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Volume: 18 Issue: 7 December 2020


Therapeutic Potential of Bama Pig Adipose-Derived Mesenchymal Stem Cells for the Treatment of Carbon Tetrachloride-Induced Liver Fibrosis

Objectives: Liver fibrosis is inevitable in the healing process of liver injury. Liver fibrosis will develop into liver cirrhosis unless the damaging factors are removed. This study investigated the potential therapy of Bama pig adipose-derived mesenchymal stem cells in a carbon tetrachloride-induced liver fibrosis Institute of Cancer Research strain mice model.

Materials and Methods: Adipose-derived mesenchymal stem cells were injected intravenously into the tails of mice of the Institute of Cancer Research strain that had been treated with carbon tetrachloride for 4 weeks. Survival rate, migration, and proliferation of adipose-derived mesenchymal stem cells in the liver were observed by histochemistry, fluorescent labeling, and serological detection.

Results: At 1, 2, and 3 weeks after adipose-derived mesenchymal stem cell injection, liver fibrosis was significantly ameliorated. The injected adipose-derived mesenchymal stem cells had hepatic differentiation potential in vivo, and the survival rate of adipose-derived mesenchymal stem cells declined over time.

Conclusions: The findings in this study confirmed that adipose-derived mesenchymal stem cells derived from the Bama pig can be used in the treatment of liver fibrosis, and the grafted adipose-derived mesenchy­mal stem cells can migrate, survive, and differentiate into hepatic cells in vivo.

Key words : Animal model, Carbon tetrachloride-treated mice, Hepatic differentiation


The liver is the largest solid organ in the human body and is responsible for multiple, essential functions.1 Exposure of livers to xenobiotics, drugs, and viruses eventually leads to chronic hepatitis and liver cirrhosis. These diseases are most likely to cause hepatocellular carcinoma, even organ damage, and eventually lead to inflammation, fibrosis, and loss of an ability of regeneration. More than 450 million people will develop cirrhosis as a consequence of these diseases, which demonstrates the relevance of liver pathologies as causes of mortality and public health expenses.

At present, liver transplant is the best treatment for cirrhosis and liver fibrosis. However, the shortage of organ donors, huge cost of treatment, and lifelong immunosuppression may limit this option. Stem cell transplant has been widely used in the treatment of liver diseases because of its advantages such as abundant source, low immunogenicity, less trauma, and simple operation. Stem cell grafting is therefore considered an effective alternative to the treatment of liver fibrosis.2 Mesenchymal stromal cells (MSCs) are easily obtained, are efficiently cultured, and can functionally differentiate into hepatocytes and therefore provide an effective treatment for liver cirrhosis and fibrosis in animal models.3 Additionally, ethical and religious concerns are not likely because MSCs are obtained by autologous transplant.

Many adipose-derived cell types and experimental models have been used to test the efficacy of cell therapies for many diseases, such as spinal cord injury, neurodegenerative disease, autoimmune diseases, and metabolic diseases, leading to divergent results on the actual role of adipose-derived mesenchymal stem cells (ADSCs) as a new treatment alternative.4-7

In this study, we used the carbon tetrachloride (CCl4)-induced model of liver fibrosis to investigate the effect of Bama pig ADSC transplant on liver function in mice with hepatic fibrosis. Our objective was to open a new direction and theoretical basis for the clinical treatment of liver fibrosis.

Materials and Methods

Research animals were 6-week-old male mice of the Institute of Cancer Research strain. Mice were purchased from Beijing HFK Bioscience ( All protocols in the experiments were in accordance with the relevant provisions of the Institutional Animal Ethics Committee.

Isolation, culture, and purification of adipose-derived mesenchymal stem cells
Adipose tissues were recovered from the visceral (omental) region of the pig under aseptic conditions. Briefly, the extracellular matrix was dissociated with 0.1% (mg/mL) type I collagenase (Sigma). After centrifugation, the supernatant was discarded and the deposits were dissolved in the complete medium. When the cells reached 70% to 80% confluence, digestion and passage were carried out with 0.125% trypsin. The cells are usually homogenous after 3 passages.

