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

Abstract

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.

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

“…In this study, the establishment of a culture system to stably grow DILI patient-derived hepatocytes enabled us to elongate hepatocyte lifespan to more than 30 population doublings with the maintenance of hepatic progenitor progenitor-like phenotypes. Usage of Wnt3a/R-Spondin 1 in combination with the feeder cells for elongation of hepatocyte lifespan is in line with the previous report that WNTs are important for liver regeneration and in vitro propagation of hepatocytes and that irradiated MEFs are useful for maintaining the expression and proliferative capacity of hepatic progenitors [20,32,33].…”

“…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).…”

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells

“…Puromycin equalized hepatocytes with diverse functions such as drug metabolism after the in vitro propagation. The stable proliferative capacity of primary hepatocytes and pluripotent stem cell-derived hepatocytes requires WNT, R-sponsin1, and feeder cells 23,[33][34][35][36] . Resistance to puromycin may be due to the high metabolic activities of CYP3A4.…”

Characterization of human embryonic stem cells in animal component-free medium

“…Another issue that arises when considering the clinical application of regenerative medical products is how to prepare large quantities of cells quickly. SEES2 cells, a strain of human embryonic stem cells, are starting to be used in clinical practice [7,8]. Akutsu et al have studied a culture system without animal-derived components, and have reported that it is possible to culture cells stably in that culture system [9].…”

“…The SEES2 cell line, an hESC lines, has been established at the National Center for Child Health and Development (NCCHD) for clinical applications and regenerative medicine research [6]. The SEES cell lines are presently being applied in clinical practice, including successful clinical trials in congenital urea cycle disorders [7,8]. It is necessary to establish a safer and more stable method of culturing SEES2 cells for clinical applications in the future.…”