Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

Manami Nishiwaki,Masashi Toyoda,Akihiro Umezawa,Yoshie Oishi,Mureo Kasahara,Takashi Ohkura,Shin-Ichi Ohno,Atsuko Nakazawa,Tohru Kimura,Hatsune Makino-Itou,Shin Enosawa,Shin-Ichiro Horiuchi,Tohru Kiyono,Hidenori Akutsu,Seiichi Ishida

doi:10.1038/s41598-020-73992-3

Abstract

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Highlights

Hepatocytes are an important tool for in vitro toxicology testing
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, and converted from other somatic cells such as fibroblasts[1,2,3,4,5,6]
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[14,15]

Summary

Introduction

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies. HepG2, a hepatoblastoma cell line, exhibits hepatocyte-like features with a limited expression of hepatocyte-associate markers such as albumin and cytochrome P450 (CYP) 10. 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. 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

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Results

Conclusion