Cirrhosis is a chronic liver disease that impairs hepatic function and causes advanced fibrosis. Mesenchymal stem cells have gained recent popularity as a regenerative therapy since they possess immunomodulatory functions. We found that injected adipose tissue-derived stem cells (ADSCs) reside in the liver. Injection of ADSCs also restores albumin expression in hepatic parenchymal cells and ameliorates fibrosis in a nonalcoholic steatohepatitis model of cirrhosis in mice. Gene expression analysis of the liver identifies up- and down-regulation of genes, indicating regeneration/repair and anti-inflammatory processes following ADSC injection. ADSC treatment also decreases the number of intrahepatic infiltrating CD11b+ and Gr-1+ cells and reduces the ratio of CD8+/CD4+ cells in hepatic inflammatory cells. This is consistent with down-regulation of genes in hepatic inflammatory cells related to antigen presentation and helper T-cell activation. Conclusion: These results suggest that ADSC therapy is beneficial in cirrhosis, as it can repair and restore the function of the impaired liver. (Hepatology 2013;53:1133–1142)
Cirrhosis is a serious, life-threatening advanced stage of chronic liver disease that leads to hepatic dysfunction. Cirrhosis frequently develops into hepatocellular carcinoma,[2, 3] which exacerbates the prognosis of patients with cirrhosis. The ultimate treatment for cirrhosis is a liver transplant, which can be lethal. The number of donor livers, however, is not sufficient to meet the needs of all transplant patients. Thus, a novel therapy for cirrhosis needs to be developed to improve cirrhotic liver prognosis.
The underlying pathogenesis of chronic liver disease is persistent inflammation. Advanced disease is marked by advanced fibrosis concomitant with distorted liver architecture characterized by regenerative nodules and impaired hepatic function. Advanced fibrosis in the cirrhotic liver is also a risk factor for the development of hepatocellular carcinoma. Treatment of cirrhosis suppresses inflammation by eradicating hepatitis virus infection or reducing liver steatosis in nonalcoholic steatohepatitis (NASH). Decreasing liver inflammation and restoring hepatocyte function improves the prognosis.
Pluripotent mesenchymal stem cells (MSCs) differentiate into adipocyte, chondrocyte, and osteocyte lineages. These cells can also differentiate into other lineages, including neurons and hepatocytes.[9, 10] MSCs can also regulate the immune response. Thus, MSCs attract attention as a therapeutic target in the regeneration or repair of various impaired organs. Mesenchymal tissue from bone marrow, umbilical cord, and adipose tissue are relatively enriched with pluripotent stem cells. Since the pathophysiological features of liver cirrhosis are a consequence of chronic hepatic inflammation, MSCs are especially suited to enhance regeneration and/or repair of damaged cirrhotic liver.
We have established a clinically relevant NASH cirrhotic murine model by feeding animals an atherogenic high-fat (Ath+HF) diet. In this study we examined whether adipose-tissue-derived stem cells (ADSCs) can regenerate and/or repair the cirrhotic liver. We observed that injected ADSCs resided in the liver and expressed albumin, leading to restored albumin expression in hepatic parenchymal cells. ADSCs also ameliorated advanced fibrosis. Moreover, ADSCs suppressed the underlying persistent inflammation contributed by granulocytes, phagocytic cells, and T cells. These results suggest that treatment of patients with cirrhosis with ADSCs is a potentially novel approach for regenerating and/or repairing damaged cirrhotic liver tissue to restore hepatic function.
Materials and Methods
Culture of ADSCs
ADSCs were prepared as described. Briefly, adipose tissue was obtained from the inguinal subcutaneous region of 10-week-old GFP-Tg male mice (a gift from Professor Okabe, Osaka University, Japan). The stem cell fraction was isolated from adipose tissue using type-I collagenase (Wako Pure Chemical Industries, Osaka, Japan) and cultured in Dulbecco's modified Eagle's medium: nutrient mixture F-12 supplemented with 10% heat-inactivated bovine serum albumin and 1% antibiotic-antimycotic solution. Cell culture reagents were purchased from Life Technologies (Carlsbad, CA).
