Abstract
More than 300 million people worldwide are diagnosed with a chronic hepatitis B virus (HBV) infection. Nucleos(t)ide viral polymerase inhibitors are available on the market and can efficiently treat patients with chronic HBV. However, life-long treatment is needed as covalently closed circular DNA (cccDNA) persists in the hepatocyte nucleus. Hence, there is a high demand for novel therapeutics that can eliminate cccDNA from the hepatocyte nucleus and cure chronically infected HBV patients. The gold standard for in vitro HBV studies is primary human hepatocytes (PHHs). However, alternatives are needed due to donor organ shortage and high batch-to-batch variability. Therefore, human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are being explored as an in vitro HBV infection model. We recently generated hPSC lines that overexpress three transcription factors (HC3x) and that, upon differentiation in a high amino-acid supplemented maturation medium, generate a more mature hepatocyte progeny (HC3x-AA-HLCs). Here, we demonstrate that HBV can efficiently infect these HC3x-AA-HLCs, as was shown by the presence of HBV core (HBc) and surface antigens. A clear increasing release of HBV surface and e antigens was detected, indicating the formation of functional cccDNA. Moreover, back-titration of culture supernatant of HBV-infected HC3x-AA-HLCs on HepG2-NTCP cells revealed the production of novel infectious HBV particles. Additionally, an increasing number of HBc-positive HC3x-AA-HLCs over time suggests viral spreading is occurring. Finally, the HC3x-AA-HLC model was validated for use in antiviral drug studies using the nucleoside reverse-transcriptase inhibitor, lamivudine, and the HBV entry inhibitor, Myrcludex B.
Highlights
Hepatitis B virus (HBV) is a non-cytopathic virus that belongs to the family Hepadnaviridae and is transmitted via blood or sexual contact [1]
At the end of the hepatocyte differentiation, hESC and hiPSC HC3x-amino acids (AA)-hepatocyte-like cells (HLCs) expressed increased levels of the transcription factors (TFs) HNF1α, PROX1 and forkhead box protein A3 (FOXA3), to similar levels observed in Primary human hepatocytes (PHHs), indicating that the overexpression cassette was functional upon doxycycline induction from day 4 onwards (Figure 1B)
We assessed the levels of hepatocyte marker genes, such as ALB, AAT, and HNF4α, as well as the levels of NTCP, which is described as the entry receptor for hepatitis B virus (HBV) into the hepatocyte [9], during the differentiation process of hESC
Summary
Hepatitis B virus (HBV) is a non-cytopathic virus that belongs to the family Hepadnaviridae and is transmitted via blood or sexual contact [1]. Prophylactic vaccines are available on the market, almost 300 million people have a chronic HBV infection and are at a high risk of developing liver fibrosis; liver cirrhosis; and, eventually, hepatocellular carcinoma (HCC) [2,3]. NRTIs, such as lamivudine, entecavir, and tenofovir, inhibit viral replication; patients need life-long treatment as the covalently closed circular DNA (cccDNA). Treatment with pegylated IFNα is effective only in a subgroup of patients and may cause severe side effects [4]. Due to these limitations, there is still a high need for novel anti-HBV antivirals that can cure rather than just control HBV infection in chronically infected patients
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