Identification of adipose-derived mesenchymal stem cells with surface marker detection
Cells were incubated in the following antibodies (from Abcam): CD29 (1:100), CD44 (1:100), CD105 (1:100), and vimentin (1:100). Then, we stained the cells with fluorescein isothiocyanate-conjugated or Cy5-conjugated secondary antibodies (Bioss) and used a confocal microscope (Nikon model TE-2000-E) to examine the stained cells.

Identification of adipose-derived mesenchymal stem cells with reverse transcription-polymerase chain reaction
We used TRIzol reagent (Invitrogen) to obtain RNA from cells of passages 4, 14, 20, and 26, and we used gel electrophoresis to examine the products.

Identification of adipose-derived mesenchymal stem cells with flow cytometry analysis
We used surface markers and flow cytometry to establish the characteristics of the cells. Cells at passage 8 were incubated with the following antibodies: CD29, CD44, CD105, vimentin, and CD34. Cells were washed with phosphate-buffered saline and then stained with fluorescein isothiocyanate-conjugated or Cy5-conjugated secondary antibody. For controls, there were no second antibodies. The results were obtained by an EPICS-XL flow cytometer.

Identification of adipose-derived mesenchymal stem cells with karyotype analysis
Cells at passage 8 were treated in a hypertonic condition and stained with Giemsa. We used the oil-immersion technique to examine numbers and shape of chromosomes.

Multipotential differentiation of adipose-derived mesenchymal stem cells: adipogenic differentiation and confirmation
When the cells reached 70% confluence, we changed the medium to adipocyte-inducing medium. Control group cells were fed with a DMEM medium. Ten days later, the induced cells were stained with Oil Red O, and the adipogenic-specific genes for lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor gamma (PPARγ) were detected by polymerase chain reaction (PCR).

Insulin-secreting cells differentiation and confirmation
When the cells reached 80% confluence, the medium was replaced with an induced medium with 90% Dulbecco’s modified Eagle medium nutrient mixture F-12, 10 ng/mL hepatocyte growth factor (PeproTech), 10 ng/mL activin A (Sigma), 1 mmol/L β-mercaptoethanol (Sigma), 10 mmol/L nicotinamide, and 2% B-27 supplement (Gibco). Control group cells were cultured with a complete medium. The insulin gene (INS) and the insulin promoter factor gene (PDX1) were evaluated by reverse transcriptase-PCR (RT-PCR).

Neurogenic differentiation of adipose-derived mesenchymal stem cells and confirmation
When the cells reached 70% confluence, we changed the medium to the neurogenic induction medium. Induction was divided into 2 steps. Briefly, in step 1, the cells were incubated with the first neurogenic induction medium for 7 days. Then, in step 2, the cells were incubated with the second neurogenic induction medium for 7 days. The cells were confirmed by immunofluorescent assays of neural markers for the genes for microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and β-tubulin. The neural-specific genes for neurofibromatosis-related protein (NF) and MAP2 were examined by RT-PCR.

Chronic liver fibrosis model in mice and model evaluation
The experimental group mice were treated with 200 μL of 10% CCl4 solution twice weekly by an intraperitoneal injection, whereas the control group mice were injected with 200 μL mineral oil in the same form twice weekly.8

Four weeks later, some mice were used for serological tests and immunohistochemical assay. Samples of 3 normal mice were set as a control group, and the results were set as a normal range of the relevant parameter. This range was calculated by using the mean values (±2SD) for each test.

To assess the extent of liver fibrosis under the enzymatic level, serum alanine transaminase, aspartate transaminase, albumin-to-globulin ratio, albumin, total protein, and globulin levels were measured. Serum was obtained after centrifugation at -80 °C. Livers from the experimental group and control group were obtained. For the immunohistochemical assay, 8-μm liver tissue sections were embedded in paraffin and stained with the antibody for smooth muscle actin, hematoxylin-eosin, and Masson stain. To examine the results, we used a confocal microscope (Nikon) and a light microscope (Nikon Eclipse model 90i) according to standard protocols.

