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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 1  |  Page : 236-242

Mesenchymal stem cell applications on the chronic liver disease


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Submission03-Oct-2018
Date of Decision08-Dec-2018
Date of Acceptance15-Dec-2018
Date of Web Publication25-Mar-2020

Correspondence Address:
Eman A.Z. Kotb
Berkitt El.Sabah, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_301_18

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  Abstract 

Objective
The aim was to evaluate the effect of application of bone marrow mesenchymal stem cells (BMMSCs) after transplantation into mice with carbon tetrachloride-induced chronic liver.
Background
Chronic disease causes hepatocyte fibrosis, with subsequent morbidity and mortality. Stem cells have emerged an alternative therapy for liver fibrosis, which can be harvested from different sources, such as mesenchymal stem cells (MSCs).
Materials and methods
This study was conducted on 55 mice and 18 human bone marrow samples. Primarily, liver fibrosis was induced in five mice, and after the fourth week of CCl4induction, they were killed, and liver biopsy was done to estimate fibrosis occurrence. The remaining 50 mice were classified as 25 mice injected with 0.9% saline to be used as control (group I) and 25 mice were infused intravenously with 1 × 106 human BMMSC (group II) after continuous CCl4administration in both groups. After 4 weeks of MSCs infusion, serum alanine transaminase, aspartate transaminase, albumin, and total bilirubin were determined for all 50 mice. Moreover, liver histology was performed to determine the fibrosis degree as well as α-fetoprotein immunohistochemistry, and RT-PCR was done for α-smooth muscle actin expression. Human BMMSCs were collected and isolated by bone marrow culture and evaluated by flow cytometric characterization of MSCs using CD45-negative and CD90-positive cell markers.
Results
MSCs-treated group revealed significant lower alanine transaminase, aspartate transaminase, and albumin; higher total bilirubin levels; decreased histopathological fibrosis; diffuse α-fetoprotein immunostaining; and lower α-smooth muscle actin gene expression when compared with the control group.
Conclusion
MSCs could be used as a therapeutic tool in end-stage liver disease.

Keywords: α-fetoprotein, α-smooth muscle actin, bone marrow, carbon tetrachloride, mesenchymal stem cells


How to cite this article:
Montaser LM, El-Azab DS, Kotb EA. Mesenchymal stem cell applications on the chronic liver disease. Menoufia Med J 2020;33:236-42

How to cite this URL:
Montaser LM, El-Azab DS, Kotb EA. Mesenchymal stem cell applications on the chronic liver disease. Menoufia Med J [serial online] 2020 [cited 2020 Jun 6];33:236-42. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/236/281299




  Introduction Top


Liver fibrosis is defined as an extracellular matrix excess which emerges from an imbalance between augmented synthesis and decreased degradation in response to sustained noxious stimuli associated with distortion of liver parenchyma and progressive function deterioration[1]. Subsequent complications such as portal hypertension, variceal bleeding, and hepatocellular carcinoma risk, lead to the emergence of a concept of early intervention with new therapeutic approaches[1],[2].

To date, liver transplantation is the only golden treatment option in patients with cirrhosis; however, increased prevalence, wide donor-recipient gap, life-long immunosuppression dependence, and poor outcome after liver transplantation, produce an immense effort to develop less invasive substitutes[2].

Some studies reported application of human hepatocytes for cell therapy in clinical trials of various liver disorders[3],[4],[5]. The lack of availability of healthy donors as well as difficulties in hepatocyte long-term maintenance is a major problem in using these specialized cells[6].

Mesenchymal stem cells (MSCs) are a type of adult stem cells that are more suitable for cell therapy because of their adequate availability, strongest homing capacity to injured tissue, rapid proliferation, multipotent differentiation, and successful integration and immunological tolerance in the host tissue. Meanwhile, MSCs could be derived from a patient's own tissues rather than blastocyst or embryos and are considered more appropriate for clinical use[7],[8],[9]. A large number of in vitro studies indicate that bone marrow (BM) MSCs can express the liver-specific marker α-fetoprotein (AFP), cytokeratin 18 (CK18), and albumin[10],[11],[12],[13].

