Compact bone-derived mesenchymal stem cells ...

International Immunopharmacology 42 (2017) 67–73

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Compact bone-derived mesenchymal stem cells attenuate nonalcoholic steatohepatitis in a mouse model by modulation of CD4 cells differentiation Huafeng Wang a,⁎,1, Dong Wang b,1, Luhong Yang a,1, Yanxia Wang a, Junli Jia a, Dongchen Na a, Huize Chen a, Yongping Luo a, Chengfang Liu c,d,⁎⁎ a

Modern College of Arts and Science, or School of Life Science, Shanxi Normal University, Linfen, China Central Blood Station of Tianjin, Tianjin, China c Department of Human anatomy, Shanxi Medical University, Taiyuan, China d Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, China b

a r t i c l e

i n f o

Article history: Received 15 August 2016 Received in revised form 3 November 2016 Accepted 14 November 2016 Available online xxxx Keywords: Compact bone MSCs MCD diet NASH CD4+ lymphocytes

a b s t r a c t Increasing evidence has accrued which indicates that mesenchymal stem cells (MSCs) have a potential clinical value in the treatment of certain diseases. Globally, nonalcoholic steatohepatitis (NASH) is a widespread disorder. In the present study, MSCs were isolated successfully from compact bone and a mouse model of NASH was established as achieved with use of a methionine-choline deficient (MCD) diet. Compact bone-derived MSCs transplantation reduced MCD diet-induced weight loss, hepatic lipid peroxidation, steatosis, ballooning, lobular inflammation and fibrogenesis. It was shown that MSCs treatment hampered MCD diet-induced proliferation of CD4+ IFN-γ+ and CD4+ IL-6+ T spleen cells. In addition, CD4+ IL-17+ lymphocytes that associated with anti-inflammation show little change in MCD as well as in MCD + MSCs splenocytes. We conclude that MSCs may have a potential clinical value upon NASH, through their capacity to suppress activation of CD4+ IFN-γ+ and CD4+ IL6+ lymphocytes. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

1. Introduction Nonalcoholic steatohepatitis (NASH), progressive form of non-alcoholic fatty liver disease (NAFLD), is a significant predisposing factor for the development of cryptogenic cirrhosis and hepatic failure [1,2]. The prevalence of NAFLD correlates with the rising incidence of obesity and metabolic syndromes in the western world [2]. In the United States, the National Health and Nutrition Examination Surveys from 2009 to 2010 reported obesity rates of 35.5% among men and 35.8% among women [3]. In Asia, prevalence of NAFLD has been found to be in the range of 15–30% in the general population and exceeds 50% in patients with diabetes and metabolic syndromes [4]. In the general population of the United States, the prevalence of NASH is approximately 3% but increases to N25% in obese individuals [5]. Since the mid-1990s, ultrasound surveys for assessing fatty liver (any cause) were first published in mainland China [6,7]. From these surveys, the median

⁎ Corresponding author. ⁎⁎ Correspondence to: C. Liu, Department of Human Anatomy, Shanxi Medical University, Taiyuan, China. E-mail addresses: [email protected] (H. Wang), [email protected] (C. Liu). 1 These authors contributed equally to this work.

prevalence of ultrasonographic steatosis in the Chinese populations was found to be 10%, but ranged from 1% to N30% [6,8]. Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues [9]. Within the past few years, many studies have focused on the therapeutic effects of different types of stem cells, which can improve liver function via distinct mechanisms [10]. Bone marrow-derived MSCs transplantation was shown to be effective in rescuing experimental liver failure induced by carbon tetrachloride gavage [11]. It was also reported that MSCs can indirectly protect against hepatocyte death and increase survival in fulminant hepatic failure. Such effects resulted from the secretion of large fractions of chemotactic, cytokines or chemokines by MSCs [12]. Others have reported the hepatic engraftment of transplanted MSCs as observed in sublethal animal models of liver injury, further signifying the clinical potential for MSCs in the treatment of liver diseases [13–15]. However, the exact effects of MSCs upon NAFLD remain poorly understood. For a long time we have been interested in liver diseases. We have demonstrated that adiponectin-derived active peptide ADP355 protected the mice from hepatic inflammation and fibrosis [16]. We have also been interested in the isolation and characterization of mesenchymal stem cells (MSCs) and in the potential application of these cells to liver diseases especially [17,18]. We have used compact bone-

