Expansion, in vivo-ex vivo cycling and genetic manipulation of primary human hepatocytes

Primary human hepatocytes (PHH) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture and resistance to genetic manipulation have limited their use. Here we show that retrorsine improves human hepatocyte repopulation of chimeric mice to levels where poor donor PHH can be isolated for ex vivo cultures. Mouse-passaged (mp)PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months, as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized and then re-cultured as spheroids or re-transplanted to create highly humanized mice carrying a genetically altered hepatocyte graft. Together, these advances provide flexible tools for studying human liver disease and evaluating hepatocyte-targeted gene therapy approaches Overall design: Stranded RNA-seq libraries were prepared from cryopreserved commercial primary human hepatocytes (PHH-two samples) and mouse passaged primary human hepatocytes (mpPHH) from the same donor. The mpPHH were generated from two independed transplant experiments, and each sample was generated from a separate mouse: 2 mice in the first experiment and 3 mice in the second experiment.