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

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

doi:10.1038/s41598-021-90708-3

Abstract

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.

Highlights

Ammonia has a cytotoxic effect and can be used as a selection agent for enrichment of zone I hepatocytes
We propagated hepatocytes or hepatocyte-like cells derived from human pluripotent stem cells (PSCs), i.e. embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and enriched for zone I hepatocytes with a mmonia[16,17]
This study introduces an alternative strategy to obtain large numbers of human hepatocytes by a combination of ammonia selection and subsequent in vitro propagation

Summary

Introduction

Ammonia has a cytotoxic effect and can be used as a selection agent for enrichment of zone I hepatocytes. We propagated hepatocytes or hepatocyte-like cells derived from human PSC, i.e. ESCs and iPSCs, and enriched for zone I hepatocytes with a mmonia[16,17]. This heterogenous population of ESC-derived hepatocyte-like cells was exposed to ammonia for 2 days, which kills most cells.

Results

Conclusion