An efficient method for differentiation of human induced pluripotent stem cells into hepatocyte-like cells retaining drug metabolizing activity

Abstract

The use of human induced pluripotent stem (iPS) cells would be of great value for a variety of applications involving drug development studies. Several reports have been published on the differentiation of human iPS cells into hepatocyte-like cells; however, the cells were insufficient for application in drug metabolism studies. In this study, we aimed to establish effective methods for differentiation of human iPS cells into hepatocytes. Two human iPS cell lines were differentiated by addition of activin A, dimethyl sulfoxide, hepatocyte growth factor, oncostatin M, and dexamethasone. The differentiated cells expressed hepatocyte markers and drug-metabolizing enzymes, revealing that the human iPS cells were differentiated into hepatocyte-like cells. Expression of CYP3A4 and UGT1A1 mRNAs increased with treatment with typical inducers of the enzymes, and the response of the cells against the inducers was similar to that of human hepatocytes. Furthermore, the drug-metabolizing activity of CYP3A4, as monitored by testosterone 6β-hydroxylase activity, was elevated by these inducers. In conclusion, we established methods for differentiation of hepatocyte-like cells expressing drug metabolizing activity from human iPS cells. The hepatocyte-like cells derived from human iPS cells will be useful for drug metabolism studies.

Fig. 1.. Differentiation into hepatocytes from 2 human iPS cell lines

Two human iPS cell lines (Fetch and Lollipop) were differentiated into endoderm cells by addition of 100 ng/mL activin A for 5 days, and then into hepatocytes by the addition of 1% DMSO for 7 days. The hepatocytes were then matured by the addition of 10 ng/mL HGF, 20 ng/mL OSM, and 10⁻⁷ M DEX for 9 days. For the final 4 days, the cells were cultured in modified Lanford medium alone, without HGF, OSM, or DEX.

Fig. 2.. Expression levels of liver marker protein mRNAs

The expression levels of HNF4α, ALB, and AFP mRNAs in undifferentiated human iPS cells (i) and hepatocyte-like cells differentiated from two human iPS cell lines (Fetch and Lollipop) were analyzed using real-time PCR. Collagen I (c) or Matrigel (m) was used for the differentiation as the extracellular matrix. A, B, and C represent HepG2 cells, human adult liver, and hepatocytes, respectively, as positive controls. Each bar represents the mean ± SD from triplicate experiments. Values were normalized to the level of GAPDH mRNA. The graph represents the relative gene expression level when the level in the liver was taken as 1. nd, not detected.

Fig. 3.. Expression levels of CYP mRNAs

The expression levels of CYP1A2, CYP1B1, CYP2C9, CYP3A4, CYP3A5, and CYP3A7 mRNAs in undifferentiated human iPS cells (i) and hepatocyte-like cells differentiated from 2 human iPS cell lines (Fetch and Lollipop) were analyzed using real-time PCR. Collagen I (c) or Matrigel (m) was used for the differentiation as the extracellular matrix. A, B, and C represent HepG2 cells, human adult liver, and hepatocytes, respectively, as positive controls. Each bar represents the mean ± SD from triplicate experiments. Values were normalized to the levels of GAPDH mRNA. The graphs represent the relative gene expression level when the level in the liver was taken as 1. nd, not detected.

Fig. 4.. Microarray analysis of phase I and II enzymes

Human iPS cells (Fetch) were differentiated into hepatocyte-like cells. After differentiation, total mRNA was extracted from the cells. The expression levels of phase I and II enzymes were analyzed by microarray analysis as described in Materials and Methods. The expression levels of fetal liver cells, hepatocyte-like cells differentiated from human iPS cells, and adult hepatocytes are presented in the left, center, and right columns, respectively.

Fig. 5.. Effects of drugs on expression of CYP3A enzymes and UGT1A1 mRNAs in the hepatocyte-like cells

The cells differentiated from human iPS cells (Fetch) were treated with OME, DEX, and RIF for 72 h. The total mRNA was extracted from the cells. The expression of CYP3A and UGT1A1 mRNAs were analyzed by microarray analysis as described in Materials and Methods. Each bar represents the mean ± SD from triplicate experiments. Values were normalized to the levels of GAPDH mRNA. The graphs represent the relative gene expression level when the levels in the hepatocyte-like cells using collagen I and DMSO were assigned a value of 1.

Fig. 6.. Effects of drugs on testosterone 6β-hydroxylase activity in hepatocyte-like cells

The cells differentiated from human iPS cells (Fetch) were treated with OME, DEX, and RIF for 72 h and then cultured with the medium containing testosterone for 6h. 6β-Hydroxytestosterone was analyzed using liquid chromatography coupled with tandem mass spectrometry as described in Materials and Methods. Each bar represents the mean ± SD from triplicate experiments. The graphs represent the relative activity ratios when the value in the hepatocyte-like cells using collagen I and DMSO were assigned a value of 1.