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. 2016 Dec 9:6:38694.
doi: 10.1038/srep38694.

Functional human induced hepatocytes (hiHeps) with bile acid synthesis and transport capacities: A novel in vitro cholestatic model

Xuan Ni  1   2 , Yimeng Gao  3 , Zhitao Wu  1   2 , Leilei Ma  1   2 , Chen Chen  1   2 , Le Wang  1   2 , Yunfei Lin  1   2 , Lijian Hui  3 , Guoyu Pan  1   2
Affiliations

Functional human induced hepatocytes (hiHeps) with bile acid synthesis and transport capacities: A novel in vitro cholestatic model

Xuan Ni et al. Sci Rep. .

Abstract

Drug-induced cholestasis is a leading cause of drug withdrawal. However, the use of primary human hepatocytes (PHHs), the gold standard for predicting cholestasis in vitro, is limited by their high cost and batch-to-batch variability. Mature hepatocyte characteristics have been observed in human induced hepatocytes (hiHeps) derived from human fibroblast transdifferentiation. Here, we evaluated whether hiHeps could biosynthesize and excrete bile acids (BAs) and their potential as PHH alternatives for cholestasis investigations. Quantitative real-time PCR (qRT-PCR) and western blotting indicated that hiHeps highly expressed BA synthases and functional transporters. Liquid chromatography tandem mass spectrometry (LC-MS/MS) showed that hiHeps produced normal intercellular unconjugated BAs but fewer conjugated BAs than human hepatocytes. When incubated with representative cholestatic agents, hiHeps exhibited sensitive drug-induced bile salt export pump (BSEP) dysfunction, and their response to cholestatic agent-mediated cytotoxicity correlated well with that of PHHs (r2 = 0.8032). Deoxycholic acid (DCA)-induced hepatotoxicity in hiHeps was verified by elevated aspartate aminotransferase (AST) and γ-glutamyl-transferase (γ-GT) levels. Mitochondrial damage and cell death suggested DCA-induced toxicity in hiHeps, which were attenuated by hepatoprotective drugs, as in PHHs. For the first time, hiHeps were reported to biosynthesize and excrete BAs, which could facilitate predicting cholestatic hepatotoxicity and screening potential therapeutic drugs against cholestasis.