NASH Murine Model
Female 8-week-old C57Bl/6J mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). Mice were fed an Ath+HF diet composed of cocoa butter, cholesterol, cholate, and corticotropin-releasing factor-1 (Oriental Yeast Co., Tokyo, Japan) to induce steatohepatitis as reported previously. Our Institutional Review Board approved the care and use of laboratory animals in all experiments.
ADSC Treatment of NASH Mice
ADSCs were harvested after six to eight passages in culture by treatment with trypsin/EDTA (Life Technologies) and passed through a 100-μm Cell Strainer mesh (BD Biosciences, San Jose, CA). Laparotomy was performed to inject 1 × 105 ADSCs or phosphate-buffered saline (PBS) into the splenic subcapsule. After ADSC treatment, the mice were anesthetized with pentobarbital (40 mg/kg; Kyoritsu Seiyaku, Tokyo, Japan), after which the liver was perfused with PBS and dissected. A portion of liver tissue was homogenized and incubated with type I collagenase (Wako Pure Chemical Industries), and hepatic parenchymal cells and inflammatory cells were separated with Percoll (GE Healthcare UK, Buckinghamshire, UK). CD4+ T cells were isolated from hepatic inflammatory cells using a magnetic sorting system, the CD4+ T cell Isolation Kit II (Miltenyi Biotec, Gladbach, Germany).
Histology and Immunohistochemical Staining
Liver tissue was preserved with formalin for paraffin embedding or embedded in OCT compound and frozen for sectioning (Sakura Finetek Japan, Tokyo, Japan). The frozen liver sections were fixed in acetone and endogenous peroxidase activity blocked with 3% hydrogen peroxide solution. After washing in PBS, the sections were incubated with a rabbit anti-CD11b antibody (BD Pharmingen, San Diego, CA) and a rabbit anti-Gr-1 antibody (eBioscience, San Diego, CA) overnight at 4°C. The slides were then washed and incubated with Histofine mouse MAXPO (Nichirei Bioscience, Tokyo, Japan) for 1 hour at room temperature. The immune complex was visualized by incubating with diaminobenzidine for 5 minutes. The paraffin-embedded sections were stained with a rabbit-anti-GFP antibody (Millipore, Billerica, MA), a rabbit anti-α-smooth muscle actin (α-SMA) antibody (Abcam, Cambridge, UK), and a rabbit anticollagen IV antibody (Abcam). Secondary antibody development was performed with diaminobenzidine as described above. In some experiments, the sliced sections were double-stained with a combination of a goat antimouse serum albumin antibody (Abcam) and a rabbit anti-GFP antibody followed by the secondary antibody and development as described above. To quantify fibrosis, paraffin-embedded sections were stained with Azan and viewed microscopically, after which the stained area was calculated using an image-analysis system (BIOREVO BZ-9000 and BZ-H1C, Keyence Japan, Osaka, Japan).
Isolated hepatic inflammatory cells were incubated in PBS supplemented with 2% bovine serum albumin (Sigma-Aldrich, St. Louis, MO) for 10 minutes at 4°C. The cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience) and phycoerythrin (PE)-conjugated anti-CD8 antibodies (eBioscience) for 30 minutes at 4°C before examination using a FACSCalibur cytometer (BD Biosciences). Similarly, ADSCs were incubated with PE-conjugated CD90 (Beckman Coulter, Fullerton, CA), or PE-conjugated CD105 (Miltenyi Biotec). The data were analyzed using the FlowJo software (Tree Star, Ashland, OR).
DNA Microarray Analysis
Isolated RNAs were amplified and labeled with Cy3 using a QuickAmp Labeling Kit (Agilent Technologies, Santa Clara, CA) in accordance with the manufacturer's protocol. cRNA (825 ng) was hybridized onto a Whole Mouse Genome 4 × 44K Array (Agilent Technologies). The hybridized microarray slide was scanned using a DNA microarray scanner (model G2505B; Agilent Technologies).
Gene expression analysis was carried out using GeneSpring analysis software (Agilent Technologies). Each measurement was divided by the 75th percentile of all measurements in that sample to normalize per chip. Hierarchical clustering and principal component analysis of gene expression was performed. Welch's t test with Benjamini and Hochberg's false-discovery rate were used to identify differentially expressed genes in the groups of interest. Analysis of biological processes was performed using the MetaCore software suite (GeneGo, San Diego, CA). BRB array tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) were also used for unsupervised clustering or one-way clustering analysis. Microarray data were deposited in the NBCI Gene Expression Omnibus (GSE ID: GSE40395).