Cell labeling with CM-DiI indocarbocyanine dye
To analyze biodistribution after injection, we used CM-DiI indocarbocyanine dye to label the ADSCs and injected 1.5 × 107 labeled ADSCs into the experimental group.9 Three mice were evaluated at 1, 2, and 3 weeks after cell injection. We applied an immunohistochemical method to detect the transplanted cells.

Transplant of adipose-derived mesenchymal stem cells to carbon tetrachloride-injured mice of the Institute of Cancer Research strain
For cell transplant, 1.5 × 107 ADSCs (P8) suspended in 50 μL of phosphate-buffered saline were injected intravenously into mice that were treated with CCl4 for 4 weeks (n = 30). For control, 50 μL of phosphate-buffered saline were injected (n = 15). The mice were given an additional 1-, 2-, and 3-week injection of CCl4 after cell transplant. Animal livers and peripheral blood samples were collected at different intervals.

In vivo detection of differentiated adipose-derived mesenchymal stem cells
Adipose-derived mesenchymal stem cells are known to differentiate into hepatic cells in vivo. Adipose-derived mesenchymal stem cells were positive for the antibody CD44 and negative for the antibody CK18, whereas hepatic cells were positive for the antibody CK18 and negative for the antibody CD44. We used an immunohistochemistry assay to evaluate the differentiated hepatic cells. We used a confocal microscope (Nikon) to examine the results, according to standard protocols.

Statistical analyses
We used analysis of variance to analyze the data. We used the Tukey-Kramer test to evaluate the statistical significance. P < .05 was considered significant. We used JMP Software (SAS Institute) to perform all statistical analyses.


Characterization of mesenchymal stem cells derived from pig adipose tissue
Cells recovered from pig adipose tissue by adherence showed a typical characterization of ADSCs, especially with regard to the proliferation pattern and the phenotype as assessed by cell morphology, RT-PCR, flow cytometry, colony-forming units, karyotype, immunofluorescence, and multiple differentiation potential.

Primary cells began to adhere to plates and extend toward the spindle after 48 hours. Cells expanded rapidly, and the transplant reached 90% confluence 7 days later (Figure 1). When the cells proliferated to passage 28, signs such as vacuolization and karyotype began to appear. Passages 4, 14, 20, and 26 were detected by RT-PCR, and these were positive for CD29, CD44, CD71, CD73, CD90, and CD105 but negative for the endothelial marker CD31 (Figure 2). The results of flow cytometry showed phenotypic characteristics of homogeneous cell populations. The results showed that 81.43% of the cells were positive for CD29 (Figure 3A), 88.88% of the cells were positive for CD44 (Figure 3B), 18.38% of the cells were positive for CD105 (Figure 3C), and 85.85% of the cells were positive for vimentin (Figure 3D), whereas CD34 expression was 11.6% (Figure 3E). The results of immunofluorescence revealed that the ADSCs expressed CD29, CD44, CD105, and vimentin (Figure 4). The results of karyotyping showed that the number of diploid chromosomes was 2n = 38, including a pair of sex chromosomes. There was no missing or broken diploid chromosome ploidy (Figure 5).

Differentiation potential of adipose-derived mesenchymal stem cells
We evaluated the multipotential differentiation of ADSCs by inducing the cells into 3 germ layers. For mesoderm, adipogenic differentiation was observed. For the ectoderm, neurogenic differentiation was observed. For the entoderm, insulin-secreting differentiation was observed. There were some lipid droplets in adipogenic differentiation, and the results were confirmed by Oil Red O staining and RT-PCR (Figure 6A and 6B). Insulin-secreting differentiation was shown by RT-PCR (Figure 7). Neurogenic differentiation was detected by RT-PCR and immunofluorescence (Figure 8).

