Abstract

Significance Major obstacles for using human hepatocytes to study hepatitis B virus (HBV) pathobiology are rapid loss of hepatocyte function after plating and the variability between hepatocyte donors. We show that micropatterning and coculturing of primary human hepatocytes with fibroblasts (MPCC format) maintains prolonged infection that is restricted by the innate immune response, and can be further boosted by suppression of this response. To address the problem of donor variability, we show that induced pluripotent stem cells (iPSC) differentiated into hepatocyte-like cells support HBV infection in a differentiation-dependent manner. Our study opens an avenue for using these systems to study virus–host interactions and test antiviral drugs, and suggests HBV permissiveness as a surrogate reporter to assess the degree of differentiation of candidate iPSC-derived hepatocyte-like cells.