This study aimed to focus on the effect of bone marrow mesenchymal stem cells (BMMSCs) transplant on mice having carbon tetrachloride (CCl4)-induced liver fibrosis and to determine the improved liver function after MSCs transplantation.


  Materials and Methods Top


Experimental animal model

A total of 55 mice of local strain with average weight of 25–30 g were used in this study. All the experiments were done in compliance with the guide for the care and use of laboratory animals approved by the local Ethics Committee of the Menoufia University. Liver fibrosis was induced in five mice by CCl4 induction, and liver biopsy was done after 4 weeks to estimate the occurrence of liver fibrosis. The remaining 50 mice were divided into the following two groups:

  1. Group I (control group): it included 25 mice that were subjected to a single tail intravenous injection of 0.9% saline
  2. Group II (HBMMSC group): it included 25 mice that were infused intravenously by 1 × 106 HBMMSC in the mice tail.


Induction of chronic liver fibrosis

Chronic liver fibrosis was experimentally induced by intraperitoneal injection of CCl4 in a dose of 1 ml/kg diluted by olive oil 1: 1 twice a week for 8 weeks.

Separation of BMMSCs

Under complete aseptic conditions, 8–10 ml of the human BM sample was withdrawn by heparinized syringe and then diluted with sterile PBS (one volume of blood to one volume of PBS). The diluted sample was layered in two conical centrifuged tubes (15 ml of each) containing ficoll–hypaque solution (Bichrom AG, Leonorenstr, Berlin), as two volume of diluted blood to one volume of ficoll, followed by 20 min centrifugation at 1800 rpm. The mononuclear cells fraction was collected by a Pasteur pipette to be washed twice in sterile PBS, and cell pellet was resuspended in 1 ml DMEM-media (catalog no. 11965-092; Thermo Scientific, Fremont, California, USA).

Cell counting and viability

Cell counting and viability were assessed by the vital stain trypan blue dye 0.4% (Sigma, Minneapolis, MN 55406, USA) using a hemocytometer, and each mouse received 1 × 106 cells with viability greater than 95%.

Isolation and primary culture of MSCs

Mononuclear cell suspensions of 1 × 106 cells/ml concentration were plated and allowed to adhere to 0.2-μm vent cap flasks (Corning, Washington, DC, USA) of horizontal position and incubated in a humidified incubator at 37°C and 5% CO2. The used nutrient media constituted the following: low glucose DMEM (DMEM-LG, Biowest, Nuaillé, France) with 2 mmol l-glutamine (Lonza), 10% FBS (Lonza, Swiss Firm, Switzerland), 2% penicillin-streptomycin (10 000 Ul/ml and 10 000 μg/ml) (Sigma), and 1% fungizone (20 mg/ml) (Bioscience, Palo Alto, CA, USA). The first change of the media was accomplished on the third–fifth day to remove nonadherent cells; the adherent cells were kept in the flask and were fed fresh complete nutrient media. The media was changed twice weekly until reaching 70–90% confluence and then the cells were detached using trypsin-EDTA 0.25% solution.

Flow cytometric analysis of HBMMSCs

The harvested MSCs from primary culture were evaluated by flow cytometric characterization of MSCs using CD45 and CD90 according to the international guidelines for stem cell identification.

Biological markers

Blood samples were collected from the mice retro-orbital venous plexus using a fine heparinized capillary tube and left for clotting then centrifuged at 3000 rpm for 20 min for serum separation. Alanine transaminase (ALT), aspartate transaminase (AST), albumin, and total bilirubin were determined by autoanalyzer (Cobas Integra 400 plus, No. 500281, R&D Systems, Minneapolis, MN, USA).

Histological examination

Liver histology was performed 4 weeks after HBMMSCs transplantation to determine the extent of liver fibrosis. The tissue wasembedded in paraffin and sectioned into 4-μm slices forhematoxylin and eosin stain and confirmatory Masson trichrome stain. Histological liver damage was evaluated by experiant pathologist blinded to the experimental groups.

