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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 4  |  Page : 1496-1500

Applications of bone marrow-derived cells in acute liver disease


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

Date of Submission15-Oct-2015
Date of Decision25-Nov-2015
Date of Acceptance30-Nov-2015
Date of Web Publication31-Dec-2019

Correspondence Address:
Sara A Saied
Department of Clinical Pathology, National Liver Institute, Menoufia University, Shebin El Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_393_15

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  Abstract 


Objective
To evaluate the effects of bone marrow mononuclear cells (BMMCs) transplantation in rats with toxic acute liver damage induced by carbon tetrachloride (CCL4).
Background
Acute liver failure is a severe syndrome which need rapid and effective treatment. The present study was to examine whether BMMCs might be useful in the management of acute liver failure in an animal model or not.
Materials and methods
BMMCs were obtained from 8 to 10 ml of human bone marrow (10 × 106 cells) injected into the portal vein of rats 24 h (n = 32) after liver damage with CCL4. Control group (n = 32) was injected with only saline in CCL4-treated animals. Transaminases and survival were measured at 24 h, 72 h, 1, 2, 3, 4 weeks. Liver histology was observed 2 and 4 weeks after injury.
Results
Transplanted BMMCs were found to populate the damaged liver. Animals that received BMMCs also showed a trend toward improved liver enzymes as well enhanced survival rates, relative to the control.
Conclusion
This study results indicate that BMMC transplantation has potential as a new therapeutic option for acute liver disease and suggest that these cells may contribute to hepatic recovery.

Keywords: acute liver failure, bone marrow mononuclear cells, carbon tetrachloride


How to cite this article:
Montaser LM, El-Azab DS, Tawfeek GA, Saied SA. Applications of bone marrow-derived cells in acute liver disease. Menoufia Med J 2019;32:1496-500

How to cite this URL:
Montaser LM, El-Azab DS, Tawfeek GA, Saied SA. Applications of bone marrow-derived cells in acute liver disease. Menoufia Med J [serial online] 2019 [cited 2020 Jan 20];32:1496-500. Available from: http://www.mmj.eg.net/text.asp?2019/32/4/1496/274270




  Introduction Top


Acute liver failure (ALF) is a syndrome characterized by rapid loss of hepatic mass and function leading to coagulopathy, increased ammonia levels and progression to encephalopathy, coma, and death if not promptly treated [1]. The cause of ALF varies according to patient age, geographic distribution, medical, and social practices within the community [2]. It may occur in patients with partly compensated, acquired, or hereditary liver diseases when a precipitating event leads to an acute decompensation of liver function [3].

The current medical treatment is limited to patient support until the liver recovers spontaneously or a replacement liver becomes available [3].

Stem cells are cells found in all multicellular organisms. They are characterized by the ability to renew themselves through mitotic cell division and differentiate into a diverse range of specialized cell types [4]. Bone marrow is a reservoir of various stem cells including hematopoietic stem cells and mesenchymal stem cells (MSCs), while MSCs have been shown to be capable of mesodermal and neuroectodermal differentiation, they have the potential of endodermal differentiation; their differentiation into functional hepatocyte-like cells has also been demonstrated [5].

The application of bone marrow mononuclear cells (BMMCs) is considered a promising cell therapy for various diseases. Donor BMMCs transplanted into animal models of various diseases have been shown to differentiate into various cell types, including bone, cartilage, cardiac muscle, vascular endothelial, and neuronal cells [6].

Autologous BMMC infusion therapy in patients with liver cirrhosis concluded that BMMC therapy should be considered as a novel treatment for patients with decompensated liver cirrhosis [7].

Aim

The aim of the study is to evaluate the effects of BMMC transplantation in rats with toxic acute liver damage induced by carbon tetrachloride (CCL4).


  Materials and Methods Top


Experimental design

In all, 64 adult albino rats of local strain weighing 150–200 g each were used in this study. ALF was induced and the animals were divided into two groups. Each group constituted of 32 rats, 20 rats were maintained for 4 weeks for the measurement of liver enzymes and survival rate; six rats were killed at 2 weeks and the other six rats were scarified at 4 weeks for histopathology.

Control group (32 rats): in this group, the rats were subjected to a single intravenous injection of 0.9% saline in the rat tail vein for 24 h after CCL4 injection.

BMMC-treated group (32 rats): in this group, the animals were infused with 10 × 106 cells via tail vein for 24 h after CCL4 injection.

Induction of acute liver injury

ALF was experimentally induced by a single intraperitoneal injection of CCL4 (3.0 ml/kg dissolved in vegetable oil).

Isolation of bone marrow mononuclear cells

A measure of 8–10 ml of human bone marrow was aseptically collected in a sterile heparinized vacutainer tubes to be handled within 6 h. Under complete aseptic conditions (laminar flow work area) the bone marrow was diluted with sterile PBS at a ratio of 1: 1 and they were mixed well. The diluted blood was layered on Ficoll-Hypaque (Biochrom AG, Leonorenstr, Berlin) in 15 ml conical centrifuged tube at a ratio of one volume of Ficoll: two volumes of blood and then the tubes were centrifuged for 20 min at 1800 rpm. The mononuclear cells fraction was collected carefully. Cell pellet was resuspended in 1 ml DMEM (Cat. 11965–092; Thermo Scientific, Fremont, California, USA) and counted using a hemocytometer. The process was carried out in the Clinical Pathology Department, Menoufia University Hospital.

