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

Abstract

Primary human hepatocytes (PHHs) 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 the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These 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 recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

A Combination of Human Broadly Neutralizing Antibodies against Hepatitis B Virus HBsAg with Distinct Epitopes Suppresses Escape Mutations

“…Both supernatant and cells were harvested 7 days after infection for analysis. Neutralization assays in primary human hepatocytes were performed as above using hepatocytes from livers of highly humanized mice that were harvested and seeded on collagen-coated plates in hepatocyte defined medium (Corning) (Michailidis et al, 2020).…”

Human hepatocyte PNPLA3 148M exacerbates rapid non-alcoholic steatohepatitis development in chimeric mice

“…To investigate how the human graft in liver chimeric mouse models responds to a hypercaloric diet, we first created humanized mice by transplanting primary human hepatocytes (PHH) into preconditioned immunocompromised Fah -/-NOD Rag1 -/-Il2rg null (FNRG) mice as described previously. 24,25 Cycling the protective drug nitisinone 26 resulted in at least 50% humanization after which mice were subjected to an ad libitum Western-style diet (WD), which consists of 60% fat in diet and 10% sucrose in drinking water (Fig 1a). WD feeding did not result in graft loss as determined by persistently high [27][28][29] human serum albumin (hAlb) levels (Suppl Fig S1a).…”

Public broadly neutralizing antibodies against hepatitis B virus in individuals with elite serologic activity

“…The 50% inhibitory concentration (IC 50 ) values were calculated based on HBsAg/HBeAg ELISA or immunofluorescence staining for HBcAg expression (Michailidis et al, 2017) (Figure 5C). Neutralizing activity was further verified by in vitro neutralization assays using primary human hepatocytes (Michailidis et al, 2020) (Figure 5C and 5D). Fourteen of the 20 antibodies tested showed neutralizing activity with IC 50 values as low as 5 ng/ml (Figure 5C).…”