http://dx.doi.org/10.1016/j.intimp.2016.11.012 1567-5769/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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H. Wang et al. / International Immunopharmacology 42 (2017) 67–73

derived method to isolate and culture mouse MSCs (mMSCs) [19]. MSCs and their secreted molecules exhibited therapeutic potency in the models of carbon tetrachloride-induced liver fibrosis and thioacetamide-induced fulminant hepatic failure (FHF) by function as immuno-suppressor [19]. In the present study, we examined the effects of MSCs within NAFLD. To accomplish this goal, a mouse model of NASH was induced by treatment with a methionine-choline deficient (MCD) diet, whose histological changes occur rapidly and are morphologically similar to those observed in human NASH [1]. MSCs were isolated from compact bone according to procedures described previously [20]. We found that NASH induced by a MCD diet was attenuated when treated with MSCs, as indicated by reductions in weight loss, hepatic steatosis, hepatocyte ballooning, liver inflammation and fibrosis. The probable mechanism of this MSC-mediated immunomodulation likely results from an inhibition in the activation of CD4+ IFN-γ+ and CD4+ IL-6+ lymphocytes.

marrow being flushed and discarded with PBS by a syringe, the compact bones were excised into chips of about 1 mm, washed with PBS until the released cells were removed. Two weeks of being incubated in α-MEM containing 10% FBS later, mMSCs sprouted from chips and permeated in the dishes. Harvest the cells by a cell scraper. The primary obtained cells were then passaged by 0.25% trypsin-EDTA (GIBCO) digestion. After 5–8 passages, cells were used for the following experiments.

2. Materials and methods

Livers were perfused with PBS, removed, weighed and sliced into small sections (0.5 × 0.5 cm). Liver sections were embedded in paraffin after being fixed in 4% (weight/volume) paraformaldehyde and were cut into 6-μm-thick sections. Paraffin sections were stained with hematoxylin–eosin (HE) for assessing inflammation and steatosis and Picrosirius red for assessing fibrosis. Evaluation of the extent of resultant NASH [2] was performed using the following scaling scores:

2.1. Animals and diet (Fig. 1) Six- to eight-week-old male C57BL/6 mice were purchased from the Academy of Military Medical Science (Beijing, China) and were maintained on a lipogenic diet deficient in methionine and choline (MCD) as obtained from TROPHIC Animal Feed High-tech Co., Ltd. (Nantong, Jiangsu, China). The diet has made into solid by manufacturer and administrated randomly. At first, the mice were randomly divided into 2 groups: (i) Normal - controls fed with a regular diet, n = 6; (ii) MCD mice fed with the MCD diet, n = 12. Six weeks later, MCD - mice were randomized into 2 groups again: (ii) MCD - mice as before, n = 6; (iii) MCD + MSC - mice fed with the MCD diet supplemented with an intravenously injection of 1 × 106 MSCs administered at 6 and 7 weeks of the 10-week experiment, n = 6. At 10 weeks, all mice were euthanized and tissue was harvested. This experiment was approved by the Animal Ethics Committee of Tianjin Medical University. 2.2. Isolation and culture of bone-derived MSCs The way to isolate and culture mMSCs from murine compact bone was modified from the previously described one [19]. In brief, we first pulled down the skin and cut many of the muscles that attached to the hindlimbs and humeri by surgical scissors, and then disconnected them from the trunk and at axillaries. Use two forceps to remove remnants of the muscles and tendons attached to the bone. After bone

2.3. Examination of immunophenotypic features of MSCs MSCs prepared as described above were stained with the following fluorescein-linked antibodies: CD29, CD44, CD11b, CD45 or CD135 (all from eBioscience, CA, USA). Cells were analyzed using a FACScalibur (BD Biosciences, USA). Flow cytometry data were analyzed using Flow Jo 7.6 software (BD Biosciences, USA). 2.4. Histology of livers

