Author Affiliations: The Combination of Chinese Medicine with Western Medicine Gulou Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing 210008, China (Zhu W); Department of Anesthesiology (Zhu W and Ma ZL); Department of Hepatobiliary Surgery (Shi XL, Xiao JQ and Ding YT), Affiliated Drum-Tower Hospital, Nanjing University Medical School, Nanjing 210008, China; Jiangsu Province's Key Medical Center for Hepatobiliary Disease, Nanjing 210008, China (Shi XL, Xiao JQ, Gu GX and Ding YT)

 

Corresponding Author: Zheng-Liang Ma, MD, Department of Anesthe-siology, Affiliated Drum-Tower Hospital, Nanjing University Medical School, Nanjing 210008, China (Tel: 86-25-83105502; Fax: 86-25-83317016; Email: mazhengliang1964@yahoo.com.cn)

 

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60007-7

 

Contributors: DYT and MZL contributed equally to this work. ZW, DYT and MZL participated in research design. ZW, XJQ and GGX accomplished this research. SXL and XJQ drafted the paper. MZL is the guarantor.

Funding: This work was supported by a grant from the National Natural Science Foundation of China (30901431).

Ethical approval: All animal procedures were approved by the Animal Care Ethics Committee of Nanjing University Medical School and Nanjing Drum-Tower Hospital.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Adipose-derived stem cells (ADSCs) are particularly attractive in future clinical applications of stem cell-based therapy for acute-on-chronic liver failure (ACLF). This study was undertaken to evaluate the therapeutic potential of ADSCs on ACLF.

 

METHODS:  ADSCs isolated from porcine fat tissue were expanded and labeled with BrdU. Rabbit models of ACLF were created by administration of D-Gal following CCl₄-induced cirrhosis. One day after administration of D-Gal, rabbits of the ACLF/ADSCs group (n=15) were received ADSCs transplantation, while those in the ACLF/saline group (n=15) were treated with the same volume of saline. Biochemical parameters and histomorphological scoring were evaluated; the distribution and characteristics of transplanted ADSCs as well as the pathology of the liver were examined.

 

RESULTS: ADSCs transplantation improved the survival rate and the liver function of rabbits with ACLF. Biochemical parameters of the ACLF/ADSCs group were improved compared with those of the ACLF/saline group, and histomorphological scoring of the ACLF/ADSCs group was significantly lower than that of the ACLF/saline group. ADSCs were identified in the periportal region of the liver after cell transplantation.

 

CONCLUSION:Xenogenic ADSCs have therapeutic efficacy in the ACLF rabbit model.

 

(Hepatobiliary Pancreat Dis Int 2013;12:60-67)

 

KEY WORDS: adipose-derived stem cells; acute-on-chronic liver failure; cell transplantation

Acute-on-chronic liver failure (ACLF) is the most common type of liver failure in China. In a large-scale study from 2002 to 2007 in China, 1813 (91.7%) of 1977 patients with liver failure were diagnosed with ACLF.^{[1, 2]} Patients with this disease have a poor outcome because most of them will develop into multi-organ failure following liver failure. Liver transplantation (LT) is the only curative option but limited by organ donor shortage, financial consideration, and treatment of life-long immunosuppression.^[3]

 

Recent studies^[4-9] have shown that a number of different sources of stem cells can differentiate into hepatocytes in vitro, suggesting that stem cells may offer an alternative therapy for liver failure. Mesenchymal stem cells (MSCs) have been isolated from many adult and fetal tissues, including bone marrow,^[4] adipose tissue,^[5] amniotic fluid (AF),^[6] scalp tissue,^[7] placenta,^[8] and umbilical cord blood.^[9] Particularly, bone marrow derived mesenchymal stromal cells (BMSCs) have been studied extensively because of their superior multipotency.^[10-14] However, the amount of available bone marrow is usually not efficient, and the procurement procedure is invasive. On the other hand, adipose-derived stem cells (ADSCs) with biological properties similar to BMSCs^[15] can also be induced to differentiate into various mesenchymal cell types^[16-18] including hepatocytes.^{[19, 20]} Unlike BMSCs, ADSCs are present abundantly in the body, and they can be harvested repeatedly according to the procedure which is simple and minimally invasive. These cells can be easily expanded and manipulated in vitro. Therefore, ADSCs represent a potential source for cell therapy on liver diseases.

