Highly efficient and expedited hepatic differentiation from human pluripotent stem cells by pure small-molecule cocktails

The advent of human-induced pluripotent stem cells holds great promise for producing ample individualized hepatocytes. Although previous efforts have succeeded in generating hepatocytes from human pluripotent stem cells in vitro by viral-based expression of transcription factors and/or addition of growth factors during the differentiation process, the safety issue of viral transduction and high cost of cytokines would hinder the downstream applications. Recently, the use of small molecules has emerged as a powerful tool to induce cell fate transition for their superior stability, safety, cell permeability, and cost-effectiveness. In the present study, we established a novel efficient hepatocyte differentiation strategy of human pluripotent stem cells with pure small-molecule cocktails. This method induced hepatocyte differentiation in a stepwise manner, including definitive endoderm differentiation, hepatic specification, and hepatocyte maturation within only 13 days. The differentiated hepatic-like cells were morphologically similar to hepatocytes derived from growth factor-based methods and primary hepatocytes. These cells not only expressed specific hepatic markers at the transcriptional and protein levels, but also possessed main liver functions such as albumin production, glycogen storage, cytochrome P450 activity, and indocyanine green uptake and release. Highly efficient and expedited hepatic differentiation from human pluripotent stem cells could be achieved by our present novel, pure, small-molecule cocktails strategy, which provides a cost-effective platform for in vitro studies of the molecular mechanisms of human liver development and holds significant potential for future clinical applications.