Enhanced metabolizing activity of human ES cell-derived hepatocytes using a 3D culture system with repeated exposures to xenobiotics.

Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the cell-based therapy of liver disease and the development of effective in vitro toxicity screening tools. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or in vitro applications. Here, we describe a highly optimized hepatic differentiation protocol, which produces > 90% (BGO1 and CHA15) albumin-positive HLCs with no purification process from human embryonic stem cell lines. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating 3D spheroidal aggregate of HLCs, compared with 2D HLCs. The 3D differentiation method increased expression of nuclear receptors (NRs) that regulate the proper expression of key hepatic enzymes. Furthermore, significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids, compared with 2D HLCs. HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated further functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic metabolizing ability of stem cell-derived HLCs as a cell source for in vitro high-throughput hepatotoxicity models.