Steatosis. Hepatocytes containing fat vacuoles were subjectively visualized and graded according to the following scale: 0 - normal, none of hepatocytes were affected, 1 - tiny, b 5% of hepatocytes were affected, 2 - mild, 5%–33% of hepatocytes were affected, 3 moderate, 34% - 66% of hepatocytes were affected and 4 - severe, N66% of hepatocytes were affected. Ballooning. 0 - none, 1 - rare or few, 2 - moderate and 3 - many. Lobular inflammation. 0 - None, 1 - 1-2 foci/20 × field, 2 - 2-4 foci/ 20 × field and 3 - N 4 foci/20 × field. Stages of NASH. 0 - none, 1 - extensive zone 3 perisinusoidal fibrosis, 2 - zone 3 perisinusoidal and portal or periportal fibrosis, 3 - bridging fibrosis and 4 - cirrhosis. 2.5. Hepatic lipid peroxidation Malondialdehyde (MDA) is one of major aldehydic metabolites of lipid peroxidation and has been used to reflect lipid peroxidation [21]. Lipid Peroxidation MDA Assay Kit (Beyotime, China) was used to measure MDA in the liver.

Fig. 1. Diagram of the experimental protocol. The mice were randomly divided into 3 groups: (i) Normal - controls fed with a regular diet, (ii) MCD - mice fed with the MCD diet only and (iii) MCD + MSC - mice fed with the MCD diet supplemented with an intravenously injection of 1 × 106 MSCs administered at 6 and 7 weeks of the 10-week experiment. At 10 weeks, all mice were euthanized and tissue was harvested.


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2.6. Analysis of spleen leukocytes

2.7. Statistical analysis

Single-cell suspensions derived from splenic tissues (splenocytes) were prepared by mechanical disruption, filtered through a 40-μm cell strainer (BD, USA), followed by lysis of red blood cells. For intracellular cytokine staining, splenocytes were resuspended in complete RPMI 1640 medium (Gibco, Auckland, NZ) supplemented with 1 mmol/l MEM sodium pyruvate, 0.1 mmol/l MEM nonessential amino acids, 2 mM L-glutamine, 100 IU penicillin, 100 mg/ml streptomycin and 4.5 × 10− 5 M 2-mercaptoethanol. The cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA), 1 μg/ml ionomycin (Enzo life sciences, Farmingdale, USA) and 3 μg/ml brefeldin A (eBioscience, CA, USA) for 5 h. Next, cells were stained for surface markers using rat anti-mouse CD4-APC (eBioscience, CA, USA). For intracellular staining, the cells were fixed and permeabilized with Cytofix/Cytoperm buffers (BD Biosciences, USA) for 20 min at 4 °C and then washed with permeabilization wash buffer (BD Biosciences, USA). The Fc receptors were blocked with 2% rat serum and 10% bovine serum albumin prior to intracellular cytokine staining followed by staining with rat anti-mouse IFN-γ-FITC, IL-17A-PE or IL-6-FITC (eBioscience, CA, USA). The cells were then analyzed using a FACSCalibur and the data generated were analyzed using FlowJo 7.6.1 software (Tree Star, Inc., USA).

Data were expressed as means ± SDs (standard deviations). Graph Pad PRISM (version 5.0) software were used for statistical analyses.

3. Results 3.1. Cells isolated from compact bone are characterized as MSCs As described above, MSCs were obtained from 2 to 3-week-old female C57BL/6 mice euthanized by cervical dislocation. Hematopoietic cells were depleted and released cells discarded by flushing with PBS or α-MEM [20]. After 1–2 weeks in culture, cells were observed to grow out from proximity of compact bone chips and appeared to be fibroblast-like (MSCs), but not polygon shaped (osteocytes) (Fig. 2A). To further identify adherent cells, immunophenotypic features were analyzed. As shown in the upper panel of Fig. 2B, the cells expressed surface markers characteristic of MSCs, such as CD29 and CD44; while negative surface markers of MSCs, such as CD11b, CD45 and CD135 were not expressed (Fig. 2B, lower panel). Taken together, these findings indicate that these cells were MSCs.