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Figures

Figure 1
Figure 1. The expression levels of BA synthases in hiHeps and PHHs.
(A) The typical epithelial morphologies of hiHeps and PHHs, as determined by light phase contrast microscopy. (B) The mRNA expression levels of BA synthases (i.e., CYP7A1, CYP8B1, and CYP27 A1) and the upstream nuclear factors (i.e., FXR, CAR, and PXR) in hiHeps. as determined by qRT-PCR. The data are expressed as the mean ± SD (n = 3). *p < 0.05 relative to PHHs. (C) Comparison of the protein expression levels of CYP7A1 and FXR among hiHeps, PHHs and SCHHs by western blotting (left) and gray intensity analysis (right). β-ACTIN was used as a reference control.
Figure 2
Figure 2. The expression levels of BA transporters in hiHeps and human hepatocytes.
(A) The mRNA levels of BA efflux (i.e., BSEP, MRPs and MDRs) and influx (i.e., NTCP, OATP1B1 and OATP1B3) transporters in hiHeps determined by qRT-PCR. The data are expressed as the mean ± SD (n = 3). *p < 0.05 relative to PHHs. (B) Comparison of the protein expression levels of MRP2, BSEP and NTCP among hiHeps, PHHs and SCHHs by western blotting (left) and gray intensity analysis (right). β-ACTIN was used as a reference control.
Figure 3
Figure 3. Comparison of the activities of BA transporters between hiHeps and human hepatocytes.
(A) The efflux transporter activities of BSEP and MRP2 were determined by calculating the BEI values of d8-TCA (the substrate of BSEP) and methotrexate (the substrate of MRP2) for hiHeps (white columns) and SCHHs (black columns). The data are expressed as the mean ± SD (n = 3). (B) The polarized locations of efflux transporters in the bile canaliculi of both hiHeps and SCHHs, as determined by fluorescence microscopy. In the absence or presence of MK571 (20 μmol/L), an MRP2 inhibitor, bile canaliculi were labelled with an MRP2 fluorescent substrate, CDF, which was formed from non-fluorescent CDFDA via intracellular esterases. (C) The influx transporter activity of NTCP was determined by accumulation assay of an NTCP substrate, d8-TCA. Troglitazone (10 μmol/L) was used as a positive control. The data are expressed as the mean ± SD (n = 3). *p < 0.05 vs control in hiHeps, #p < 0.05 vs control in PHHs.
Figure 4
Figure 4. Comparisons of the BA concentrations in cell lysates and supernatants of hiHeps and SCHHs
. (A) The concentration of each BA (i.e., CA, GCA, TCA, CDCA, GCDCA, TCDCA, DCA, GDCA, LCA, GLCA and TLCA) in both the cell lysates and supernatants of hiHeps and SCHHs was determined by LC-MS/MS. (B) The total BA concentrations in the cell lysates and supernatants were measured using a total BA reagent kit.
Figure 5
Figure 5. Cholestatic agent-induced BSEP inhibition and cytotoxicity in hiHeps and human hepatocytes.
(A) BSEP function was inhibited by cholestatic drugs in hiHeps compared with SCHHs, resulting in a decrease in the BEI value of d8-TCA. The cholestatic drugs included 10 μmol/L troglitazone, 30 μmol/L ketoconazole, 25 μmol/L rifampicin, 25 μmol/L bosentan, 10 μmol/L glibenclamide and 100 μmol/L omeprazole. The data are expressed as the mean ± SD (n = 3). (B) BSEP expression was inhibited by cholestatic drugs in hiHeps (white columns) compared with SCHHs (grey columns). The data are expressed as the mean ± SD (n = 3). *p < 0.05 vs control. (C) Correlation analysis of hiHeps and human hepatocytes based on the log(IC50) determined by MTT assay. The calculated r2 value of the linear correlation between hiHeps and human hepatocytes was 0.8032.
Figure 6
Figure 6. Comparison of the BA concentration-dependent cytotoxicities between hiHeps and PHHs.
The BAs included CA, TCA, and GCA (top row); CDCA, TCDCA, and GCDCA (middle row); and DCA and LCA (bottom row). The data are expressed as the mean ± SD (n = 5).
Figure 7
Figure 7. Blood biochemical analysis of DCA-mediated hepatotoxicity in hiHeps and PHHs.
(A) The enzymatic activities of ALT, AST, ALP and γ-GT in hiHeps. The data are expressed as the mean ± SD (n = 3), *p < 0.05 vs 0 group. (B) The enzymatic activities of ALT, AST, ALP and γ-GT in PHHs. The data are expressed as the mean ± SD (n = 2), *p < 0.05 vs 0 group.
Figure 8
Figure 8. The mechanisms of DCA-mediated toxicity in hiHeps and PHHs.
The mechanism of DCA-mediated toxicity was examined by performing ATP (A), MMP (B), ROS (C), apoptosis (D) and LDH (E) assays. The data are expressed as the mean ± SD (n = 3–5). *p < 0.05 in hiHeps, #p < 0.05 in PHHs.
Figure 9
Figure 9. Hepatoprotective effects of therapeutic drugs in hiHeps and PHHs.
The hepatic roles of the hepatoprotective drugs quercetin (40 μmol/L), silymarin (25 μmol/L), curcumin (15 μmol/L) and metformin (200 μmol/L) in hiHeps and PHHs were elucidated via MTT (A), ATP (B), MMP (C), ROS (D), apoptosis (E) and LDH (F) assays. The data are expressed as the mean ± SD (n = 3–5). *p < 0.05 vs DCA group in hiHeps, #p < 0.05 vs DCA group in PHHs.
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