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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.

Hepatocyte-like cells (HLCs) generated from human pluripotent stem cells (PSCs) exhibit hepatocytic properties in vitro; however, their engraftment and functionality in vivo remain unsatisfactory. Despite optimization of differentiation protocols, HLCs did not engraft in a mouse model of liver injury. In contrast, organ-derived hepatocytes reproducibly formed colonies in the liver injury mouse model. As an extension of the phenomenon observed in hematopoietic stem cells giving rise to colonies within the spleen, commonly referred to as “colony-forming units in spleen (CFU-s“, we hypothesize that “colony-forming units in liver (CFU-L)“ serves as a reliable indicator of stemness, engraftment, and functionality of hepatocytes. The uniform expression of the randomly inactivated gene in a single colony, as reported by Sugahara et al. 2022, suggests that the colonies generated by isolated hepatocytes likely originate from a single cell. We, therefore, propose that CFU-L can be used to quantify the number of “hepatocytes that engraft and proliferate in vivo“ as a quantitative assay for stem cells that utilize colony-forming ability, similar to that observed in hematopoietic stem cells.

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.

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.

Many drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can accurately evaluate drug-mediated CYP3A4 induction as the gold standard for in vitro hepatic toxicology test, but their lot variation is an issue to be solved. Only a limited number of immortalized hepatocyte cells have been reported. In this study, we generated an immortalized cell expressing CYP3A4 from a patient with drug-induced liver injury (DILI). To generate DILI-derived cells with a high expression of CYP3A4, we employed a three-step approach: 1. Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); 2. Immortalization of the differentiated cells; 3. Selection of the cells with puromycin. We hypothesize that cells with a high expression of cytochrome P450 genes can survive even after exposure to cytotoxic antibiotics because of high drug-metabolism activity. Puromycin, one of the cytotoxic antibiotics, was used in this study because of its rapid cytocidal effect at a low concentration. Phenotypic studies in vitro revealed that the puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at an extremely high level, and continued to proliferate at least up to 34 population doublings for more than 250 days. The expression profiles were independent of population doublings. Drug-mediated induction test revealed that the cells significantly increased CYP3A4 after exposure to rifampicin, suggesting that the immortalized cells would serve as another useful source for in vitro examination of drug metabolism and CYP3A4 induction.

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 ammonia-selected 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.