LAUSR

Background and Aims: Cell fate can be directly reprogrammed from accessible cell types (e.g., fibroblasts) into functional cell types by exposure to small molecule stimuli. However, no chemical reprogramming method has been reported to date that successfully generates functional hepatocyte‐like cells that can repopulate liver tissue, casting doubt over the feasibility of chemical reprogramming approaches to obtain desirable cell types for therapeutic applications. Approach and Results: Here, through chemical induction of phenotypic plasticity, we provide a proof‐of‐concept demonstration of the direct chemical reprogramming of mouse fibroblasts into functional hepatocyte‐like cells using exposure to small molecule cocktails in culture medium to successively stimulate endogenous expression of master transcription factors associated with hepatocyte development, such as hepatocyte nuclear factor 4a, nuclear receptor subfamily 1, group I, member 2, and nuclear receptor subfamily 1, group H, member 4. RNA sequencing analysis, metabolic assays, and in vivo physiological experiments show that chemically induced hepatocytes (CiHeps) exhibit comparable activity and function to primary hepatocytes, especially in liver repopulation to rescue liver failure in fumarylacetoacetate hydrolase−/−recombination activating gene 2−/−interleukin 2 receptor, gamma chain−/− mice in vivo. Single‐cell RNA‐seq further revealed that gastrointestinal‐like and keratinocyte‐like cells were induced along with CiHeps, resembling the activation of an intestinal program within hepatic reprogramming as described in transgenic approaches. Conclusions Our findings show that direct chemical reprogramming can generate hepatocyte‐like cells with high‐quality physiological properties, providing a paradigm for establishing hepatocyte identity in fibroblasts and demonstrating the potential for chemical reprogramming in organ/tissue repair and regeneration therapies.