Fig. 2. Morphology and immunophenotypic features of MSCs isolated from mouse compact bone. (A) Photograph of MSCs morphology. Fibroblast-like MSCs grew out from the proximity of compact bone chips. (B) Immunophenotypic features of MSCs. As shown in the upper panel of B, the cells expressed surface markers characteristic of MSCs, such as CD29 and CD44; while negative surface markers of MSCs, such as CD11b, CD45 and CD135 were not expressed (B, lower panel). Representative images are shown for all panels.

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3.2. MCD diet-induced weight losses and oxidative stress were relieved by MSCs treatment

associated with anti-inflammation show little change in MCD as well as in MCD + MSCs splenocytes (Fig. 6 C).

MCD diet-induced NASH has been reported to be accompanied by weight loss [1]. Here, as compared with Normals, we also observed weight loss in MCD mice when recorded at weeks 6 and 7 of the experiment, prior to treatment with MSCs. Following MSC treatment, the MCD + MSC mice began to show weight gains (Fig. 3A). As the aldehydic metabolites of lipid peroxidation, MDA is often used to reflect lipid peroxidation [21]. It was demonstrated that MCD diet induced-hepatic MDA accumulation was hampered with MSCs treatment (Fig. 3B).

4. Discussion

3.3. MCD diet-induced steatosis, ballooning and lobular inflammation were attenuated by MSCs treatment Hepatocytes containing fat vacuoles can be easily visualized as clear bubbles following HE staining [2]. When compared with Normals, MCD diet induced lipid deposition and inflammation within the liver, effects that were reduced by MSCs treatment (Fig. 4A). As indicated by scores obtained from the scales described above, increases in steatosis (Fig. 4B), ballooning (Fig. 4C) and lobular inflammation (Fig. 4D) as induced by MCD were all decreased in response to MSC treatment. 3.4. MSCs treatment ameliorated MCD diet-induced liver fibrosis Progression of NASH is often accompanied with fibrosis [2]. Therefore, we also assessed the stages of fibrosis in these cells as achieved with picrosirius red staining. MCD fed mice showed a progression into extensive fibrogenesis within their livers whereas MSCs treatment reduced hepatic fibrogenesis as observed in MCD + MSC mice (Fig. 5A). Differences in fibrosis stage scores and fibrosis area between MCD and MCD + MSCs mice were significantly different (Fig. 5B, C). 3.5. MSCs treatment reduced percent of CD4+ IFN-γ+ and CD4+ IL-6+ T spleen cells MSCs can interact with both innate and adaptive immune system cells, leading to the modulation of several different effector functions [9]. Analysis of spleen leukocytes was performed to explore the effect of MSCs on immunological responses. We found that as compared with Normals, percent of CD4+ IFN-γ+ and CD4+ IL-6+ lymphocytes that were associated with pro-inflammation were decreased with MSCs (Fig. 6 A, B). However, percent of CD4+ IL-17+ lymphocytes that

MSCs, also known as multipotent mesenchymal stromal cells, were originally isolated by Friedenstein and his colleagues from bone marrow in 1976 [22], and have subsequently been found to exist in other organs and tissues [20]. However, the method established by Friedenstein as based on plastic adherence, has proved unsuccessful for mouse MSCs (mMSCs) due to the low frequency of MSCs and the contamination of hematopoietic cells in the culture [23]. A recent adaption of this protocol, which has offered an easy and reproducible method to harvest mouse MSCs, has been provided by Heng Zhu [20]. In the present study, we utilized this method and successfully isolated MSCs that were fibroblast-like and expressed putative surface markers of MSCs such as CD29 and CD44, but not CD11b, CD45 and CD135, which was consistent with a previous report [20]. Increasing evidence has been accrued which indicate that MSCs have a potential clinical value in the treatment of certain diseases [9]. Interestingly, there is limited data available that show any promising or desirable therapeutic effects of MSCs in ameliorating fulminant hepatic failure, chronic liver fibrosis and cirrhosis. Findings from a previous report have demonstrated that fulminant hepatic failure can be induced in Sprague-Dawley rats with an intraperitoneally administration of two injections of 1.2 g/kg hepatotoxin, D-galactosamine. When these rats were treated 24 h later with an intravenous injection of MSCs lysates, cellular lysates showed an increased survival trend [12]. In the carbon tetrachloride-induced hepatic fibrosis model, Flk1 mouse MSCs were systemically infused immediately or at 1 week after mice were challenged with carbon tetrachloride. In these mice, mMSCs administration immediately and significantly reduced carbon tetrachloride-induced liver damage and collagen deposition [14]. In a previous study, we indicated that compact bone-derived mMSCs ameliorated gross and microscopic liver histopathology and reduced death of mice in thioacetamide-induced FHF and also suppressed carbon tetrachlorideinduced chronic liver fibrosis [19]. In addition, we also demonstrated that MSC-CM tendentiously improves chronic liver fibrosis [19]. Recently, it was reported by Boeykens that MSC-therapy could have a limited effects on NASH, which was observed that MCD diet model both serum CHE and TG levels of non-treated and cell-treated rats with PHx after MCD remained significantly lower as compared to those of control rats [24]. Despite this, Boeykens also found that enhanced the recovery to normal body weight, liver weight and NAS

