Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Oct 26, 2021; 13(10): 1595-1609
Published online Oct 26, 2021. doi: 10.4252/wjsc.v13.i10.1595
Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells
Francesco Bellanti, Giorgia di Bello, Rosanna Tamborra, Marco Amatruda, Aurelio Lo Buglio, Michał Dobrakowski, Aleksandra Kasperczyk, Sławomir Kasperczyk, Gaetano Serviddio, Gianluigi Vendemiale
Francesco Bellanti, Giorgia di Bello, Rosanna Tamborra, Marco Amatruda, Aurelio Lo Buglio, Gaetano Serviddio, Gianluigi Vendemiale, Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
Michał Dobrakowski, Aleksandra Kasperczyk, Sławomir Kasperczyk, Department of Biochemistry, Medical University of Silesia, Zabrze 41-808, Poland
Author contributions: Bellanti F and Vendemiale G designed and coordinated the study; Bellanti, F, di Bello G, Tamborra R, Lo Buglio A, Amatruda M, Dobrakowsky M, and Kasperczyk A performed the experiments, acquired, and analyzed data; Bellanti, F, di Bello G, Tamborra R, Amatruda M, Lo Buglio A, Dobrakowsky M, Kasperczyk A, Kasperczyk S, Serviddio G, and Vendemiale G interpreted the data; Bellanti F wrote the manuscript; Kasperczyk S, Serviddio G, and Vendemiale G supervised the manuscript; All authors approved the final version of the article.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board at the University of Foggia.
Conflict-of-interest statement: All authors have nothing to disclose.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Francesco Bellanti, MD, PhD, Doctor, Associate Professor, Department of Medical and Surgical Sciences, University of Foggia, viale Pinto 1, Foggia 71122, Italy. francesco.bellanti@unifg.it
Received: April 26, 2021
Peer-review started: April 26, 2021
First decision: May 12, 2021
Revised: June 14, 2021
Accepted: August 23, 2021
Article in press: August 23, 2021
Published online: October 26, 2021
Processing time: 182 Days and 11.5 Hours
Abstract
BACKGROUND

Senescence is characterized by a decline in hepatocyte function, with impairment of metabolism and regenerative capacity. Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism, liver diseases, and organ regeneration. The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not yet known.

AIM

To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells.

METHODS

We compared the transdifferentiation capacity of cells over 10 (passage 10 [P10]) vs P20. Aging was evaluated by senescence-associated (SA) beta-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]), quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT], and carcinoembryonic antigen [CEA]), and functional analyses (albumin secretion, CYP3A4, and γ-GT). Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were also measured.

RESULTS

SA β-galactosidase staining was higher in P20 than P10 HepaRG cells; in parallel, the comet assay showed consistent DNA damage in P20 HepaRG cells. With respect to P10, P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM, and CK19 after the induction of transdifferentiation. Furthermore, lower gene expression of albumin, CYP3A4, and γ-GT, as well as 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. Of note, both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells. Finally, both ATP and NAD+/NADH were depleted in P20 cells with respect to P10 cells.

CONCLUSION

SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells, with consequent impairment of metabolic and regenerative properties, which may alter applications in basic studies.

Keywords: Senescence; HepaRG cells; Transdifferentiation; Mitochondria; Regeneration; Nicotinamide adenine dinucleotide

Core Tip: The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not known. We characterized the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of HepaRG cells. By using a replication protocol, we described higher senescence-associated markers and lower transdifferentiation markers in passage 20 (P20) than in P10 cells. Cellular and mitochondrial oxygen consumption, and ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were lower in P20 than in P10 transdifferentiated cells. To conclude, senescence-associated mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells.