Enrichment of Pluripotent Stem Cell-Derived Hepatocyte-Like Cells by Ammonia Treatment

et al. 2020

Preprint

Zone I and zone III hepatocytes metabolize ammonia through urea cycle and drug by cytochrome P450, respectively. Ammonia has a cytotoxic effect, and can therefore be used as a selection agent for enrichment of hepatocytes. Besides, isolated hepatocytes from livers can be propagated ex vivo under appropriate condition. However, it has not been investigated so far whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed the ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human pluripotent stem cells (PSCs) that are embryonic stem cells (ESCs) and induced pluripotent stem cells. Hepatocyte-like cells after exposure to ammonia highly expressed the CPS1 gene that metabolizes ammonia to carbamoyl phosphate. The resistance to cytotoxicity or cell death by ammonia is probably attributed to the metabolic activity of ammonia in the cells. In addition to the ammonia metabolism-related genes, ammonia-selected PSC-derived hepatocytes increased expression of the CYP3A4 gene, one of the cytochrome P450 genes, that is mainly expressed in zone III hepatocytes. Ammonia-selected hepatocyte-like cells derived from both ESCs and iPSCs can be propagated in vitro up to 30 population doublings for more than 190 days without affecting expression of the liver-associated genes, implying that the ammoniaselected cells have immortality or equivalent life span on the appropriate feeder cells like ESCs and iPSCs. The long-term cultivation of ammonia-selected hepatocyte-like cells resulted in the increased expression of hepatocyte-associated genes such as the CPS1 and CYP3A4 genes. The ammonia selection method to enrich a hepatocyte population was also applicable to immortalized cells from the liver. Ammonia treatment in combination with in vitro propagation will be used to obtain large amounts of hepatocytes or hepatocyte-like cells for pharmacology, toxicology and regenerative medicine.

Section: Ammonia-based Enrichment Of Zone I Hepatocytesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Tsuneishi

et al. 2020

Preprint

“…Ammonia has been used as a selection agent for enrichment of hepatocytes because of its cytotoxic effect (Tanimizu et al, 2016; Tomotsune et al, 2016; Tsuneishi et al, 2021). Puromycin has been used to select pluripotent stem cell-derived hepatocytes with a high expression of CYP3A4 (Miyata et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

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

Akiyama

et al. 2021

Preprint

The liver plays many important roles in homeostasis, including drug detoxification, metabolism, and bile production. Hepatocytes, which are the main constituent cells of the liver, play an important role in the pathogenesis of liver diseases, identification of candidate compounds in drug discovery research, pharmacokinetic studies, and toxicity evaluation. Human hepatocytes are, however, difficult to grow in normal in vitro culture systems, making it difficult to secure cell numbers. As an alternative, evaluation systems using animal models and hepatocellular carcinoma cells have been established, but interspecies and interracial differences and low hepatic function have been pointed out as problems. Therefore, there is still a need for a highly stable method to prepare human hepatocytes with sufficient functionality. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of the functionality of human hepatocytes to stably supply human hepatocytes. In the established culture system, the stable proliferation of human hepatocytes was achieved by co-culturing hepatocytes with mouse fetal fibroblasts to dedifferentiate them into hepatic progenitor-like cells. Furthermore, we succeeded in purifying human hepatocytes by puromycin with a rapid cytocidal effect and proliferating them to over 30 population doublings for more than 200 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytotoxic antibiotics because of enhanced drug-metabolizing activity. These results show that the above culture system enables simple and efficient hepatocyte proliferation, and is considered to be an effective method for stable supply of hepatocytes and significant cost reduction in drug discovery research.

“…The escape of PSC-derived differentiated cells from ammonia is therefore explained by endogenous ammonia metabolism, i.e. expression of urea cycle-related genes such as OTC and CPS1 13 . To enrich for cells that can metabolize ammonia, selection with cell surface markers such as CD13 by flow cytometric analysis and magnetic cell sorting can be used.…”

Section: Discussionmentioning

confidence: 99%

“…Hepatocytes can be selected by ammonia because hepatocytes actively metabolize ammonia 12 . Likewise, hepatocyte-like cells differentiated from human PSCs can be enriched with ammonia 13 . Hepatocytes can be maintained in vitro without the introduction of any genes 14 , 15 .…”

Section: Introductionmentioning

confidence: 99%

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

Tsuneishi

et al. 2021

Sci Rep

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.