Liver Portal Fibroblasts Induce the Functions of Primary Human Hepatocytes In Vitro

In vitro human liver models are critical to mitigate the species-specific differences observed for toxicology, disease modeling, and regenerative medicine. Interactions with mesenchyme (i.e., fibroblasts), as in liver development, can promote the survival and phenotypic functions of primary human hepatocytes (PHHs) in culture; however, the use of liver-derived fibroblasts in such co-cultures remains elusive. Portal fibroblasts (PFs) in and around the portal liver triad influence bile duct formation during liver development, but their role in regulating homeostatic hepatic functions is unknown. Here, we show that human liver PFs, but not hepatic stellate cells, induced long-term and high levels of phenotypic functions in PHHs within industry-standard micropatterned co-cultures and spheroids. While PF-conditioned media induced some hepatic functions, partly via insulin-like growth factor binding protein-5 signaling, direct contact co-culture was necessary to induce optimal functional levels. Inhibiting Notch signaling reduced progenitor-like characteristics of PHHs and further enhanced their functions in co-cultures. Lastly, inhibiting transforming growth factor-β signaling suppressed the overgrowth and inflammation in higher passage PFs while enabling similar inductive effects on PHH functions as with the lower passage PFs. Overall, this work demonstrates a unique role for PFs in modulating hepatic functions and provides all-human and all-liver co-culture strategies for applications.