Basic Study
Copyright ©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Sep 26, 2023; 15(9): 876-896
Published online Sep 26, 2023. doi: 10.4252/wjsc.v15.i9.876
Interferon-γ priming enhances the therapeutic effects of menstrual blood-derived stromal cells in a mouse liver ischemia-reperfusion model
Qi Zhang, Si-Ning Zhou, Jia-Min Fu, Li-Jun Chen, Yang-Xin Fang, Zhen-Yu Xu, Hui-Kang Xu, Yin Yuan, Yu-Qi Huang, Ning Zhang, Yi-Fei Li, Charlie Xiang
Qi Zhang, Si-Ning Zhou, Jia-Min Fu, Li-Jun Chen, Yang-Xin Fang, Hui-Kang Xu, Yin Yuan, Yu-Qi Huang, Ning Zhang, Yi-Fei Li, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
Qi Zhang, Si-Ning Zhou, Jia-Min Fu, Li-Jun Chen, Yang-Xin Fang, Hui-Kang Xu, Yin Yuan, Yu-Qi Huang, Ning Zhang, Yi-Fei Li, Research Units of Infectious Disease and Microecology, Chinese Academy of Medical Sciences, Hangzhou 310003, Zhejiang Province, China
Zhen-Yu Xu, Innovative Precision Medicine Group, Shulan Hospital, Hangzhou 311215, Zhejiang Province, China
Charlie Xiang, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
Author contributions: Zhang Q, Zhou SN, Chen LJ, Xu ZY, and Xiang C designed the research study; Zhang Q, Zhou SN, Fu JM, Fang YX, Xu HK, Yuan Y, Huang YQ, Zhang N, and Li YF performed the research; Zhang Q, Zhou SN, and Chen LJ analyzed the data and wrote the manuscript; and all authors have read and approved the final manuscript.
Supported by National Key R&D Program of China, No. 2022YFA1105603 and 2022YFC2304405; Hangzhou Science and Technology Project, China, No. 20200224; National Natural Science Foundation of China, No. 81900563; Key Research & Development Plan of Zhejiang Province, China, No. 2019C03015 and 2020C04016.
Institutional review board statement: The study was reviewed and approved by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University Institutional Review Board.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang University (ZJU-IACUC), and the ethics code is ZJU20210299.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: All data mentioned in the article can be obtained by contacting the corresponding author.
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
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: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Charlie Xiang, PhD, Director, Professor, The First Affiliated Hospital, Zhejiang University School of Medicine, No. 79 Qingchun Road, Hangzhou 310003, Zhejiang Province, China. cxiang@zju.edu.cn
Received: April 20, 2023
Peer-review started: April 20, 2023
First decision: June 7, 2023
Revised: June 21, 2023
Accepted: July 18, 2023
Article in press: July 18, 2023
Published online: September 26, 2023
Processing time: 157 Days and 18.5 Hours
Abstract
BACKGROUND

Mesenchymal stem cells (MSCs) have been used in liver transplantation and have certain effects in alleviating liver ischemia-reperfusion injury (IRI) and regulating immune rejection. However, some studies have indicated that the effects of MSCs are not very significant. Therefore, approaches that enable MSCs to exert significant and stable therapeutic effects are worth further study.

AIM

To enhance the therapeutic potential of human menstrual blood-derived stromal cells (MenSCs) in the mouse liver ischemia-reperfusion (I/R) model via interferon-γ (IFN-γ) priming.

METHODS

Apoptosis was analyzed by flow cytometry to evaluate the safety of IFN-γ priming, and indoleamine 2,3-dioxygenase (IDO) levels were measured by quantitative real-time reverse transcription polymerase chain reaction, western blotting, and ELISA to evaluate the efficacy of IFN-γ priming. In vivo, the liver I/R model was established in male C57/BL mice, hematoxylin and eosin and TUNEL staining was performed and serum liver enzyme levels were measured to assess the degree of liver injury, and regulatory T cell (Treg) numbers in spleens were determined by flow cytometry to assess immune tolerance potential. Metabolomics analysis was conducted to elucidate the potential mechanism underlying the regulatory effects of primed MenSCs. In vitro, we established a hypoxia/reoxygenation (H/R) model and analyzed apoptosis by flow cytometry to investigate the mechanism through which primed MenSCs inhibit apoptosis. Transmission electron microscopy, western blotting, and immunofluorescence were used to analyze autophagy levels.

RESULTS

IFN-γ-primed MenSCs secreted higher levels of IDO, attenuated liver injury, and increased Treg numbers in the mouse spleens to greater degrees than untreated MenSCs. Metabolomics and autophagy analyses proved that primed MenSCs more strongly induced autophagy in the mouse livers. In the H/R model, autophagy inhibitors increased the level of H/R-induced apoptosis, indicating that autophagy exerted protective effects. In addition, primed MenSCs decreased the level of H/R-induced apoptosis via IDO and autophagy. Further rescue experiments proved that IDO enhanced the protective autophagy by inhibiting the mammalian target of rapamycin (mTOR) pathway and activating the AMPK pathway.

CONCLUSION

IFN-γ-primed MenSCs exerted better therapeutic effects in the liver I/R model by secreting higher IDO levels. MenSCs and IDO activated the AMPK-mTOR-autophagy axis to reduce IRI, and IDO increased Treg numbers in the spleen and enhanced the MenSC-mediated induction of immune tolerance. Our study suggests that IFN-γ-primed MenSCs may be a novel and superior MSC product for liver transplantation in the future.

Keywords: Mesenchymal stem cells; Cell therapy; Reperfusion injury; T-lymphocytes; Autophagy; Liver

Core Tip: In this study, we identified a suitable interferon-γ (IFN-γ) priming strategy for menstrual blood-derived stromal cells (MenSCs) in the liver ischemia-reperfusion (I/R) model and proved that primed MenSCs could significantly increase the number of regulatory cells in the spleen by secreting higher levels of indoleamine 2,3- dioxygenase (IDO) and thereby exhibited better immunoregulatory potential. Besides, through metabolomics and related molecular biology experiments, we found that IDO could reduce ischemia-reperfusion injury via the AMPK-mammalian target of rapamycin-autophagy axis. Our study suggests that IFN-γ-primed MenSCs may be a novel and superior mesenchymal stem cell product for liver transplantation in the future.