 

The purpose of this study was to evaluate the therapeutic potential of xenogeneic ADSC to treat ACLF. We successfully created a rabbit model of ACLF, and gained sufficient quantities of ADSCs from swine. Porcine ADSCs then were transplanted into ACLF rabbits to investigate the effects of porcine ADSCs transplantation on ACLF.

 

 

New Zealand rabbits and outbred white pigs were obtained from the Animal Experimental Center of Nanjing Drum-Tower Hospital. All animal procedures were approved by the Animal Care Ethics Committees of Nanjing University Medical School and Nanjing Drum-Tower Hospital, and performed in accordance with Guide for the Care and Use of Laboratory Animals (NIH Publication 85-23 revised 1996, USA).^[21] All chemicals were of analytical grade and purchased from GIBCO (USA) unless otherwise stated.

 

Porcine fat tissue was obtained from male pigs and washed extensively with phosphate buffered saline (PBS). It was cut and minced into small pieces, and digested with 0.1% type I collagenase (Sigma, USA) under gentle shaking for 60 minutes at 37 ��. Then DMEM/F12 containing 10% fetal bovine serum was added to neutralize the enzyme activity. Cell suspension was filtrated sequentially through a 100-µm or 40-µm nylon mesh, washed via three centrifugations (50 g), and then resuspended in complete culture medium. Cells were incubated at 37 �� in a humidified chamber containing 5% CO₂ for 24 hours. The adherent cells were further expanded with medium change at 3-day intervals.

 

ADSCs were labeled with BrdU (Sigma, USA) according to the manufacturer's instruction. Proliferation of cells was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide colorimetric assay daily from day 1 to day 10 after the labeling and growth curves were plotted. The percentage of the BrdU-labeled ADSCs was also examined with immunofluorescence staining (described below). The viability of ADSCs was determined by trypan blue exclusion assay after they were detached with 0.25% trypsin and resuspended in PBS at 2×10⁷ cells/mL.

 

The phenotype of porcine ADSCs was evaluated by flow cytometry analysis (FACS, Becton Dickinson, USA). Antibodies against the following cell surface markers were: phycoerythrin (PE)-conjugated CD45 (Antigenix America, USA), CD90, CD105, CD44, CD34 (Becton Dickinson, USA). Isotypic antibodies served as controls.

 

ACLF models were created in rabbits by CCl₄ intraperitoneal injection for 10 weeks, followed by venous injection with D-Galactosamine (D-Gal), as described by Brandão.^[22]

 

After injection with D-Gal, ACLF rabbits were further divided into two groups: ACLF/ADSCs group (n=15) infused with 1×10⁸ porcine ADSCs on the second day, and ACLF/saline group (n=15) infused with the same volume of saline and served as controls.

 

Cell transplantation and surgical procedures were performed when rabbits were anesthetized with ether inhalation. An upper midline incision was made inferiorly from the xiphoid, and the portal vein was gently exposed with a moistened gauze. Freshly harvested porcine ADSCs (1×10⁸) was suspended in 10 mL PBS and injected into the portal vein with a 25-gauge needle connected to a 2 mL syringe.

 

At 1, 3, 5, 7, 14, 21 and 28 days after D-gal administration, blood was drawn from each rabbit into a heparin-containing tube and serum was collected after centrifugation for 10 minutes at 5000 rpm. Serum samples from all animals were taken to determine the levels of albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum ammonia (NH3), and total bilirubin (TBil) as well as prothrombin time (PT) by an automatic biochemical analyzer (Hitachi 7600, Tokyo, Japan).

