around 100-200 organoids were infected with each coronavirus at 5×10 5 TCID 50 /ml for 1 h at 37°C. The organoids were washed three times with culture medium, reembedded in Matrigel at the same conditions with the same growth medium and incubated at 37°C with 5% CO 2 . The viral titers in the culture supernatants were measured at 1, 24, 48, and 72 h after infection using the TCID 50 assay in Vero-E6 cells. Cell lysates were collected 72h after infection to assess mRNA expression of cytokines. Organoids were fixed 72 h after infection in paraformaldehyde for immunofluorescent staining.

Organoids in matrigel (from 2 HB livers) were fixed in 4% paraformaldehyde (w/v) in PBS (phosphate-buffered saline, pH 7.2) for 48 h at 4°C, dehydrated in graded series of alcohol, and cleared in xylene before being embedded in paraffin. Sections (6 µm in thickness) were prepared and mounted onto TESPA-coated microscope glass. Sections were dewaxed in xylene, hydrated in a graded series of alcohol and finally in distilled water. Antigen retrieval was performed by incubation of slides in Citrate buffer (pH 6.0) at 95°C for 10 minutes. After blocking in PBS-T (PBS with 0.1% Triton) supplemented with 1% Bovine Serum Albumin for 1 h at room All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted February 12, 2021. Table 1 (supplementary data). After PBS-T washings, sections were mounted in DAPI-containing anti-fade mounting fluid. Images were taken with Nikon Eclipse 80i microscope mounted with a SPOT RT3 microscope digital camera under fluorescence illumination. Photos were compiled using Adobe Photoshop CS6.

The RNA of infected cells was extracted at 72 h post infection using a MiniBEST universal RNA extraction kit (Takara Biotechnology). RNA was reverse-transcribed by using oligo-dT primers with RT-PCR kit (Takara). mRNA expression of target genes was performed using an ABI ViiA TM 7 real-time PCR system (Applied Biosystems). The gene expression profiles of cytokines and chemokines were quantified and normalized with β-actin as previously described [26] .

Statistical analysis was done using GraphPad Prism software version 9. Experiments with the human organoids were performed independently in two different donors each with triplicate wells. Viral titers and area under the curve (AUC) derived from viral titers and mRNA expression were compared using one-or two-way ANOVA with Tukey's multiple comparisons test. Mock infected organoids served as negative controls. Results shown in figures are the calculated mean and standard deviation of mean. Differences were considered significant at p < 0.05.

To investigate if human liver derived organoids can be used to establish an ex vivo SARS-CoV-2 infection model for intra-hepatic bile ducts, we first determined whether the organoid culture could preserve the cholangiocytes expressing ACE2 and TMPRSS2 ex vivo. We performed single-cell RNA sequencing (scRNA-seq) analysis of human liver organoids from 2 patients to interrogate the transcriptomic signatures of cells in human liver organoids. The organoids used in All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 12, 2021. in hepatoblasts, it is worth mentioning that the ACE2+ cells were co-expressing TMPRSS2 (159 out of 233) ( Figure 1B and 1C) , making this cell population potentially highly vulnerable to SARS-CoV-2 infection. Interestingly, these same cells were also found to express another serine protease, TMPRSS4. Immunostaining further verified the presence of ACE2+, TMPRSS2+ cholangiocytes (CK19+) in human liver ductal organoids ( Figure 1D ). Taken together, our data demonstrate that human liver tissue derived organoid preserves the human-specific ACE2+/TMPRSS2+ population of intra-hepatic cholangiocytes.

Next, we examined the susceptibility of human liver ductal organoids to SARS-CoV-2. We infected liver organoids with the SARS-CoV-2, and the SARS-CoV. The liver organoids from two individuals were inoculated with viruses for 1 h, re-embedded in Matrigel and cultured for 48 and 72 hrs. We performed immunostaining to identify the virus-positive cholangiocytes 48 h and 72 h post-infection (Figure 2 and data not shown) . The expression of SARS-CoV nucleocapsid protein (Sco-Np) was detected in patchy areas of human liver organoids infected with SARS-CoV-2 and SARS-CoV, whereas no signal was found in uninfected control (Mock, Figure 2A ).

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The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.10.21251458 doi: medRxiv preprint Virus tropism and replication competence of SARS-CoV-2 with SARS-CoV were compared.

Here, we showed that the viral titres of SARS-CoV-2 increased starting at 24h and with more than 2 log increase from 24h to 72h in bile duct organoids. SARS-CoV-2 replicated significantly higher than that of the SARS-CoV, with about 1 log increase in viral titer along at all-time points ( Figure 2B) . When comparing the area under the curve (AUC) derived from the viral titers from 24h to 72h, similar observations were found, in that SARS-CoV-2 had a significantly higher viral replication competence than SARS-CoV ( Figure 2C ). These data demonstrate that human bile duct organoids are susceptible to SARS-CoV-2 infection and support robust viral replication.

COVID-19, which is caused by SARS-CoV-2, has become a significant global pandemic since January 2020. Now one year on, there have been over 70,000,000 confirmed cases and 2,000,000 deaths worldwide [1] . Although the main route of SARS-CoV 2 infection remains the respiratory tract, with infected patients displaying symptoms of severe respiratory compromise,

the digestive system has been reported to be another potential portal of viral infection [7] [8] [9] 21] .

Indeed, ACE2 has now been shown to be abundantly expressed in intestinal cells and SARS-CoV-2 can invade and enter via the gastrointestinal tract [27, 28] .

In many COVID-19 patients, liver damage, as typified by deranged liver function tests, is seen during the SARS-CoV-2 infection [10, 11] . Despite this, the mechanisms of liver injury remain largely undetermined. It has been postulated that direct viral infection, drug cytotoxicity, and a bystander inflammatory immune response may play a role. Given the fact that ACE2 has also been confirmed to be present on liver cells [29] and that the liver is connected to the gastrointestinal system via the biliary tract, it is indeed a possibility that the liver may also be a potential target for SARS-CoV-2 infection. Previous report on the detection of SARS CoV in liver tissues by RT-PCR provided indirect evidence on the susceptibility of liver cells to SARS-CoV-2 infection [30] . This finding was supported by a recent report which showed the presence of SARS-CoV-2 viral particles in hepatocyte cytoplasm in two COVID-19 patients [20] . Indeed, Zhao et al recently found that SARS-CoV-2 infection impaired the barrier and bile acid transporting functions of cholangiocytes [23] . In our study, we further confirmed that human liver organoid derived intra-hepatic bile duct cells provided an excellent ex vivo model for studying SARS-CoV-2 infection. Cholangiocytes were found to co-express both ACE2 and All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted February 12, 2021. ; https://doi.org/10.1101/2021.02.10.21251458 doi: medRxiv preprint TMPRSS2 and they were highly susceptible to coronavirus infection. One interesting finding was the identification of TMPRSS4 expression on these cells. In a recent study using an enteroid model, TMPRSS4 protein was shown to be another important serine protease which could work in synergy with TMPRSS2 to release SARS-CoV-2 into the host cell, after viral binding to ACE2 [31] . Thus, similar viral interaction and kinetics with intra-hepatic bile duct cells may be expected. Further experiments are underway to determine this. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. h. Data are presented as mean ± standard deviation (SD). Statistical significances compared among viruses were calculated using one-way ANOVA with Tukey's multiple comparisons test. ***p<0·001.

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