AbstractThe ability to generate integration-free induced hepatocyte-like cells (iHeps) from somatic fibroblasts has the potential to advance their clinical application. Here, we have generated integration-free, functional and expandable iHeps from mouse somatic fibroblasts. To elicit this direct conversion, we took advantage of an oriP/EBNA1-based episomal system to deliver a set of transcription factors, Gata4, Hnf1a and Foxa3, to the fibroblasts. The established iHeps exhibit similar morphology, marker expression and functional properties to primary hepatocytes. Furthermore, integration-free iHeps prolong the survival of fumarylacetoacetate-hydrolase-deficient (Fah^−/−) mice after cell transplantation. Our study provides a novel concept for generating functional and expandable iHeps using a non-viral, non-integrating, plasmid-based system that could facilitate their pharmaceutical and biomedical application.