 

The serum level of porcine albumin was quantified by enzyme-linked immunosorbent assay (ELISA) using purified goat anti-albumin and horseradish peroxidase-conjugated antibody (Bethyl Laboratories, USA). The absorbance was measured at 490 nm with a Cytofluor multiwell plate reader (Benchmark, USA).

 

ACLF rabbits were followed up for survival 28 days after D-Gal administration. Liver tissues were removed from each group for pathological examination. The time points were day 2, 7, 14, 21, and 28, respectively. After formalin fixation and paraffin embedding, the liver samples were sectioned into 4 µm slides and stained with hematoxylin-eosin (HE). Histological assessment was performed by a blinded observer using a previously described scoring method.^[23]

 

Cultured cells or frozen tissue sections (6 µm in thickness) were incubated with either a mouse anti-BrdU antibody (dilution 1:1000; sigma, USA) alone or together with a goat anti-pig albumin antibody (dilution 1:500, bethyl, USA) at 4 �� overnight. These samples were further treated with a second antibody, FITC conjugated donkey anti-mouse IgG (H+L) (dilution 1:100; Jackson immunoResearch, USA) alone or together with a R-PE-conjugated donkey anti-goat IgG (H+L) (dilution 1:100; Jackson immunoResearch, USA). The nucleus was stained with Hoechst 33342 (1 mg/mL, beyotime, China).

 

The destination of transplanted ADSCs was assessed by immunofluorescence double staining with antibodies against pig albumin and BrdU. Fluorescent in situ hybridization (FISH) was also performed to identify the origin of cells in the liver following the Cambio protocol (Cambio, Cambridge, UK) (http://www.cambio.co.uk/).^[24]

 

Statistical analysis was performed using the SPSS version 17.0. Data were expressed as mean±SD for values obtained from three repeated experiments/measurements. Survival curves were calculated by the Kaplan-Meier method and analyzed with the Chi-square test. Statistical significance was defined as P<0.05 using two-tailed unpaired Student's t test or one-way analysis of variance or repeated measures analysis of variance.

 

 

The freshly isolated ADSCs were small and approxi-mately round cells, which then gradually grew into spindle or stellate-shaped cells (Fig. 1A). They proliferated rapidly and reached 90% confluency 3-4 days after they were passaged. After the second passage, ADSCs adopted a more uniform fibroblast-like morphology with long cell processes, and cell culture monolayer displayed the typical fingerprint-like pattern (Fig. 1B, C) similar to bone marrow stem cells.

 

After BrdU labeling and immunofluorescence, labeled cells showed uniform fluorescence signals (green) in the entire nucleus (Fig. 1D). After counter-stained with Hoechest-33342, the number of BrdU-positive cells was equal to that of Hoechest-positive cells at passage 0, and decreased over passages due to the proliferation of the labeled cells. However, at least half of the cells were still BrdU-positive after passages 5.

 

To examine whether BrdU labeling would adversely affect cell proliferation, we used the MTT assay to measure proliferation of the BrdU labeled or unlabeled cells. The proliferation rates from days 1-9 were not significantly different in the two cell groups as shown by their growth curves (Fig. 2).

 

Flow cytometric analysis revealed that few ADSCs expressed CD45 and CD34, and that over 90% of cells expressed CD44, CD90 and CD107 (Fig. 3). Such an expression profile is consistent with previous reports for porcine ADSCs.^{[25, 26]}

 

ADSCs were infused into ACLF rabbits 24 hours after D-Gal treatment. All the animals were followed up for 28 days, and the percentage of animals survived was graphed. As shown in Fig. 4, no rabbits died in the first 24 hours after D-Gal injection; at 3 days, 73% and 47% of the animals survived in the ACLF/ADSCs and ACLF/saline groups, respectively; at 7 days, the survival rate was 60% (9/15) and 27% (4/15) in the ACLF/ADSCs and ADSCs/saline groups, respectively (P<0.05).