Immunohistochemistry

The liver tissue was collected after 8 weeks and fixed in 10% buffered formalin and paraffin-embedded blocks. Sections were incubated with primary antibodies against human AFP (LabVision, NY, USA) to confirm homing and function of transplanted cells.

Real-time PCR for ASMA gene expression

RNA was extracted from liver tissue with Trizol reagent (Invitrogen, Carlsbad, California, USA) and reversely transcribed using the Gene Amp Gold RNA PCR Kit (Applied Biosystems, Monza (MI), Italy). The reverse transcriptase PCR (RT-PCR) was done as the following: PCR amplification of target DNA was performed using α-smooth muscle actin (ASMA) sequence-specific primer (forward: 5'-GTGCTATGTCGCTCTGGACTTTGA-3' and reverse: 5'-ATGAAA GATGGCTGGAAGAGGGTC-3'), and the amplified products were hybridized to SYPER Green PCR Master Mix (Applied Biosystems) and the bound products were detected by colorimetric determination in an ABI PRISM 7500HT Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. For each sample, mRNA expression level was normalized to the level of GAPD housekeeping gene (forward: 5'-GCATGGCCTTCCGTGTTC-3' and reverse: 5'-GATGTCATCATAC TTGGCAGGTTT-3'). The expression ratio was analyzed using the delta comparative cycle threshold method.

Statistical analysis

Results were fed to the computer and analyzed using IBM SPSS software package version 20.0 (2011; IBM Corp., Armonk, New York, USA). Qualitative data were described using number and percentage. The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range, mean, SD, and median. Significance of the obtained results was judged at the 5% level. The used tests were Student's t-test for normally distributed quantitative variables, to compare between two studied groups and Mann–Whitney test for abnormally distributed quantitative variables, to compare between two studied groups.


  Results Top


MSCs detection

Morphological assay

Cells were examined every 3 days by the inverted microscope for assessment of the morphology, viability of cells, and microbial contamination. By continuous changing the media, the plastic-adherent cells were observed on day 2 in the flask, and the floating cells were gradually removed. Colonies of adherent cells with fibroblastoid morphology started to appear on firth to seventh day in between many rounded cells. The fibroblastoid cells became much more pure and reached confluence OF 30% at 10 days, and then 80% at 15 days with disappearance of the rounded cells [Figure 1].
Figure 1: Inverted microscope image ×200 of mesenchymal stem cells culture flask showing 30% confluence on the 10th day while the fibroblastoid cells become much more pure and reach confluence of 80–90% confluence on the 15th day with disappearance of the rounded cells.

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Immunophenotyping profile by flowcytometry

The analytical region was selected by gating and analyzed for the forward and side characteristics of BMSCs. These cells positively expressed the MSCs marker (CD90) and did not expressed the HSCs marker (CD45).

Experimental animal result

Liver function assessment

The mean values of ALT, AST, albumin, and total bilirubin were significantly lower (P < 0.001) in HBMMSCs-treated group (group II) compared with their value in the control group (group I) [Table 1].
Table 1: Comparison between nonmesenchymal stem cells-treated (group I) and mesenchymal stem cells-treated (group II) groups regarding alanine transaminase, aspartate transaminase, albumin, and total bilirubin

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Histological examination

After 4 weeks of MSCs transplantation, liver biopsy examination confirmed the improvement of liver damage in group II mice which shows milder periportal fibrosis, compared with diffuse extensive cirrhotic nodules in non-MSCs transfused mice (group I) [Figure 2].
Figure 2: A photomicrograph images ×400 of liver tissue section of nonmesenchymal stem cells-treated mice (group I) after 8 weeks with noticed cirrhotic nodules (hematoxylin and eosin) and porto-portal link (Masson trichrome) whereas the mesenchymal stem cells-treated group showed mild periportal fibrosis (hematoxylin and eosin) and (Masson trichrome).

