Cell Transplantation, Vol. 21, pp. 2753–2764, 2012 Printed in the USA. All rights reserved. Copyright 2012 Cognizant Comm. Corp.
0963-6897/12 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X652959 E-ISSN 1555-3892 www.cognizantcommunication.com
Adipose-Derived Stem Cells Can Abrogate Chemical-Induced Liver Fibrosis and Facilitate Recovery of Liver Function Horng-Jyh Harn,*†1 Shinn-Zong Lin,‡§1 Shih-Hsiao Hung,¶ Yi-Maun Subeq,# Yuan-Sheng Li,¶ Wan-Sin Syu,¶ Dah-Ching Ding,** Ru-Ping Lee,# Dean-Kuo Hsieh,†† Po-Cheng Lin,‡‡ and Tzyy-Wen Chiou¶ *Department of Pathology, China Medical University Hospital, Taichung, Taiwan, ROC †Department of Medicine, China Medical University, Taichung, Taiwan, ROC ‡Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan, ROC §Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin, Taiwan, ROC ¶Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, ROC #Department of Nursing, Tzu Chi University, Hualien, Taiwan, ROC **Department of Obstetrics and Gynecology, Buddhist Tzu Chi General Hospital, Tzu Chi University, Hualien, Taiwan, ROC ††Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan, ROC ‡‡Department of Research and Development, Gwo Xi Stem Cell Applied Technology Co., Ltd., Hsinchu, Taiwan, ROC
Adipose-derived stem cells (ADSCs) are easy to harvest and have the ability for self-renewal and to differentiate into various cell types, including those of the hepatic lineage. Studies on the use of ADSCs for liver transplanta tion are, however, limited. The objective of this study was to investigate the feasibility of using human ADSCs and to better understand their mechanism of action for the repair of liver damage in a thioacetamide (TAA)induced model of chronic liver damage in the rat. To induce liver damage, 200 mg/kg TAA was injected intraperitoneally into Wistar rats every 3 days for 60 days. For cell therapy, 1 × 106 human ADSCs suspended in 300 ml of phosphate-buffered saline were transplanted into each experimental rat by direct liver injection. Immunohistochemistry showed that the transplanted ADSCs differentiated into albumin- and a-fetoproteinsecreting liver-like cells 1 week after transplantation. In addition, liver function recovered significantly, as determined by biochemical analyses that analyzed total bilirubin, prothrombin time, and albumin levels. The Metavir score, derived from histopathological analysis, also showed a significant decrease in liver fibrosis and inflammatory activity after ADSC transplantation. Finally, we found a reduction in the expression of a-smooth muscle actin, a marker of hepatic stellate cells, which produce collagen fiber, and an increase in the expression of matrix metalloproteinase-9, which degrades collagen fiber, after ADSC transplantation. These findings are consistent with abrogation of liver fibrosis in the ADSC therapy group. Consequently, these results suggest that ADSC transplantation may facilitate recovery from chronic liver damage and thus may have clinical applications. Key words: Adipose-derived stem cells (ADSCs); Thioacetamide; Liver fibrosis; Matrix metalloproteinase-9 (MMP-9)
INTRODUCTION Liver fibrosis is a consequence of severe liver damage and follows many forms of chronic liver damage, including viral infection, autoimmune liver disease, and sustained alcohol abuse (1). It is a chronic progressive disorder with no definitive treatment. The main goals of liver-directed cell therapy are the replacement of diseased hepatocytes and the stimulation of endogenous or exogenous stem cell regeneration. In a rat model of liver injury, Petersen et al. first demonstrated that
liver stem cells are derived from the bone marrow (BM) (16). Subsequently, BM stem cells, including mesenchymal stem cells (MSCs), were proposed as a reservoir for liver repair. Although MSCs were first identified in the BM, they can also be isolated from human umbilical cord blood, synovium, placenta, periosteum, skeletal muscle, and adipose tissue (20). Adipose-derived stem cells (ADSCs) are characterized as being similar to BM-derived MSCs. They can differentiate into neurogenic, myogenic, osteogenic, and chondrogenic lineages (24,25). More importantly, in