GraphPad Prism (v. 5.0; GraphPad Software, La Jolla, CA) was used to perform a Mann-Whitney U test to compare data between two groups, and differences were considered statistically significant at P < 0.05.
All other materials and methods are described in the Supporting Information.
Characteristics of the NASH Mouse Model
The pathological and clinical features of cirrhosis in patients are not well replicated by the majority of chemically induced murine cirrhotic liver models. We have established steatohepatitis as a cirrhotic liver mouse model by feeding mice an Ath+HF diet. When mice were fed this diet for 34 weeks, hepatocytes developed steatosis, Mallory-Denk bodies, and ballooning (Fig. 1A,B), which are identical to typical pathological features of clinical NASH. Albumin expression in parenchymal cells of the cirrhotic liver significantly decreased in mice fed the Ath+HF diet for 24 weeks (Fig. 1C), while alpha-fetoprotein (AFP) expression was not affected (Fig. 1D). Fibrosis developed and reached maximal levels after 34 weeks of feeding the Ath+HF diet (Fig. 1E,F). Immunohistochemical staining for immunomodulatory cells showed an increased number of Gr-1+ cells in the liver of the steatohepatitis mice fed the Ath+HF diet for 12, 34, and 70 weeks (Fig. 2A,B). The number of CD11b+ cells in the liver also increased and reached maximal levels after 34 weeks of feeding the Ath+HF diet (Fig. 2C,D). Thus, the murine cirrhosis model established by an Ath+HF diet mimics the features of clinical NASH.
Effect of ADSCs Treatment on Liver Albumin Expression and Fibrosis
Adipose tissue contains MSCs, which have the potential to differentiate into several types of cell lineages[10, 14] and to act as immunomodulators. In this study, we isolated stromal cells from inguinal adipose tissue of GFP-expressing transgenic (GFP-Tg) mice as ADSCs and expanded them in culture. The majority of these cells expressed CD90 and CD44, known surface markers of mesenchymal cells (Supporting Fig. 1A). A proportion of the expanded ADSCs also expressed CD105 (Supporting Fig. 1B), which has been recognized as a representative surface marker of MSCs.
We evaluated whether ADSCs could provide a therapeutically beneficial treatment for liver cirrhosis in steatohepatitis mice. We injected 1 × 105 GFP-ADSCs by way of the spleen/portal vein in mice fed the Ath+HF diet for 32 weeks. We observed that the GFP-ADSCs resided in all lobes of the liver at 3, 7, and 14 days after injection (Fig. 3A,B). Importantly, immunohistochemical staining showed that GFP+ cells in the cirrhotic liver expressed higher levels of albumin than did the surrounding parenchymal cells (Fig. 3C).
We also injected 1 × 105 or 2 × 104 GFP-ADSCs twice every 2 weeks by way of the splenic/portal vein in mice fed an Ath+HF diet for 32 or 36 weeks, respectively. Two weeks after the last injection the mice were euthanized and the therapeutic effects were assessed. The expression of albumin (Fig. 4A) was restored in hepatic parenchymal cells of cirrhotic mice at 2 weeks after the last injection, suggesting that ADSC treatment restored parenchymal cell function. The expression of AFP was also increased by ADSC treatment (Fig. 4B), implying enhanced regeneration of hepatic parenchymal cells. Similar effects were observed with a reduced number of (2 × 104) GFP-ADSCs (Supporting Fig. 2A,B).
We also assessed the effect of ADSC injection on fibrosis in cirrhotic mice. Liver tissue stained with Azan and anticollagen type IV antibody showed that ADSC administration reduced fibrosis compared to control animals (Fig. 5A,B; Supporting Fig. S3A,B). We also evaluated immunohistochemical staining of α-SMA, a marker of stellate cells, which are largely responsible for developing fibrosis. These results demonstrated that the number of α-SMA+ cells was reduced by ADSC treatment (Fig. 5C-E), suggesting that ADSCs suppress the activity of stellate cells and ameliorate liver fibrosis.