Fig. 3. MCD diet-induced weight loss and hepatic lipid oxidation stress was hampered by MSCs treatment. (A) Effects of MCD diet on body weight growth. P value was examined by repeated measures ANOVA. (B) MSCs treatment with an intravenously injection of 1 × 106 MSCs administered at 6 and 7 weeks reversed MCD diet-induced weight loss. P value was examined by repeated measures ANOVA. (C) Hepatic lipid oxidation stress was determined by Lipid Peroxidation MDA Assay Kit. *P b 0.05 with t-TEST.


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Fig. 4. MCD diet-induced steatosis, ballooning, and lobular inflammation were ameliorated by MSCs treatment. (A) HE staining of mice liver sections. Fat vacuoles (black arrows) and inflammation foci (green arrows) were reduced by MSCs treatment in MCD + MSCs mice. Scale scores of steatosis (B), ballooning (C) and lobular inflammation (D) were all significantly decreased in MCD + MSCs versus MCD mice. Representative images are shown for all panels. The data are presented as the means ± SDs. *P b 0.05; **P b 0.01 with t-TEST.

score [24]. In the present study, we demonstrated that MSCs treatment significantly reduced MCD diet-induced weight loss, steatosis, ballooning and lobular inflammation, which consistent with the report described above. Altogether, it could be yet proposed that MSCs may possess a potential clinical value upon NASH. A primary basis for disease protection afforded by MSCs likely involves their potent anti-inflammatory and immunomodulatory activities [25]. Mice injected with MSCs (5 × 106 MSCs/kg body weight) showed that host T-cell responses to donor alloantigens were decreased in the majority of recipients [26]. There are data which indicate that MSCs did not preferentially target any T-cell subset, and their capacity for inhibition extended to B cells, with the result being that MSC-mediated inhibition induces an unresponsive T-cell profile. Such findings are consistent with that observed in division arrest energy [27]. In our previous study, we also demonstrated the immunosuppressive effects of MSC treatment, converting the body into an anti-inflammatory state by up-regulating anti-inflammatory Treg cells and reducing pro-inflammatory Th1 and Th7 cells in thioacetamide-induced FHF and carbon tetrachloride -induced chronic liver fibrosis [19]. In the present study, among potential spleen leukocytes, we selected CD4+ T cells to explore the immunomodulatory effects of MSCs on NASH. We found that MSCs treatment suppressed multiplication of CD4+ IFN-γ+ and CD4+ IL-6+ T

spleen cells, which was consistent with previous reports [25]. The molecular mechanism may be due to the molecules secreted by MSCs [19]. Accordingly, we conclude that MSCs-mediated immunosuppression accounts for the ameliorative effects upon NASH. In summary, MSCs isolated from compact bone have potential clinical value upon NASH (summarized in Fig. 7). MSCs transplantation enhances the resistance to MCD diet-induced NASH, as indicated by several parameters including reductions in MCD diet-induced weight loss, steatosis, ballooning and lobular inflammation. As based upon these findings, the ameliorative effects upon NASH by MSCs likely results from suppression of activation of CD4+ IFN-γ+ and CD4+ IL-6+ lymphocytes. However, the effectiveness and mechanics of treatment in regards to gender and age of animals are unclear and remain to explore in the future.