Received November 13, 2011; final acceptance February 25, 2012. Online prepub date: July 5, 2012. 1 These authors provided equal contribution to this work. Address correspondence to Tzyy-Wen Chiou, Ph.D., Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, No. 1, Sec. 2, Da Hsueh Road, Shoufeng, Hualien 97401, Taiwan, Republic of China. Tel: +886-3-8630398; E-mail:
[email protected]
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comparison with other types of adult stem cells, ADSCs are easily accessible and abundant. As a consequence, many experiments with ADSCs are now under way. In a model of liver fibrosis, Kiso and colleagues (8) demonstrated a decrease in hepatic fibrosis after transplantation of ADSCs that were pretreated with basic fibroblast growth factor (bFGF) into C57BL6/J mice injured by carbon tetrachloride. Furthermore, in the rat carbon tetrachloride liver injury model, intravenous injection of ADSCs into recipient livers significantly decreases serum levels of liver enzymes and improves serum levels of albumin (11). In addition, the phosphorylation of extracellular signalrelated kinases 1/2 (ERK1/2) is significantly upregulated during the hepatogenic differentiation of ADSCs (11). We recently demonstrated that BM-derived MSCs and Wharton’s jelly stem cells contribute to improved liver function of chemical-induced liver injury with progressive fibrosis (3,12). Stem cells derived from adipose tissue are an alternative source that can be obtained by a less invasive method and in larger quantities than stem cells derived from bone marrow (9). Moreover, our previous work involved the administration of stem cells into the portal vein, which is a tedious method that requires a well-trained surgeon. These considerations led us to investigate the possibility of using ADSC transplantation to abrogate liver fibrosis in a rat model of chemical-induced liver injury. In the current study, we used naive ADSCs and direct administration of ADSCs into the thioacetamide (TAA)-induced liver. We investigated the effects of the engrafted ADSCs on the function of recipient livers. We also analyzed the migration of the implanted ADSCs and the potential of transplanted ADSCs to differentiate into hepatic-like cells. In addition, we used immuno histochemistry to examine the expression of a-smooth muscle actin (a-SMA), a marker of stellate cells, and matrix metalloproteinase-9 (MMP-9), which degrades collagen fiber, to investigate the possible mechanisms through which the transplanted ADSCs are able to abrogate liver fibrosis. MATERIALS AND METHODS Patient ADSC Donors Female donors who underwent gynecological surgery were enrolled in this study. The ADSC03 cell line (AD03) was derived from a 65-year-old female, and the ADSC27 cell line (AD27) was derived from a 27-year-old female. ADSCs at passages 3–5 were used in this study. The Research and Ethics Committee of China Medical University Hospital approved all procedures used in this study, and informed consent was obtained from each subject before tissue collection. Isolation of Human ADSCs Human adipose tissue was harvested from the subcutaneous fat (1 cm3) of the abdominal wall during gynecologic surgery. Tissue samples were placed in Ca2+/
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Mg2+-free phosphate-buffered saline (PBS) and immediately transferred to the laboratory. Human adipose tissue was removed from the transport medium, placed in a Petri dish, and cut into small pieces (1–2 mm3) in the presence of Ca2+/Mg2+-free PBS. The tissues were dissociated with 0.1 mg of collagenase IA (Sigma-Aldrich, St. Louis, MO) and incubated for 60 min at 37°C. After enzymatic digestion, the resultant cells were collected and cultured in knockout serum-free medium (Invitrogen-Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT), L2 ascorbic acid, and phosphate. The supernatant and debris were removed from the culture dish on day 2 of culture. The resultant ADSC culture was denoted as passage 0. To prevent spontaneous differentiation, cultures were maintained at subconfluent levels (