Gene Expression Profiling of Cirrhotic Livers Following ADSC Treatment
We examined the gene expression profile of the livers in the NASH mouse model of cirrhosis by DNA microarray to determine whether administration of ADSCs was therapeutically beneficial. We identified expression of 1,249 gene probes that were significantly affected by ADSC injection. Clustering analysis of gene expression using these gene probes distinguished between ADSCs-treated mice and PBS-treated mice (Fig. 6A). Among 1,249 genes, 797 were up-regulated and 452 were down-regulated by ADSC treatment. Regarding matrix metalloproteinase (MMP), expressions of MMP-8 and MMP-9 were enhanced in the liver of NASH mice treated with PBS compared to the wild type; this enhancement was removed by ADSC treatment (Supporting Fig. S4). Biological process analysis indicated that the down-regulated genes were primarily related to inflammation and the immune response (Fig. 6B), and the up-regulated genes were related to tissue construction and development (Fig. 6C). Thus, gene expression analysis of liver tissue demonstrated that ADSCs treatment caused anti-inflammatory effects, as well as regeneration/repair effects, in the livers of a NASH mouse model of cirrhosis.
Anti-inflammatory Effects of ADSC Treatment
The fundamental underlying pathophysiology of steatohepatitis-induced cirrhosis is persistent hepatic inflammation caused by steatosis in hepatocytes. We examined how ADSCs affected persistent inflammation of the liver in NASH mice at 2 weeks after the last injection of ADSCs. Immunohistochemical staining showed that the number of CD11b+ cells accumulating in the livers of cirrhotic mice decreased with ADSC treatment compared to those of PBS-treated mice (Fig. 7A). The number of Gr-1+ cells in cirrhotic liver also decreased with ADSC treatment (Fig. 7A), suggesting that ADSCs affect granulocytes and antigen-presenting cell lineage.
We further examined whether ADSC treatment affected the lymphocyte lineage of T cells, since they also play an important role in immune regulation of steatohepatitis. We isolated lymphocytes from the livers of mice treated with ADSCs and examined the CD4+ and CD8+ T cells using flow cytometry. CD8+ T cells were found predominantly in cirrhotic mice treated with PBS (Fig. 7B,C). However, when the mice were treated with ADSCs the number of CD4+ T cells increased and was comparable to that of CD8+ T cells, indicating that ADSC treatment affected T-cell subpopulations.
Gene Expression Profiling of Hepatic Inflammatory Cells Following ADSC Treatment
We further examined how injected ADSCs affected hepatic inflammatory cell gene expression by using DNA microarrays. By filtering the results from 5,065 gene probes, completely discernible clusters of gene expression were formed between ADSC- and PBS-treated animals (Fig. 8A). We identified the expression of 873 genes that were significantly up-regulated at least 2-fold with ADSC injection and 658 genes that were down-regulated. Most of the chemokines and cytokines whose expression was significantly affected by ADSCs were down-regulated (Supporting Table S1). Using the publicly available gene expression database for hematopoietic cells (GSE27787) and various types of helper T cells (GSE14308), we examined features of these affected genes in the context of immunomodulatory cells. Among the hematopoietic cells, genes with available symbol annotation were predominately Gr-1+ and CD11b+ cells from granulocyte and macrophage lineages (Fig. 8B). Among helper T-cell populations, annotated genes included activated Th1, Th2, and Th17 cell types (Fig. 8C). We also isolated CD4+T cells from hepatic inflammatory cells obtained from NASH mice fed an Ath+HF diet for 12 weeks, then treated with ADSC. Expressions of the Th1, Th2, and Th17 cytokines, interferon-γ, interleukin (IL)−4, IL-10, and IL-17, the Th17-related cytokine transforming growth factor beta (TGF-β), and Foxp3, a representative transcription factor of regulatory T cells, were down-regulated by ADSC treatment (Supporting Fig. S5).
These results suggest that ADSC treatment suppresses inflammation in the NASH mouse model primarily by down-regulating granulocytes, antigen-presenting cells, and activated helper T cells.