 

To examine the effect of ADSC transplantation on liver function of the ACLF rabbits, we measured the levels of ALT, AST, NH3, PT, TBil and albumin in the animals with or without ADSC transplantation. As shown in Fig. 5, all the surviving animals with ACLF displayed liver function abnormality. Elevated liver function induced by D-Gal administration peaked at 3 days and then gradually recovered to almost normal levels at 7 days. However, peak levels were significantly lower in the ACLF/ADSC group than those in the ACLF/saline group (P<0.05). At the same time, D-Gal also significantly reduced albumin levels, and the lowest albumin level in the ACLF/ADSCs group was significantly higher than that in the ACLF/saline group (P<0.05). No significant differences in these parameters were observed between the two groups after 7 days. However, the serum level of porcine albumin in all groups was undetected.

 

The pathology of ACLF can be characterized by sequential changes in the liver tissue morphology with microscopic evaluation, consisting of ballooning and/or eosinophilic degeneration of hepatocytes, parenchymal necrosis or collapse with features of underlying chronic liver disease, especially fibrosis, which is different from that of normal tissues (Fig. 6A). After 10 weeks of CCl₄ treatment, all of the rabbits showed liver cirrhosis in HE-stained and Masson-stained sections (Fig. 6B, C). Two days after transplantation, liver samples from the ACLF/saline group displayed profound hepatocyte death, thickened septal fibrosis and severe distortion of tissue architecture (Fig. 6D), while those from the ACLF/ADSC group showed only minor hepatocyte death with edema, thinner septal fibrosis and certain characteristics of tissue repair such as dual-core hepatocytes (Fig. 6E, F). Liver tissues taken from the ACLF/ADSC group at 28 days after D-Gal injection revealed almost normal hepatic trabecular architecture with moderate mononuclear infiltration, indicating a recovery from acute liver failure. At 3 months post-transplantation, we did not see any tumor development or abnormality in the liver, lung, bone marrow and other organs of the rabbits injected with ADSCs.

 

Semi-quantitative histological examination on liver tissues also revealed significant differences in morphology between ACLF rabbits with or without ADSC transplantation. The average pathology scores of liver tissues obtained two days after transplantation were 1.8±0.6 and 2.9±0.8 in the ACLF/ADSC and ACLF/saline groups, respectively (P<0.05). These results demonstrated that ADSC therapy prevented histopathological changes in the liver of ACLF rabbits.

 

Using albumin as a molecular marker for hepato-cytes, immunofluorescence staining with antibody specific to porcine albumin revealed the presence of albumin positive cells derived from pig throughout the hepatic lobules at 14 and 21 days post-injection. These pig ADSC-derived hepatocytes distributed evenly among the periportal region and other parts of the liver. The number of albumin positive cells was lower at 14 days (Fig. 7A) than that at 21 days post-injection (Fig. 7B), indicating the enrichment of the differentiated cells over time. To follow the repopulation and differentiation of ADSC, FISH was performed to detect Y chromosomes in the female recipients. We found that male ADSC infused via the portal vein into female rabbit liver could engraft and differentiate into hepatocytes. Fourteen days after ADSC transplantation, some hepatocytes showed green signals in the nuclei stained with 4', 6-diamidino-2-phenylindole (DAPI) (Fig. 7C). Moreover, more donor-derived hepatocytes were found in the injured liver of rabbits 21 days after treatment (Fig. 7D).

