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

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

doi:10.1038/s41598-020-73992-3

Cited by 10 publications

(12 citation statements)

References 26 publications

(28 reference statements)

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“…Gene chip analysis was performed as previously described [ 26 ]. Total RNA was isolated using a miRNeasy mini kit (217004, Qiagen, Hilden, Germany).…”

Section: Methodsmentioning

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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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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“…Gene chip analysis was performed as previously described [ 26 ]. Total RNA was isolated using a miRNeasy mini kit (217004, Qiagen, Hilden, Germany).…”

Section: Methodsmentioning

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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“…Compared with these sophisticated approaches, successful enrichment with ammonia exposure as used in this study is simple and straightforward. Indeed, the ammonia selection method was also applicable to the HepaMN immortalized cell line 21 . It is also noteworthy that ammonia selection may increase homogeneity or decrease heterogeneity in a cell lot with regard to ammonia metabolic activity.…”

Section: Discussionmentioning

confidence: 99%

“…We also used HepaMN and SM cells for qRT-PCR analysis. HepaMN cells have been established from a liver associated with biliary atresia 21 . SM cell has been generated as an immortalized cell from a patient with drug-induced liver injury (Supplementary Experimental Procedures, “SM cells”).…”

Section: Methodsmentioning

confidence: 99%

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

Tsuneishi

Saku

Miyata

et al. 2021

Sci Rep

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Ammonia has a cytotoxic effect and can therefore be used as a selection agent for enrichment of zone I hepatocytes. However, it has not yet been determined whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed an ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human embryonic stem cells (ESCs) and from induced pluripotent stem cells (iPSCs). The resistance to cytotoxicity or cell death by ammonia is likely attributable to the metabolism of ammonia in the cells. In addition to ammonia metabolism-related genes, ammonia-selected hepatocytes showed increased expression of the cytochrome P450 genes. Additionally, the ammonia-selected cells achieved immortality or at least an equivalent life span to human pluripotent stem cells without affecting expression of the liver-associated genes. Ammonia treatment in combination with in vitro propagation is useful for obtaining large quantities of hepatocytes.

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“…We propagated them in α-MEM medium supplemented with 10% FBS (Gibco or HyClone) and 1% Pen-Strep. These cells were immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tightcyclin D1, and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses according to our previous report [17]. We further infected the immortalized cells with the lentiviruses carrying a combination of the R-WNT3A, R-SPO, and NOGGIN genes.…”

Section: Preparation Of Human Embryonic Stem Cell-derived Feeder Cellsmentioning

confidence: 99%

Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

et al. 2021

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Background Thin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro. Methods We prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells. Results Co-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model. Conclusions We herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.

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

Cited by 10 publications

References 26 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)

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

Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

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