Cell counting and viability were assessed by the vital stain trypan blue (0.4%) exclusion dye (Sigma, NY, USA):



Each rat received 10 × 106 cells with a viability more than 95%.

Biochemistry

A measure of 2 ml of blood was collected in a tube that was left for clotting at room temperature and then centrifuged at 3000 rpm for 20 min. The supernatant serum was collected in dry, clean tubes and was used for estimation of serum alanine transferase (ALT), aspartate transferase (AST).

Survival rate measurement was conducted at 72 h, 1, 2, 3, and 4 weeks after injection of BMMCs.

Histological evaluation

Hematoxylin and eosin-stained sections were analyzed under an optical microscope by a trained pathologist. Necrosis was evaluated by counting the rows of necrotic hepatocytes from the perivenular zone. Steatosis and hydropic degeneration were noted when present. The mitotic index was counted in 10 high-power fields. The inflammatory infiltrate was classified as either present or absent.


  Results Top


Serum aminotransferase activity

No statistically significant differences in serum ALT activity was found between the two groups at 24, 72 h, and after 1 week. Statistically significant differences in serum ALT activity was found between the two groups at 2, 3, and 4 weeks with a P value of 0.001, <0.001, and 0.001, respectively. ALT is significantly lower in the BMMC-treated group than the control group.

No statistically significant differences in serum AST activity was found between the two groups at 24 and 72 h. Statistically significant differences in serum AST activity was found between the two groups at 1, 2, 3, and 4 weeks with a P value of 0.04, 0.02, 0.001, and 0.04, respectively. AST is significantly lower in the BMMC-treated group than the control group [Table 1] and [Table 2].
Table 1: Comparison between control group and bone marrow mononuclear cells-injected group as regards alanine transferase

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Table 2: Comparison between control group and bone marrow mononuclear cell-injected group as regards aspartate transferase

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Survival analysis

There was no significant increase in survival rate at the end of 72 h. However, it was significantly increased at 1, 2, 3, and 4 weeks in the BMMC-treated group than in the control group with a P value of less than 0.001, 0.003, 0.008, and 0.01, respectively. Survival rate is higher in the BMMC-treated group than the control group [Table 3].
Table 3: Comparison between the control group and bone marrow mononuclear cells-injected group as regards survival

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Histological evaluation

As regards steatosis: at 2 and 4 weeks steatosis is significantly lower in the BMMC-treated group than the control group.

As regards degeneration: at 2 weeks there was a significant decrease in degeneration in the BMMC-treated group than the control group. At 4 weeks there was no significant difference between the two groups.

As regards inflammation: at 2 weeks no significant difference was seen between the two groups. But, at 4 weeks inflammation significantly decreased in the BMMC-treated group than the control group.

As regards necrosis: at 2 weeks and 4 weeks there was no significant difference between the two groups [Table 4] and [Figure 1], [Figure 2], [Figure 3].
Table 4: Comparison between the control group and bone marrow mononuclear cell-injected group as regards steatosis, degeneration, inflammation, and necrosis

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Figure 1: A photomicrograph of section in the liver of a rat Arrow snowed bridging necrosis (hematoxylin and eosin, ×200).

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Figure 2: A photomicrograph of section in the liver of a rat Arrow snowed inflammatory cells in portal tract (hematoxylin and eosin, ×400).

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Figure 3: A photomicrograph of section in the liver of a rat Arrow snowed steatosis (hematoxylin and eosin, ×200).

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  Discussion Top


ALF is a multisystemic severe disorder with a high mortality rate characterized by the onset of jaundice, hepatic encephalopathy, and coagulopathy. There is impairment of detoxifying and synthetic activities of the liver resulting from loss of functioning hepatocytes [8].

The benefits of using bone marrow as a source of cells for treating ALF are based mostly on their availability and feasibility of autotransplantation. The use of mononuclear bone marrow cells as proposed in this study may offer other advantages when compared with stem cell purified populations. Bone marrow cells contain both hematopoietic and MSCs, which are able to differentiate into hepatocytes [9].

In this study, the effect of BMMCs was evaluated for transplantation in rats with CCL4-induced acute liver damage. A marked increase in the survival rate of BMMC-treated animals was demonstrated, which could be in the near future an important clinical application [10].

So, this aimed to evaluate the feasibility of BMMCs to attenuate or improve recovery of induced ALF animal models.

The present study showed that single intraperitoneal injection of CCL4 to normal control rats caused ALF with significant rise of serum ALT and AST levels reaching mean values of about five to eight times higher than normal 24 h after injury.

These results agreed with Shizhu et al. [11]; all concluded that abnormally higher activities of serum ALT and AST after CCL4 administration is an indication of the development of hepatic injury, which is responsible for leakage of cellular enzymes into the blood. When liver plasma membrane gets damaged, a variety of enzymes normally located in the cytosol are released into the circulation [11].

