Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Nov 26, 2019; 11(11): 990-1004
Published online Nov 26, 2019. doi: 10.4252/wjsc.v11.i11.990
Ameliorating liver fibrosis in an animal model using the secretome released from miR-122-transfected adipose-derived stem cells
Kee-Hwan Kim, Jae Im Lee, Ok-Hee Kim, Ha-Eun Hong, Bong Jun Kwak, Ho Joong Choi, Joseph Ahn, Tae Yun Lee, Sang Chul Lee, Say-June Kim
Kee-Hwan Kim, Jae Im Lee, Department of Surgery, Uijeongbu St. Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul 11765, South Korea
Kee-Hwan Kim, Ok-Hee Kim, Ha-Eun Hong, Say-June Kim, Catholic Central Laboratory of Surgery, Institute of Biomedical Industry, College of Medicine, the Catholic University of Korea, Seoul 06591, South Korea
Ok-Hee Kim, Ha-Eun Hong, Bong Jun Kwak, Ho Joong Choi, Joseph Ahn, Tae Yun Lee, Say-June Kim, Department of Surgery, Seoul St. Mary’s Hospital, College of Medicine, the Catholic University of Korea, Seoul 06591, South Korea
Sang Chul Lee, Department of Surgery, Daejeon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 34943, South Korea
Author contributions: Kim SJ was responsible for planning the study, data interpretation, and manuscript preparation; Kim KH and Kim SJ wrote paper, complemented in vivo experiments, and data interpretation; Kim OK and Hong HE performed in vitro experiments; Lee JI, Kwak BJ, Choi HJ, Ahn J, Lee TR and Lee SC were involved in various in vivo experiments and data interpretation. All authors read and approved the manuscript.
Supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT), No. NRF-2015R1D1A1A01060721.
Institutional review board statement: This research was approved by Institutional Review Board, IRB No. 700069-201407-BR-002-01, of Hurim BioCell Co. Ltd. (Seoul, Korea).
Institutional animal care and use committee statement: Animal studies were carried out in compliance with the guidelines of the Institute for Laboratory Animal Research, Korea, IRB No: CUMC-2017-0317-04.
Conflict-of-interest statement: The authors have declared no potential conflicts of interest.
Data sharing statement: Requests for access to data should be addressed to 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 which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (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: Say-June Kim, MD, PhD, Professor, Department of Surgery, Seoul St. Mary’s Hospital, College of Medicine, the Catholic University of Korea, 222, Banpo-daero, Seocho-gu, Seoul 06591, South Korea. sayjunekim@gmail.com
Telephone: +82-2-22587636 Fax: +82-2-5350070
Received: March 12, 2019
Peer-review started: March 15, 2019
First decision: July 31, 2019
Revised: September 2, 2019
Accepted: September 13, 2019
Article in press: September 13, 2019
Published online: November 26, 2019
Processing time: 238 Days and 1.4 Hours
ARTICLE HIGHLIGHTS
Research background

The therapeutic potential of mesenchymal stem cells (MSCs) is known to be mediated mainly by the secretome that refers to the total collection of secretory materials from MSCs. Basically, naïve secretome has anti-inflammatory, immunomodulatory, and tissue reparative properties. To increase the amount or to reinforce the potential of naïve secretome, researchers have attempted to adjust physico-chemical environment of MSCs or genetically manipulate MSCs. The former has the advantage of being simple but lacking persistence, while the latter has a strong persistence but has the disadvantage of a safety concern in the clinical application.

Research motivation

We have been considering genetic modification as a way of persistently potentiating the therapeutic potential of naïve secretome. In addition, contrasted by the use of genetically modified MSCs, we thought that the use of the secretome could significantly lower the safety concern. We also noted miRNAs as the materials to be used for genetic manipulation, because miRNA is critically involved in the process of liver fibrosis.

Research objectives

Our aim was to determine the antifibrotic potential of the secretome released from miR-122-transfected adipose-derived stromal cells (ASCs) in the model of liver fibrosis.

Research methods

Secretory materials released from ASCs that had been transfected with antifibrotic miR-122 were collected and termed as miR122-secretome. The in vitro model of liver fibrosis was generated by treating human hepatic stellate cells (LX2 cells) with a hepatotoxin (thioacetamide; TAA), and the in vivo model of liver fibrosis was generated by subcutaneous injection of TAA (200 mg/kg, three times a week for 8 wk) into five-week male BALB/c mice. For determining in vivo effects of miR122-secretome, each secretome (miR122-secrectome and naïve secretome) was intravenously administered to the mice with liver fibrosis, respectively. The degree of liver fibrosis and other alternations in cells or tissues were determined using by molecular and histological investigations, including cell viability assay, western blotting, immunohistochemistry, serology tests, and sandwich enzyme-linked immunosorbent assays.

Research results

The addition of miR-122-secretome to fibrosis-induced LX2 cells significantly decreased the expression of fibrotic markers (MMP2, TGF-β1, TIMP-1, and α-SMA) and increased the expression of an antifibrotic marker (TIMP-1). The western blot analysis showed that miR122-secretome infusion significantly increased the expression of PCNA (a proliferation marker), significantly decreased the expression of α-SMA, TGF-β1, and MMP1 (fibrotic markers), and increased an antifibrotic marker (TIMP-1) in the livers of TAA-treated mice. In addition, miR122-secretome infusion significantly reduced collagen content in the livers, inhibited serum levels of proinflammatory cytokines, such as IL-6 and TNF-α, as well as serum levels of liver enzymes than infusion of the naïve secretome. Finally, our analysis of the components of miR-122-secretome showed that miR-122-secretome exhibited a significantly decreased concentration of essential intermediates of the TGF-β/Smad signaling, such as transgelin, PIN1, and profilin-1, compared to NCM.

Research conclusions

miR-122-secretome was found to be superior to the naïve secretome in improving liver fibrosis while minimizing inflammatory processes in mice with TAA-induced liver fibrosis. Our proteomic analysis of the miR-122-secretome also validated that miR-122-secretome had significantly lesser contents of essential intermediates of liver fibrosis. Therefore, transfecting miR-122 into ASCs is worth considering as a way of reinforcing antifibrotic properties of the secretome from ASCs.