 

 

In this study, ADSCs isolated from porcine adipose tissues were similar to other MSCs in morphology when cultured. Interestingly, they had a higher proliferation than MSCs which was also derived from adipose tissue reported by Zuk et al.^[27] After culture expansion, their yield was 40-fold higher than that of several other types of BMSCs.^[15] Various studies^{[28, 29]} have demonstrated the immunosuppressive characteristic of MSCs in vitro and in vivo. A recent study also indicated that this immunosuppression may partially due to chemokines and immune-inhibitory nitric oxide or indoleamine 2, 3-dioxygenase produced by MSCs.^[30] ADSCs showed immunosuppressive properties of inhibiting mixed lymphocyte proliferation as well, which may share the same immunosuppressive mechanism with MSCs, and the cell-cell contact was required for ADSC-mediated full immunosuppression. These results suggest that ADSCs possess the low immunogenicity and can escape from immune response.^{[28, 31]} Thus, the cells may be robustly expanded for a variety of therapeutic applications and offer a rich source for stem cell-based therapy.^{[32, 33]}

 

We studied the implantation and differentiation of ADSCs in vivo after they were labeled by BrdU, an S-phase marker that can be incorporated into newly synthesized DNA during the DNA replication. The BrdU-positive cells were detectable from half a day to 28 days post-transplantation, shown with their clear morphology by immunostainning against BrdU, suggesting that BrdU is relatively safe and stable for labeling stem cells. Banas et al^[20] observed reduction of serum ammonia level and ALT after ADSCs transplan-tation. In the current study, similar results were found that ADSC transplantation significantly improved the liver function of ACLF rabbits, and prolonged their survival time. This improvement was paralleled to the change of engrafted ADSCs quantity, suggesting that the improvement of liver function may be mediated by ADSCs engraftment. Since liver histopathology is often used for the diagnosis and assessment of ACLF, and it is also associated with the outcome in ACLF patients,^[34] we assessed liver pathology by a semi-quantitative scoring system. The average pathology score in ACLF rabbits transplanted with ADSCs was much lower than that in the ACLF/saline group. Consistent with this, microscopic examination revealed that ADSCs inhibited death and promoted regeneration of hepatocytes.

 

In our pilot experiments, ALF was induced by a single injection of D-Gal without pretreatment with CCl₄ in rabbits. In these animals, transplantation of ADSCs did not restore liver function, suggesting that cell transplantation may be not efficient in ALF conditions except ACLF. In other pilot experiments with the ACLF models, engrafted ADSCs quantity related liver function improvement was verified. In this current study, transplanted ADSCs were first concentrated in the portal areas 3 days after D-Gal injection and then more widely distributions were performed at 7, 14, 21 days post-injection (not shown), indicating their migration in vivo. We also found that some cells positive for BrdU also expressed albumin. However, we have not adequately proven hepatogenic differentiation of ADSCs in vivo. To clarify the mechanism, we will perform dual FISH for porcine DNA and albumin.

 

It is still not clear that by which mechanisms the transplanted cells improve the liver function. Hepatic regeneration may have multiple pathways to accomplish cell replacement and organ repair. The mechanisms of improved liver function may be due to cytokine production from transplanted ADSCs. It has been reported that ADSCs can secret several potentially beneficial growth factors, such as vascular endothelial growth factor, hepatocyte growth factor, basic fibroblast growth factor, transforming growth factor-β, and insulin-like growth factor-1.^[35-37] ADSCs have also shown to produce significantly more bioactive factors than BMSCs,^[38] therefore, they may have an equal or even stronger regenerative effect on implanted tissues than BMSCs. Also, researches have shown that ADSCs could inhibit the inflammatory reaction to reduce injury.^[39-42] Moreover, some studies^{[42, 43]} have shown that regenerating hepatocytes in transplant recipients may be derived from MSCs that fused with host hepatocytes. However, recent reports^{[44, 45]} have indicated that bone marrow cells and hematopoietic stem cells can convert into hepatocytes without fusion. Therefore, we considered a possibility that all situations mentioned above simultaneously happened in the ADSCs transplantation for ACLF model. It is necessary to verify this possibility in further study.

 

In conclusion, this study generated the first rabbit ACLF, and successfully transplanted ADSCs into the livers of this model. ADSCs transplantation prolonged the survival of ACLF animals by improving their liver function. Although the underlying mechanisms are not clear, these cells present a valuable source of stem cells with great potentiality and promise for future clinical applications.

 

 

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