Controlled functional zonation of hepatocytes in vitro by engineering of Wnt signalling.

Key liver functions including protein synthesis, carbohydrate metabolism and detoxification are performed by specific populations of hepatocytes which are defined by their relative position 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-signalling 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-signalling in murine and human hepatocytes using a Doxycycline dependent cassette for externally controlled, digital expression of stabilised β-catenin. Thereby, we achieved adjustable mosaic-like activation of Wnt-signalling in in vitro cultured hepatocytes that was resistant towards negative feedback loops. This allows the establishment of long-term stable periportal and pericentral phenotypes that mimick the heterogeneity observed in vivo. The in vitro zonated hepatocytes show differential expression of drug-metabolising enzymes and associated differential toxicity and higher levels of autophagy. Further, recombinant adeno-associated virus and hepatitis C virus preferentially transduce the pericentral zonation phenotype suggesting a so far unappreciated bias of these viruses. These tightly controlled in vivo-like systems will be important for studies evaluating aspects of liver zonation and for assessment of drug toxicity for mouse and man.