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Immunohistochemistry staining

The expression of AFP was evaluated by immunohistochemistry analysis (positive staining was brownish, granular and cytoplasmic). The immunohistochemistry staining for liver tissue showed a higher diffuse AFP expression in MSCs transplanted mice (group II), suggesting homing and in-vivo differentiation of transplanted MSCs toward functioning hepatocytes, whereas a negative reaction was shown in nontreated mice (group 1) [Figure 3] and [Figure 4].
Figure 3: A photomicrograph image ×200 of liver tissue section of nonmesenchymal stem cells-treated mice (group I) after 8 weeks showing negative reaction for human alpha fetoprotein expression (immunohistochemical staining).

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Figure 4: A photomicrograph image ×200 of liver tissue section of mesenchymal stem cells-treated mouse showing diffuse positive reaction for the a-fetoprotein. Cells expressing human a-fetoprotein account for approximately 25% of section (immunohistochemical staining).

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Real-time PCR ASMA gene expression

A significant lower mean value of ASMA gene expression (P < 0.001) was detected in MSC-transfused mice (group I) compared with corresponding mean values in non-MSC treated (group II) [Table 2].
Table 2: Comparison between nonmesenchymal stem cell treated (group I) and mesenchymal stem cell treated (group II) regarding α-smooth muscle actin gene expression

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  Discussion Top


Based on liver transplantation drawbacks, recent insights into cell therapy emerged as a new therapeutic strategy for chronic liver diseases[14]. Insufficient functional human hepatocyte yield, short-term maintenance, and immunologic rejection on transplantation are major obstacles in using these cells on a wide scale in regenerative tissue field[15].

Emerged studies proved differentiation potential of stem cells toward hepatocytes, among them are BMMSCs, which are considered the most potent one in hepatic differentiation both in vitro andin vivo and had attracted significant attentions by their high homing capacity into the injured liver tissue, potency, and immunological tolerance[16],[17],[18],[19].

The current study included 55 mice with average weight of 25–30 g. CCl4 injection was done in five mice (as a test control for induced fibrosis), and liver biopsy was done after 4 weeks to estimate the occurrence of liver fibrosis. Then, liver fibrosis was induced to the remaining 50 mice by intraperitoneal injection of CCl4 for 8 weeks. After that, 25 mice were injected by 0.9% saline to be used as a control group (group I), and group II included 25 mice that were infused intravenously by 1 × 106 HBMMSCs in the mice tail.

For achievement of a condition of general liver damage, persistent intraperitoneal administration of CCl4 was established in our experimental model for continues 8 weeks. Among the different ways of CCl4 administration including inhalation, subcutaneous injection, and intraperitoneal injection, the latter has several advantages, for example, fast development of cirrhosis, less toxicity, and low lethality[20]. CCl4 hepatic pathophysiology is explained by cell membrane lipid peroxidation as a resultant of CCl4 active metabolite by cytochrome P-450 radical and loss of mitochondrial Ca+2 uptake and increase its release by NADPH oxidation[21].

Biochemical analysis of liver function in 50 mice with CCl4-induced liver damage was investigated for evaluation of the therapeutic efficacy of MSCs. After 4 weeks of MSC transplant, the mean levels of ALT, AST, albumin, and total bilirubin were significantly lower in MSCs-treated group (group II) compared with their value in non-MSCs-treated control group (group I).

These results seen in this investigation were in agreement with Raafat et al.[22], Wang et al.[23], Li et al.[24], and Sinn et al.[25], who revealed that, both ALT and AST are cytoplasmic enzymes discharged into the circulation as a resultant of induced liver cell membrane injury by CCl4 toxin and the enzymatic activity normalization in MSC-treated group, reflecting their effect on hepatocyte structure preservation.

In addition, the significant decreased total bilirubin and elevated albumin in the present work after MSCs transplantation was in accordance with those of Ali and Masoud[26] and Raafat et al.[22], who reported that, CCl4-treated mice associated with edema of the hepatocyte organelles and mitochondrial dysfunction by its membrane oxidation and Ca+2 release that decrease albumin synthesis and bilirubin conjugatory enzyme activity and their improved levels are indicative of amelioration of liver dysfunction.

To assess liver damage degree, histopathological analysis was performed after 4 weeks of MSCs transplantation, which graded from the extensive disturbed hepatic architecture with cirrhotic nodules and porto-portal link fibrosis in control group to clearly demonstrated mild periportal fibrosis in MSC-treated group.

