Controlled Functional Zonation of Hepatocytes In Vitro by Engineering of Wnt Signaling.

ABSTRACT

Key liver functions, including protein synthesis, carbohydrate metabolism, and detoxification, are performed by specific populations of hepatocytes that are defined by their relative positions within the liver lobules. On a molecular level, the functional heterogeneity with periportal and pericentral phenotypes, so-called metabolic liver zonation, is mainly established by a gradient of canonical Wnt signaling activity. Since the relevant physiological cues are missing in in vitro liver models, they fail to reflect the functional heterogeneity and thus lack many liver functions. We synthetically re-engineered Wnt signaling in murine and human hepatocytes using a doxycycline-dependent cassette for externally controlled digital expression of stabilized ß-catenin. Thereby, we achieved adjustable mosaic-like activation of Wnt signaling in in vitro-cultured hepatocytes that was resistant to negative-feedback loops. This allowed the establishment of long-term-stable periportal-like and pericentral-like phenotypes that mimic the heterogeneity observed in vivo. The in vitro-zonated hepatocytes show differential expression of drug-metabolizing enzymes and associated differential toxicity and higher levels of autophagy. Furthermore, recombinant adeno-associated virus and hepatitis C virus preferentially transduce the pericentral-like zonation phenotype, suggesting a bias of these viruses that has been unappreciated to date. These tightly controlled in vivo-like systems will be important for studies evaluating aspects of liver zonation and for the assessment of drug toxicity for mouse and man.