Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes

Human pluripotent stem cells (hPSCs) represent a novel source of hepatocytes for drug metabolism studies and cell-based therapy for the treatment of liver diseases. These applications are, however, dependent on the ability to generate mature metabolically functional cells from the hPSCs. Reproducible and efficient generation of such cells has been challenging to date, owing to the fact that the regulatory pathways that control hepatocyte maturation are poorly understood. Here, we show that the combination of three-dimensional cell aggregation and cAMP signaling enhance the maturation of hPSC-derived hepatoblasts to a hepatocyte-like population that displays expression profiles and metabolic enzyme levels comparable to those of primary human hepatocytes. Importantly, we also demonstrate that generation of the hepatoblast population capable of responding to cAMP is dependent on appropriate activin/nodal signaling in the definitive endoderm at early stages of differentiation. Together, these findings provide new insights into the pathways that regulate maturation of hPSC-derived hepatocytes and in doing so provide a simple and reproducible approach for generating metabolically functional cell populations.