Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells

ARTICLE HIGHLIGHTS

Research background

The HepaRG cell line is used to study metabolism, toxicology, and the regeneration /differentiation processes, as a replacement to primary hepatocytes, HepG2, and Huh-7 cells. These cells exhibit a hepatocyte-like morphology and express hepatocyte-specific functions in defined culture conditions; furthermore, HepaRG display features of human oval ductular bipotent hepatic progenitors.

Research motivation

Cellular senescence consists in a steady cell cycle block occurring because of different harmful events, leading to defective stemness and differentiation processes, as well as changes in cell cycle regulation, signal transduction, and metabolism. The impact of senescence on HepaRG cells has not yet been investigated.

Research objectives

This study investigated whether a replication protocol would induce senescence in HepaRG cells. In addition, we characterized the effects of senescence on transdifferentiation capacity and mitochondrial metabolism.

Research methods

The transdifferentiation capacity of HepaRG cells over passage 10 (P10) vs passage 20 (P20) was compared. To stimulate transdifferentiation, HepaRG cells were treated with dimethyl sulfoxide (DMSO). Aging was evaluated by senescence-associated (SA) β-galactosidase activity and the comet assay. HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry (expression of cluster of differentiation 49a [CD49a], CD49f, CD184, epithelial cell adhesion molecule [EpCAM], and cytokeratin 19 [CK19]), by quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT] and carcinoembryonic antigen [CEA]) and functional analysis (albumin secretion, CYP3A4 and γ-GT). Mitochondrial respiration, the ATP and the NAD⁺/NADH content were also measured.

Research results

We first observed that replication induces the expression of senescence markers in HepaRG cells, since SA β-galactosidase staining was higher in P20 than in P10 HepaRG cells, and the comet assay showed a consistent DNA damage in P20 HepaRG cells. We further reported that transdifferentiation towards bipotent progenitors is altered in senescent HepaRG cells, as P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM and CK19 – with respect to P10 – after DMSO treatment. Furthermore, the lower gene expression of albumin, CYP3A4, and γ-GT, as well as the reduced albumin secretion capacity, CYP3A4, and γ-GT activity were reported in transdifferentiated P20 compared to P10 cells. By contrast, the gene expression level of CEA was not reduced by transdifferentiation in P20 cells. Finally, we show that senescence-induced impairment of HepaRG transdifferentiation is associated with mitochondrial dysfunction, since both cellular and mitochondrial oxygen consumption were lower in P20 than in P10 transdifferentiated cells, and both ATP and NAD⁺/NADH were depleted in P20 cells with respect to P10 cells.

Research conclusions

The present study demonstrates that HepaRG cells undergo replicative senescence, with consequent impairment in transdifferentiation, functional activity, mitochondrial dysfunction, and NAD⁺ depletion.

Research perspectives

Further studies will define the molecular mechanisms underlying our observations. The limitations in the transdifferentiation potential of senescent HepaRG cells, with consequent alteration of metabolic and regenerative properties, may have serious implications when this cell line is applied for basic studies.