The liver is essential for maintaining normal physiology and homeostasis and is composed of hepatocytes, endothelial cells, and stellate cells. Among these cells, hepatocytes play a key role in metabolism and detoxification. However, human hepatocytes are difficult to propagate ex vivo due to lack of appropriate culture conditions. To solve this issue, hepatocytes have been freshly isolated from livers, generated from hepatomas, induced from pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and converted from other somatic cells such as fibroblasts^. However, a steady supply of hepatocytes dissociated from livers for use as in vitro models cannot be guaranteed because of limited supply and lot-to-lot variations due to genetic and environmental backgrounds. Likewise, iPSC- or ESC-derived hepatocytes may show variation between lots, due to the difficulty of strictly controlling the differentiation process. Even when hepatocytes are generated from a bank of undifferentiated iPSCs or ESCs, the direct reprogramming technology requires complex protocols and a relatively long period for complete differentiation, and yields a limited number of mature hepatocytes among a heterogeneous population HepG2, a hepatoblastoma cell line, exhibits hepatocyte-like features with a limited expression of hepatocyte-associate markers such as albumin and cytochrome P450 (CYP)^. Likewise, HepaRG, a spontaneously immortalized cell line, from hepatocarcinoma of a female patient has more hepatic features compared to HepG2.

Hepatocytes express a series of drug metabolizing enzymes known as phase I and phase II enzymes. Phase I enzymes, such as cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), and carboxylesterase (CES), introduce a highly reactive functional group or polar moiety to lipophilic compounds. Phase II enzymes conjugate various groups, including glutathione (GSH), sulfate, glycine, or glucuronic acid to the highly reactive products produced by phase I enzyme reactions. Hepatocytes also express transporters in the basolateral membrane, including the sodium/taurocholate co-transporting peptide (NTCP), organic cation transporter (OCT), and organic anion transporter (OAT), and in the apical membrane including P-glycoprotein (P-gp), bile-salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). Primary hepatocytes are the gold standard as an in vitro model to accurately evaluate drug-metabolizing enzymes and hepatotoxicity. In addition to primary hepatocytes, only a limited number of immortalized hepatocytes has been used so far. Immortalized hepatocytes derived from normal hepatocytes would be ideal to ensure of a steady supply. From this viewpoint, HepG2 and HepaRG cells have been used for evaluating the toxicity of chemicals and drugs. The majority of small-molecule drugs commonly used by humans are metabolized by members of CYP3A family, and inhibition of CYP3A4-mediated metabolism is a common cause of drug-induced liver injury.

In this study, we generated an immortalized hepatocyte cell line, HepaMN, from a Japanese patient with biliary atresia. We applied a previously used strategy for immortalization of human keratinocyte or mammary epithelial cells—inactivation of the Rb/p16 pathway and acquisition of telomerase activity. HepaMN cells constitutively exhibited a hepatocytic phenotype both in vitro and in vivo, and showed increased CYP3A4 after exposure to rifampicin, implying that HepaMN cells can be another suitable tool for pharmaceutical studies.

Regards,

Jessica

Managing editor

Pancreatic disorder and therapy