부산대학교 도서관

Hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs) are an attractive cell source for disease modeling and drug development. Although hepatic differentiation protocols from human pluripotent stem cells (hPSCs) have been well established, current methods have still limitations in homogenous generation of HLCs from various hPSC lines. In the present study, we established an efficient protocol to generate homogenous HLCs from 2 hESC and 3 hiPSC lines by optimizing definitive endoderm (DE) induction and hepatic specification stages. We examined the effect of small molecules regulating Wnt and PI3K signaling pathway and histone deacetylation on endoderm induction of hPSCs. The combination of GSK-3β inhibitor CHIR99021 and histone deacetylase inhibitor sodium butyrate enhanced Activin A-induced DE differentiation, which results in homogeneous generation of DE cells (> 90%) from all five hPSC lines. Furthermore, we showed that the treatment of BMP4 and TGF-β inhibitor SB431542 at hepatic specification stage efficiently directed DE cells into hepatic lineage, regardless of hPSC line variation. From our optimized protocol, we obtained highly homogenous and functional HLCs having matured hepatocyte phenotype from five independent hPSC lines. Therefore, established protocol can be used in generation of functional hepatocytes efficiently.