Background and Aims: Hepatocyte transplantation is a potential therapy for acute and chronic liver diseases, but this approach has been limited by the availability of high-quality donor livers and well-characterized, functional hepatocytes. Given no methods exist to expand transplantable hepatocytes ex vivo, we hypothesized that an in vivo rat bioreactor could be used for the large-scale expansion of functional primary human hepatocytes for clinical use.

Method: An immune-deficient rat model of hereditary tyrosinemia type 1 (Fah-/-, Rag1-/-, Il2rg-/- [FRG] rats) was generated. Phenotypic, histological, and molecular characterization was performed on and off the protective drug NTBC. Transplantation of rodent and human hepatocytes were performed to determine in vivo repopulation kinetics and therapeutic efficacy. 

Results: FRG rats exhibited normal health while administered NTBC in the drinking water. FRG rats off NTBC displayed a failure-to-thrive phenotype resulting in liver and kidney damage that was associated with hypertyrosinemia and elevated succinylacetone. Transplantation of FAH+ rat hepatocytes, combined with NTBC cycling, provided selective pressure for FAH+ cells to expand and rescue the lethal phenotype. Next, immunodeficiency of FRG rats, needed for xenograft maintenance, was confirmed by flow cytometry which demonstrated severely reduced T, B, and NK cells in peripheral blood. Repopulation of FRG rat livers was achieved with multiple human hepatocyte donors based on human albumin ELISA (>5mg/ml) and FAH immunohistochemistry (>50%). Purified expanded human hepatocytes exhibited typical hepatocyte transcription profiles based on multiple analyses, including scRNA-Seq. Expanded cells retained their synthetic (e.g. albumin expression), metabolic (e.g. cytochrome P450 activity), and detoxification (e.g. ammonia clearance) functions. Finally, FRG rat-derived human cells demonstrated robust engraftment and expansion in a mouse model of hereditary tyrosinemia. Transplanted human hepatocytes normalized tyrosine and succinylacetone levels, preventing onset of liver failure, and demonstrating therapeutic efficacy of bioreactor-expanded human cells.

Conclusion: Development of the FRG rat bioreactor for large-scale expansion of primary human hepatocytes may provide the quantity of high-quality cells necessary for clinical testing of hepatocyte transplantation as an alternative to organ transplantation for severe liver diseases.