This study investigated the therapeutic effect of ADSCs in a NASH murine model of cirrhosis. This model is relevant to clinical NASH, with similar pathological features established by an atherogenic high-fat diet, including the appearance of steatosis, ballooning, and Mallory-Denk bodies in hepatocytes, infiltration of inflammatory cells, and pericellular fibrosis. Our results demonstrate that ADSC injection is therapeutically beneficial for cirrhosis in this murine model through restoration of albumin expression in hepatic parenchymal cells, amelioration of fibrosis, and suppression of persistent hepatic inflammation.
Gene expression analysis of the liver in this cirrhotic mouse model revealed that ADSC injection affects biological processes relating to anti-inflammatory and regeneration/repair pathways. The anti-inflammatory effects are mediated by ADSC targeting of Gr-1+, CD11b+, and helper T-cell lineages. In patients with clinical NASH, the ratio of neutrophils to lymphocytes increases, suggesting that granulocytes are involved in the pathogenesis of NASH. The NASH murine model used in this study produced an increased CD8+/CD4+ T-cell ratio, which is also comparable to clinical NASH patient pathology. Gene expression analysis of liver tissue and hepatic inflammatory cells from NASH mice showed that Th1-, Th2-, and Th17-related genes were down-regulated by ADSC treatment. Helper T-cell activation skewed to produce Th1 cytokines is pathogenic in steatohepatitis.[20, 21] In particular, Th17 is emerging as an important source of IL-17 family cytokines and is involved in the hepatic inflammation in NASH. Helper T cells producing Th2 cytokines such as IL-4, 5, and 13 contribute to fibrosis. We conclude that activated T helper cells are responsible for the pathogenesis of steatohepatitis in the NASH murine model used in this study and that ADSCs suppress pathogenic helper T-cell activation. However, the suppression of miscellaneous effector and regulatory helper T cells by ADSCs should be further evaluated with regard to prevention of hepatocellular carcinoma, a frequent sequela to cirrhosis, since Th1 promotes antitumor immunity and Th2 down-regulates antitumor immunity.
We also observed that ADSC treatment ameliorated fibrosis and decreased the number of α-SMA+ stellate cells in cirrhotic liver. When inflammation persists in the liver, fibrosis progresses due to these activated stellate cells, which are almost identical to myofibroblasts and produce extracellular matrix. Stellate cells are activated by miscellaneous factors including TGF-β and platelet-derived growth factor, produced mostly from Kupffer cells. Helper T cells expressing Th2 cytokines are also involved in the development of fibrosis. Gene expression analysis of the cirrhotic livers indicated that ADSC treatment suppressed Th2-type helper T cells. Although details of how these molecules mediate fibrosis development have yet to be examined in the current NASH murine model, the antifibrotic effect of ADSCs is achieved in part by suppressing Th2-type helper T cells. We found that MMP-8 and MMP-9 enhancement in the NASH-cirrhotic liver was ameliorated by ADSC treatment. MMP-9 expression is related to the inflammation typical of steatohepatitis and can ameliorate the hepatic fibrosis induced by carbon tetrachloride. Further studies are needed to clarify the role of MMPs in the pathogenesis of cirrhosis as well as to explore novel therapies for this condition.
Pluripotent MSCs differentiate into several cell lineages and are a promising avenue for regenerative therapy of various impaired organs, including the liver. Although ADSCs were observed in cirrhotic livers at up to 2 weeks after injection and expressed albumin, the numbers of resident cells were not sufficient to supplement hepatic function. Therefore, pluripotency, as well as the anti-inflammatory and antifibrotic effects of ADSCs, are important for their regenerative/repair effects in liver cirrhosis. Rather than studying the effects of ADSCs on early-stage steatohepatitis, we treated mice with endstage cirrhosis with ADSCs to observe their therapeutic effects. Our results demonstrated that ADSCs can effectively resolve chronic fibrosis and decrease inflammation, thereby restoring hepatic function in endstage cirrhotic mice, implying the usefulness of this therapy as an alternative to liver transplantation.
In conclusion, ADSCs proved therapeutically beneficial and clinically relevant in regenerative therapy of a murine steatohepatitis-cirrhosis model. Clinical application of ADSCs in the treatment of cirrhosis is expected to provide a novel alternative regenerative/repair therapy for patients with cirrhosis.