The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, HBeAg, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV infected patients.

The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system.Here we interrogate the potential of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We show that organoids derived from HBV-infected patients display an aberrant early cancer gene signature, which clusters with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue. Furthermore, we demonstrate HBV infection in healthy donor liver organoids after challenge with recombinant virus or HBV infected patient serum. Ex vivo infected liver organoids produced cccDNA, expressed intracellular HBV RNA and proteins, and produced infectious HBV. HBV replication supported by ex vivo infected liver organoids was blocked by treatment with Tenofovir, highlighting the potential of this model system as a primary differentiated hepatocyte platform for HBV drug screening. Interestingly, transgenic organoids exogenously over expressing the HBV receptor NTCP by lentiviral transduction are not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. Finally, we generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription.

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Human mesenchymal stem cells (MSCs), a promising source of stem cells for regenerative medicine, have different morphological and functional characteristics. Carbohydrate moieties on the cell surface play an important role, including cell-cell interaction and cell recognition.