Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Tsuneishi, Ruri; Saku, Noriaki; Miyata, Shoko; Akiyama, Saeko; Javaregowda, Palaksha Kanive; Ite, Kenta; Takashima, Nagisa; Toyoda, Masashi; Kimura, Tohru; Kuroda, Masahiko; Nakazawa, Atsuko; Kasahara, Mureo; Nonaka, Hiroshi; Kamiya, Akihide; Kiyono, Tohru; Yamauchi, Junji; Umezawa, Akihiro

doi:10.1038/s41598-021-90708-3

Cited by 10 publications

(8 citation statements)

References 34 publications

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“…Hepatocytes have high metabolic activity of drugs and predictably have resistance to puromycin. The escape of PSC-derived differentiated cells from puromycin is therefore explained by endogenous drug metabolic activity, i.e., expression of the cytochrome P450 genes [ 37 , 38 ]. To enrich hepatocytes, selection with cell surface markers such as EpCAM by flow cytometric analysis and magnetic cell sorting can also be used [ 39 ].…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, the puromycin-selected zone III hepatocytes start to express the ALB gene. This coordinated change of these genes is aligned with the selection with ammonia, another cytotoxic molecule [ 37 ]. In the ammonia-selected cells, not only ammonia metabolizing enzymes are upregulated, but also the expression of cytochrome P450 genes characteristic of zone III hepatocytes is upregulated.…”

Section: Discussionmentioning

confidence: 99%

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Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

et al. 2022

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Background Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI). Methods To generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations. Results The hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen. Conclusions iPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

et al. 2022

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“…Ammonia has been used as a selection agent for enrichment of hepatocytes because of its cytotoxic effect [ 19 – 21 ]. Puromycin has been used to select pluripotent stem cell-derived hepatocytes with a high expression of CYP3A4 [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture

et al. 2022

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Background The liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of hepatocytes is used for the understanding of liver physiology as well as for the drug development. Hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. Alternatively, systems using animal models and hepatocellular carcinoma cells have been established, but interspecies differences, variation between human cell sources and limited hepatic functions are among the challenges faced when using these models. Therefore, there is still a need for a highly stable method to purify human hepatocytes with functional sufficiency. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. Methods We first established a growth culture system for hepatocytes derived from patients with drug-induced liver injury using fetal mouse fibroblasts and EMUKK-05 medium. We then evaluated the morphology, proliferative capacity, chromosome stability, gene and protein expression profiles, and drug metabolic capacity of hepatocytes in early, middle and late passages with and without puromycin. In addition, hepatic maturation in 3D culture was evaluated from morphological and functional aspects. Results In our culture system, the stable proliferation of human hepatocytes was achieved by co-culturing with mouse fetal fibroblasts, resulting in dedifferentiation into hepatic progenitor-like cells. We purified human hepatocytes by selection with cytocidal puromycin and cultured them for more than 60 population doublings over a span of more than 350 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytocidal antibiotics because of enhanced drug-metabolizing activity. Conclusions These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.

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“…Puromycin equalized hepatocytes with diverse functions such as drug metabolism after the in vitro propagation. The stable proliferative capacity of primary hepatocytes and pluripotent stem cell-derived hepatocytes requires WNT, R-sponsin1, and feeder cells 23,[33][34][35][36] . Resistance to puromycin may be due to the high metabolic activities of CYP3A4.…”

Section: Discussionmentioning

confidence: 99%

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

Akiyama

Saku

Miyata

et al. 2023

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As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 2^20-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

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Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

Cited by 10 publications

References 34 publications

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

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