Salvianolic Acid B Enhances Hepatic Differentiation of Human Embryonic Stem Cells Through Upregulation of WNT Pathway and Inhibition of Notch Pathway

Hepatocytes differentiated from human embryonic stem cells (ESCs) could provide a powerful tool for enabling cell-based therapies, studying the mechanisms underlying human liver development and disease, and testing the efficacy and safety of pharmaceuticals. However, currently most in vitro protocols yield hepatocytes with low levels of liver function. In this study, we investigated the potential of Salvianolic acid B (Sal B), an active pharmaceutical compound present in Salvia miltiorrhiza, which has been shown to have an antifibrotic effect in previous studies, to enhance hepatocyte differentiation from human ESCs. After treatment with Sal B, albumin expression and secretion were consistently increased, indicating that Sal B could promote hepatocyte differentiation process. Expression of a large number of important phase 1 and 2 metabolizing enzymes and phase 3 transporters was also increased in treated cells, indicating an enhanced biotransformation function. Our investigations further revealed the activation of Wnt pathway in treated cells, as determined by upregulation of Wnts, which increased amounts of nuclear beta-catenin. This increased nuclear beta-catenin led in turn to the enhanced expression of T cell factor (TCF) 3 and lymphoid enhancer-binding factor (LEF) 1 which upregulated their downstream targets, cyclin D1 and c-Myc. Notch receptors (Notch1, Notch3), Notch ligand (Jagged2), and Notch receptor targets [hairy and enhancer of split (Hes) 1, 5] were downregulated in treated cells, suggesting that Notch pathway was inhibited. Consistent with the inhibition of Notch pathway, expression of cholangiocyte marker, CK7, was significantly reduced by treatment with Sal B. Numb, a direct transcriptional target of Wnt pathway and a negative regulator of Notch pathway, was upregulated, consistent with activation of Wnt signaling and suppression of Notch signaling. In conclusion, our study demonstrated that Sal B enhanced hepatocyte differentiation from human ESCs through activation of Wnt pathway and inhibition of Notch pathway. Therefore, this study suggests that Sal B can be used as a potential agent to generate more mature hepatocytes for cell-based therapeutics and pharmaceutical studies.