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

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.

Stem cell-derived polarized hepatocytes

“…Pol-HLCs synthesized and released the two primary bile acids, CA and CDCA, mainly from the apical side into the top compartment. Of note, unlike nonpol-HLCs 24 , pol-HLCs secreted both bile acid species and their conjugates at a similar ratio ( Fig. 3f and g).…”

“…PHHs mainly secrete glycine or taurin conjugates of cholic acid (CA) and chenodeoxycholic acid (CDCA), whereas HepG2 hepatoma cells secrete mainly precursors and unconjugated bile acids 23 . HLCs, probably owing to their immature nature, were reported to secrete unconjugated CA and CDCA 24 . We therefore used liquid chromatography-mass spectrometry (LC-MS) to profile released bile acid species.…”

Leflunomide Increases Hepatic Exposure to Methotrexate and Its Metabolite by Differentially Regulating Multidrug Resistance–Associated Protein Mrp2/3/4 Transporters via Peroxisome Proliferator–Activated Receptor α Activation

“…Western blot analysis was conducted as previously described (Ni et al, 2016). Briefly, total protein was extracted using radioimmunoprecipitation assay buffer containing the protease inhibitor phenylmethanesulfonyl fluoride.…”

Alternative Cell Sources for Liver Parenchyma Repopulation: Where Do We Stand?

“…In these transdifferentiation studies, the hepatocyte phenotype is induced by overexpressing different sets of TFs, combined with hepatocyte supportive media. Two possible avenues are used: (1) somatic cells can be directly transdifferentiated towards hepatocytes [117][118][119], or (2) somatic cells are first partially dedifferentiated, followed by differentiation into the hepatic lineage [120][121][122].…”