Long-term HBV infection of engineered cultures of induced pluripotent stem cell-derived hepatocytes.

Abstract

Background: HBV infects ~257 million people and can cause hepatocellular carcinoma. Since current drugs are not curative, novel therapies are needed. HBV infects chimpanzee and human livers. However, chimpanzee studies are severely restricted and cost-prohibitive, while transgenic/chimeric mouse models that circumvent the species barrier lack natural HBV infection and disease progression. Thus, in vitro human models of HBV infection are useful in addressing the above limitations. Induced pluripotent stem cell-derived hepatocyte-like cells mitigate the supply limitations of primary human hepatocytes and the abnormal proliferation/functions of hepatoma cell lines. However, variable infection across donors, deficient drug metabolism capacity, and/or low throughput limit iHep utility for drug development.

Methods: We developed an optimal pipeline using combinations of small molecules, Janus kinase inhibitor, and 3',5'-cAMP to infect iHep-containing micropatterned co-cultures (iMPCC) with stromal fibroblasts within 96-well plates with serum-derived HBV and cell culture-derived HBV (cHBV). Polyethylene glycol was necessary for cell-derived HBV but not for serum-derived HBV infection.

Results: Unlike iHep monocultures, iMPCCs created from 3 iHep donors could sustain HBV infection for 2+ weeks. Infected iMPCCs maintained high levels of differentiated functions, including drug metabolism capacity. HBV antigen secretion and gene expression patterns in infected iMPCCs in pathways such as fatty acid metabolism and cholesterol biosynthesis were comparable to primary human hepatocyte-MPCCs. Furthermore, iMPCCs could help elucidate the effects of interferons and direct-acting antiviral drugs on the HBV lifecycle and any hepatotoxicity; iMPCC response to compounds was similar to primary human hepatocyte-MPCCs.

Conclusions: The iMPCC platform can enable the development of safe and efficacious drugs against HBV and ultimately help elucidate genotype-phenotype relationships in HBV pathogenesis.