RT Journal Article SR Electronic T1 Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.03.20.999680 DO 10.1101/2020.03.20.999680 A1 Ruri Tsuneishi A1 Noriaki Saku A1 Shoko Miyata A1 Palaksha Kanive Javaregowda A1 Kenta Ite A1 Masashi Toyoda A1 Tohru Kimura A1 Masahiko Kuroda A1 Atsuko Nakazawa A1 Mureo Kasahara A1 Hidenori Nonaka A1 Akihide Kamiya A1 Tohru Kiyono A1 Junji Yamauchi A1 Akihiro Umezawa YR 2020 UL http://biorxiv.org/content/early/2020/03/20/2020.03.20.999680.abstract AB 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.