Author contributions H.W. and D.W. conceived and designed the experiments. D.W., J.J., H.C., C.L. and Y. L. performed the experiments. D.N., C.L., Y. W. and L.Y. collected and analyses data. H.W., and L.Y. wrote the manuscript. H.W. and C.L. contributed to the guidance of experiments and contributed to the final manuscript. All authors reviewed the manuscript.

Fig. 5. Improvement of MCD diet-induced liver fibrogenesis by MSCs. (A) Picrosirius red staining of mice liver sections. (B) Scale scores of liver fibrosis. (C) Quantification of fibrosis area. Representative images are shown for all panels. The data are presented as the means ± SDs. *P b 0.05 with t-TEST.

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Fig. 6. Effects of MSCs on CD4+ T cells differentiation. For intracellular cytokine staining, splenocytes were treated with 50 ng/ml PMA, 1 μg/ml ionomycin and 3 μg/ml brefeldin A for 5 h. The cells were stained for surface markers using rat anti-mouse CD4. For intracellular staining, the cells were fixed and permeabilized with Cytofix/Cytoperm buffers for 20 min at 4 °C and then washed with permeabilization wash buffer. The Fc receptors were blocked with 2% rat serum and 10% bovine serum albumin prior to intracellular cytokine staining. Next, the cells were stained with rat anti-mouse IFN-γ, IL-6 and IL-17A. (A) CD4+ IFN-γ+, (B) CD4+ IL-6+ and (C) CD4+ IL-17A+ lymphocytes was analyzed by flow cytometry. Representative data are shown for all panels.

Fig. 7. Compact bone-derived MSCs have potential clinical value upon NASH. MSCs transplantation enhances the resistance to MCD diet-induced NASH, as indicated by several parameters including reductions in MCD diet-induced weight loss, steatosis, ballooning and lobular inflammation. As based upon these findings, the ameliorative effects upon NASH by MSCs likely results from suppression of activation of CD4+ IFN-γ+ and CD4+ IL-6+ lymphocytes.


Conflicts of interest No potential conflict of interest relevant to this article was reported. Acknowledgements This work was supported by One College One Policy Project, Modern College of Arts and Science, Shanxi Normal University (2016KJYJ-6); Scientific Research Foundation for Doctor, Shanxi Normal University (0505/02070293); Shanxi Provincial University Science and Technology Innovation Project (20161107); The Natural Science Foundation of Shanxi Province (2015021177). References [1] M.E. Rinella, M.S. Elias, R.R. Smolak, T. Fu, J. Borensztajn, R.M. Green, Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet, J. Lipid Res. 49 (2008) 1068–1076. [2] M. Ahmed, Non-alcoholic fatty liver disease in 2015, World J. Hepatol. 7 (2015) 1450–1459. [3] K.M. Flegal, M.D. Carroll, B.K. Kit, C.L. Ogden, Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999–2010, JAMA 307 (2012) 491–497. [4] V.W.S. Wong, Nonalcoholic fatty liver disease in Asia: a story of growth, J. Gastroenterol. Hepatol. 28 (2013) 18–23. [5] G. Vernon, A. Baranova, Z. Younossi, Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults, Aliment. Pharmacol. Ther. 34 (2011) 274–285. [6] J. Fan, Steatohepatitis studies in China, World Chinese Journal of Digestology 9 (2001) 6–10. [7] J.G. Fan, G.C. Farrell, Epidemiology of non-alcoholic fatty liver disease in China, J. Hepatol. 50 (2009) 204–210. [8] H. Zhang, H. Zhuang, X. Liu, [Advances in the epidemiological study of fatty liver]. Zhonghua liu xing bing xue za zhi=, Zhonghua liuxingbingxue zazhi. 25 (2004) 630–632. [9] A. Uccelli, L. Moretta, V. Pistoia, Mesenchymal stem cells in health and disease, Nat. Rev. Immunol. 8 (2008) 726–736. [10] E.S. Gilchrist, J.N. Plevris, Bone marrow-derived stem cells in liver repair: 10 years down the line, Liver Transpl. 16 (2010) 118–129. [11] T.K. Kuo, S.P. Hung, C.H. Chuang, C.T. Chen, Y.R.V. Shih, S.C.Y. Fang, et al., Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells, Gastroenterology 134 (2008) 2111–2121, e3.