Adipose-derived mesenchymal stem cell transplant decreased carbon tetrachloride-induced chronic fibrosis in mouse livers
To discover whether the transplanted ADSCs could mitigate liver injury, we compared the histological difference of liver tissue in transplanted and nontransplanted CCl4-treated mice. As shown in Figure 9, the mice without transplant showed more severe fibrous liver dysfunction compared with normal mice. After transplant of ADSCs, mice with liver injury showed reduced vacuolar degeneration. Using immunohistochemistry, we found out that necrosis caused by CCl4 treatment was mitigated by ADSC injection. Staining with Masson stain and hematoxylin-eosin showed that the transplant of ADSCs could ameliorate CCl4-induced fibrous deposition (Figure 9A).

Serological analysis showed that the transplanted cells significantly improved the impaired liver function (Figure 9B). Colorimetric assays showed that injection of the cells remarkably reduced aspartate transaminase, alanine transaminase, the albumin-to-globulin ratio, albumin, total protein, and globulin content and collagen production in the CCl4-damaged liver tissues. In brief, these results suggested that cell transplant may inhibit CCl4-induced fibrous deposition and may have antifibrotic and anti-inflammatory functions.

Distribution of adipose-derived mesenchymal stem cells after intraportal transplant
We analyzed a group of 3 mice at 1, 2, and 3 weeks after transplant. Injected cells were confirmed by immunohistochemistry (Figure 10). One week after transplant, 50% of the cells were observed on each slide. We observed that most cells were around the portal vein, although we also observed aggregated cells. After 2 weeks, only 20% of cells were observed in the liver slices. After 3 weeks, the number of cells dropped to 5% (Figure 10A). We did not detect the cells in the vascular system, and this may be because these cells were not integrated into the parenchyma.

Adipose-derived mesenchymal stem cells differentiated into hepatic-like cells in injured liver
To test whether ADSCs were able to differentiate into hepatic-like cells after injection into the injured liver, we incubated liver samples with pig-specific antibodies to CD44 and CK18 (Figure 10B). We compared the number of positive cells to CD44 and CK18 in liver tissues of CCl4-injured mice with ADSC transplant during 5, 6, and 7 weeks. As presented in Figure 4C, after 1 week of infusion, the number of CK18-positive cells increased. Over time, the number of CK18-positive cells declined at 6 and 7 weeks postinfusion, which suggested that the differentiated ability of ADSCs decreased over time.


We studied the biological characteristics of ADSCs and analyzed the transdifferentiated ability of ADSCs in the injured liver model. For the first part, we displayed the following 2 features of ADSCs: a strong ability to self-renew and proliferate, which allows these cells to be a valuable resource for cell therapy; and the ability to differentiate in vitro into 3 germ layers including ectoderm, mesoderm, and entoderm, which indicates a powerful therapeutic potential for these cells,10,11 particularly in paralytic stroke, meniscus repair, and myocardial infarction. Recently, reports have been published on the ability of ADSCs to differentiate into hepatocytes in vitro and in vivo. Our results presented here were based on these findings.12-15

Adipose-derived mesenchymal stem cells were obtained easily from adipose tissue and demonstrated large-scale in vitro proliferation; also, these cells were induced into functional hepatic-like cells in an efficient manner. All these factors were important in regeneration treatments. The differentiated cells can express the specific genes ALB and AFP and can be positive for the antibody ALB, with albumin production and urea secretion. In addition, the cells have a glycogen storage capacity that balances glucose in vivo.

We analyzed the distribution of ADSCs after tail vein injection by CM-DiI indocarbocyanine dye labeling and immunofluorescence (with pig-specific antibodies to CD44 and CK18). At 1, 2, and 3 weeks after the transplant, the results showed the persistence of injection into the parenchyma of liver, whereas the cells remaining in the liver were obviously reduced over time, which revealed that most ADSCs were removed from the liver. Therefore, the reason for failure of cells to enter the liver parenchyma may be attributable to the lack of adhesion molecules.