These results were in agreement with Raafat et al.[22], Wang et al.[23], Rabani et al.[27], and Tanimoto et al.[28], who referred the ability of MSCs to degrade excess ECM directly, by secreting matrix metalloproteinases, or indirectly, by stimulating the immune cells to increase the production of these enzymes and decrease the production of their inhibitors such as tissue-induced metalloproteinase inhibitor (TIMPI).

To confirm homing and in-vivo differentiation of transplanted HBMMSCs, immunohistochemical analysis of formalin and paraffin-embedded liver sections was investigated for human AFP expression. Immunohistochemistry staining for liver tissue revealed higher diffuse protein reaction with 25% AFP expression in MSCs group, whereas a negative reaction was shown in nontreated mice. These results go hand in hand with those of Raafat et al.[22], Wang et al.[23], and Ali and Masoud[26], who mentioned that MSCs exhibit numerous characteristics, such as distinctive CYP 450 activity as well as expression of AFP.

Based on Zoubek et al.[14] and Li et al.[24], who stated that, the activated hepatic stellate cell (aHSCs) are major players mediating liver fibrogenesis process and could be identified as a base of the expression of ASMA in the experimental animal models to detect liver fibrosis degree, the present study determined ASMA gene expression by RT-PCR analysis in the two different studied groups.

ASMA has a critical role in aHSC behavior, during liver injury. It is an important ERK signaling pathway regulator which is involved in phosphorylation of focal adhesion kinase, caplain2, and paxilliin signaling partners, leading to turnover of extracellular matrix binding proteins such as integrins with resultant stellate cells motility and migration to injury site[25].

This study revealed an increase of ASMA gene expression with hyperplastic fibrous tissue caused by CCl4, whereas following MSCs therapy, an obvious alleviation of liver damage by histopathological examination associated with parallel decrease of ASMA gene expression confirming their antifibrotic property in MSCs treated mice. This decrease was statistically lower compared with its level in non-MSC treated mice, suggesting no fibrosis improvement in this group. These findings are in agreement with Raafat et al.[22], Sinn et al.[25], and Ali and Masoud[26], who reported that, following CCl4-induced liver injury, transforming growth factor-β mediates the proliferation aHSC which displays specific phenotype markers such as collagen I, ASMA, α-actinin and myosin heavy chain proteins, and the increase of ASMA gene expression gives a reflection of increased HSCs activity and correlates with the disease progression. It had been reported that, MSCs could directly inhibit the activation of HSCs, via MSC-derived interleukin-10 and tumor necrosis factor-α, and may also induce their apoptosis through the Fas/FasL pathway[16],[22].

On the contrary, several studies failed to observe significant improved liver function after MSCs therapy in chronic liver injury model. It was reported that MSCs might contribute to the liver fibrosis by differentiating into profibrogenic myofibroblast-like cells[29]. Furthermore, it was demonstrated that, in-vivo hepatic differentiation of transplanted MSCs had limited engraftment to the site of injection, promotion of tumor growth by suppression of the antitumour immune response, and tumor invasion by secretion of angiogenic growth factors, including vascular endothelial factor, fibroblast growth factor, platelet derived growth factor, and stromal-derived factor-1[16].

So, future studies are needed to solve the limitations of MSCs use. In addition, MSC therapy needs to be further evaluated for longer follow-up periods to detect their long-term efficiency. Moreover, for optimizing their therapeutic effects and engraftment, standardization of their delivery route into the liver, the number of their injections and concentrations must be taken into consideration.


  Conclusion Top


Based on the current work findings, MSCs could be considered as a suitable option for the treatment of end-stage liver disease in human after success in experimental mice. The reversible fibrogenesis mechanisms, even at late stage, make a new evolution in using the stem cells for liver regenerative medicine.

So, future studies are needed to solve the limitations of MSCs use. In addition, MSC therapy needs to be further evaluated for longer follow-up periods to detect their long-term efficiency. Moreover, for optimizing their therapeutic effects and engraftment, standardization of their delivery route into the liver, the number of their injections, and concentrations must be taken in consideration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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