73

[12] B. Parekkadan, D. Van Poll, K. Suganuma, E.A. Carter, F. Berthiaume, A.W. Tilles, et al., Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure, PLoS One 2 (2007), e941. [13] Y. Sato, H. Araki, J. Kato, K. Nakamura, Y. Kawano, M. Kobune, et al., Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion, Blood 106 (2005) 756–763. [14] B. Fang, M. Shi, L. Liao, S. Yang, Y. Liu, R.C. Zhao, Systemic infusion of FLK1+ mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice, Transplantation 78 (2004) 83–88. [15] I. Aurich, L.P. Mueller, H. Aurich, J. Luetzkendorf, K. Tisljar, M.M. Dollinger, et al., Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers, Gut 56 (2007) 405–415. [16] H. Wang, H. Zhang, Z. Zhang, B. Huang, X. Cheng, D. Wang, et al., Adiponectin-derived active peptide ADP355 exerts anti-inflammatory and anti-fibrotic activities in thioacetamide-induced liver injury, Sci. Rep. 6 (2016) 19445. [17] B. Huang, G. Li, X.H. Jiang, Fate determination in mesenchymal stem cells: a perspective from histone-modifying enzymes, Stem Cell Research & Therapy 6 (2015) 35. [18] C. Ke, H. Biao, L. Qianqian, S. Yunwei, J. Xiaohua, Mesenchymal stem cell therapy for inflammatory bowel diseases: promise and challenge, Current Stem Cell Research & Therapy 10 (2015) 499–508. [19] B. Huang, X. Cheng, H. Wang, W. Huang, H.Z. la Ga, D. Wang, et al., Mesenchymal stem cells and their secreted molecules predominantly ameliorate fulminant hepatic failure and chronic liver fibrosis in mice respectively, J. Transl. Med. 14 (2016) 45. [20] H. Zhu, Z.K. Guo, X.X. Jiang, H. Li, X.Y. Wang, H.Y. Yao, et al., A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone, Nat. Protoc. 5 (2010) 550–560. [21] Y. Wang, L.M. Ausman, R.M. Russell, A.S. Greenberg, X.-D. Wang, Increased apoptosis in high-fat diet–induced nonalcoholic steatohepatitis in rats is associated with c-Jun NH2-terminal kinase activation and elevated proapoptotic Bax, J. Nutr. 138 (2008) 1866–1871. [22] A.J. Friedenstein, J. Gorskaja, N. Kulagina, Fibroblast precursors in normal and irradiated mouse hematopoietic organs, Exp. Hematol. 4 (1976) 267–274. [23] D.G. Phinney, G. Kopen, R.L. Isaacson, D.J. Prockop, Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation, J. Cell. Biochem. 72 (1999) 570–585. [24] N. Boeykens, P. Ponsaerts, A. Van der Linden, Z. Berneman, D. Ysebaert, K. De Greef, Injury-dependent retention of intraportally administered mesenchymal stromal cells following partial hepatectomy of steatotic liver does not lead to improved liver recovery, PLoS One 8 (2013), e69092. [25] D.C. Ding, W.C. Shyu, S.Z. Lin, Mesenchymal stem cells, Cell Transplant. 20 (2011) 5–14. [26] K.J. Beggs, A. Lyubimov, J.N. Borneman, A. Bartholomew, A. Moseley, R. Dodds, et al., Immunologic consequences of multiple, high-dose administration of allogeneic mesenchymal stem cells to baboons, Cell Transplant. 15 (2006) 711–721. [27] S. Glennie, I. Soeiro, P.J. Dyson, E.W. Lam, F. Dazzi, Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells, Blood 105 (2005) 2821–2827.