Through immunofluorescence, we observed a morphological improvement of the liver after cell transplant. Transforming growth factor-β, smooth muscle actin, and collagen I staining demonstrated the reduction of the injured area. These results revealed that these cells could improve liver fibrosis. Stem cell therapy has great potential for use in the treatment of liver fibrosis and tissue regeneration.16,17

We also used biochemical assay to analyze the therapeutic potential of ADSCs. Parameters such as aspartate transaminase, alanine transaminase, albumin/globulin ratio, albumin, total protein, and globulin declined in the injured group after cell injection, showing that ADSCs could regenerate the liver. Parekkadan and colleagues stated that engrafted ADSCs did not usually differentiate into hepatocytes.18 To detect the possibility of transdifferentiation in mice, we conducted immunofluorescence staining for pig-specific antibodies CD44 and CK18. Adipose-derived mesenchymal stem cells were positive for CD44 (green), and hepatocytes were positive for CK18 (red). The results showed that ADSCs could transdifferentiate into hepatocytes, but the numbers would reduce over time.

Mesenchymal stem cells have been shown to secrete a wide range of chemokines, growth factors, and active cytokines and therefore may mediate a congenital immune system function and regulate liver regeneration in vivo.18-21 Furthermore, MSCs have been reported to inhibit proliferation and fibrogenesis of activated hepatic stellate cells.18 Several questions remain. How do the factors participate in the recovery of liver injury? How many signal pathways are involved during the recovery and what are they? Which factors play the main function during the recovery? What are the differences between cell therapy and tissue self-recovery? It is clear that further research is required.

We propose that ADSCs may be a new source of adult stem cells. Compared with bone marrow MSCs, ADSCs have an advantage because ADSCs originate from “waste” tissue. However, more analyses are needed before ADSCs may be used as a standard alternative source of stem cells; for example, further studies are needed to evaluate the efficacy of ADSCs for the treatment of disease. We evaluated the efficacy of ADSCs in the treatment of liver regeneration. First, ADSCs have a selective recruitment characteristic and these cells could be induced into hepatic-like cells that are positive for CK18 in the injured liver. Second, we observed the decline of liver enzyme levels, inflammatory factors, and recovery of liver fibrosis following infusion of ADSCs. Third, we confirmed ADSCs could differentiate into hepatocytes in mice, but the numbers are reduced over time. However, further assessment of the mechanisms is required. In brief, the data showed the transplant of ADSCs to be functional. However, the mouse liver-infused mechanism of donor cells and recipient liver cells and the principle of in vivo homing and differentiation potential of injected ADSCs are not yet fully clear. More work is required to identify the cellular and molecular mechanism(s) involved in the animal model.


We investigated the effectiveness of Bama pig ADSCs on the treatment of CCl4-induced liver fibrosis. The mouse liver fibrosis model was successfully induced by CCl4 and evaluated by staining with Masson stain and hematoxylin-eosin. The characterization and pluripotent differentiation of ADSCs were analyzed experimentally, and ADSCs can differentiate into functional hepatocytes with a high inductive rate. At 1, 2, and 3 weeks after ADSC transplant, we evaluated liver function by serological test, and the results showed that the transplant of ADSCs could ameliorate liver injury caused by CCl4. These findings offer novel insight regarding the usefulness of MSCs as a resource for clinical therapy for liver fibrosis.


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Volume : 18
Issue : 7
Pages : 823 - 831
DOI : 10.6002/ect.2020.0108

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From the 1Department of Animal Genetic Resources, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, China; the 2Scientific Experimental Research Center, Harbin Sport University, Nangang District, Harbin, Heilongjiang Province, China; and the 3Sport and Exercise Sciences Centre, University of Malaya, Kuala Lumpur, Malaysia
Acknowledgements: *Xinran Wu and Shuang Zhang contributed equally to this work. This research was supported by the National Natural Science Foundation of China (Grant No. 31472099), the Agricultural Science and Technology Innovation Program (cxgc-ias-01), and the National Infrastructure of Animal Germplasm Resources project (2016). Other than described above, the authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no further declarations of potential interest.
Corresponding author: Weijun Guan, Department of Animal Genetic Resources, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, China