The mechanism of CCL4 hepatotoxicity was explained by Fujimoto and Iimuro [12]; the mechanism of action is thought to involve the P450 cytochrome enzyme with the production of an active metabolite. It has been suggested that procedures that induced P450 enzymes would increase CCL4 toxicity. The mechanism has been postulated to involve free radicals [12].

Transaminases were measured after 24, 72 h, 1, 2, 3, and 4 weeks; there were significant decline in the mean values of serum ALT and AST in BMMC-treated groups when compared with the corresponding mean values in the nontreated groups, this decline began at 72 h for ALT and AST.

Fortunately, Carvalho et al. [13] have found that ALT and AST returned to normal levels in cell-treated earlier than nontreated groups. This indicate that soon after aggression is interrupted, damage markers tend to normalize, which is expected since fewer cells will die and these enzymes will no longer be released into the circulation [13].

The results of the present study agreed with Shizhu et al. [11], who reported that transplanted BMMCs differentiate into functional hepatocytes that can compensate for ALF in rats. Serum ALT and AST levels also markedly declined in response to transplantation of BMMCs, confirming that liver deterioration can be ameliorated by transplanted BMMCs [11].

The survival rate at each time point was significantly higher in the differentiated BMMC-treated group than the non-treated group.

Baldo et al. [10] stated that the survival rates were calculated in the experimental and control groups at different time points for 72 h, 1, 2, 3, and 4 weeks, respectively, and were significantly different between the experimental and control groups at corresponding time points. At each time point, the number of rats surviving the induction of ALF was greater in the BMMC-treated versus the control group [10].

In the present study, hematoxylin and eosin-stained sections from ALF nontreated group showed both degenerative and necrotic changes in hepatocytes. These changes were marked in the centrilobular areas where there was marked necrosis, disorganization, and loss of hepatocytes in areas adjacent to the central vein. The necrotic changes were either in the form of pyknotic or karyolitic nuclei, in addition to cytoplasmic changes or complete loss of the cells with distortion of the hepatocyte plate architecture. Also, the percentages of affected hepatocytes were significantly increased when compared with the corresponding mean values in the treated group.

CCL4-induced rapid histological changes in the rat liver, including bridging necrosis, steatosis, and hydropic degeneration as soon as 24 h after damage, coinciding with ALT peak. Later on after few weeks, histology and ALT levels were almost normal, indicating that the liver was already in its regenerative process.

The results of histological changes agreed with Belardinelli et al. [2], who proved that acetaminophen causes fatty changes in liver parenchyma and areas of centrilobular necrosis and vascular congestion involving the portal triad and dilatation of the central vein indicating backflow of the circulation [2].

Yang et al. [14] reported that livers of ALF mice showed serious hepatic congestion, hepatocyte degeneration, disordered hepatocyte cords, as well as multiple and extensive areas of cellular necrosis and inflammatory cell infiltration. This liver damage was ameliorated after stem cell treatment [14].

Baldo et al. [10] reported that necrosis was reduced to almost null levels, but mild levels of steatosis and hydropic degeneration were still present [10].

Cao et al. [15] reported that necrosis reduction was also detected in rats with CCL4-induced acute liver injury after BMMCs transplantation via the portal vein. The general condition of the rats in the treatment group also improved markedly [15].


  Conclusion Top


So in this study, we were able to show the presence of transplanted cells in the liver and a significant increase in survival rate using bone marrow-derived cells in a model of ALF induced by CCL4 and we have demonstrated the feasibility and benefit of BMMCs transplantation as an alternative therapeutic approach to ameliorate ALF. BMMC transplantation may be useful for the treatment of ALF as well as the restoration of liver function in human clinical situations in the future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Polson J, Lee WM. AASLD position paper: the management of acute liver failure. Hepatology 2005; 41:1179–1197.  Back to cited text no. 1
    
2.
Belardinelli MC, Pereira F, Baldo G, Vicente Tavares AM, Kieling CO, Silveira TR et al. Adult derived mononuclear bone marrow cells improve survival in a model of acetaminophen-induced acute liver failure in rats. Toxicology 2008; 247:1–5.  Back to cited text no. 2
    
3.
O'Grady J. Modern management of acute liver failure. Clin Liver Dis 2007; 11:291–303.  Back to cited text no. 3
    
4.
Ahmedy E, Kandel S, Rizk S, Gabr H, Khalifaa K, Kamal S. Methods and applications for mesenchymal stem cells. Menouf Med J 2013; 26:71–77.  Back to cited text no. 4
    
5.
Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M, Bagheri M, Bashtar M, Ghanaati H, et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Arch Iranian Med 2007; 4:459–466.  Back to cited text no. 5
    
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Atala A, Koh CJ. Tissue engineering applications of therapeutic cloning. Ann Rev Biomed Eng 2004; 6:27–40.  Back to cited text no. 6
    
7.
Terai S, Ishikawa T, Omori K, Aoyama K, Marumoto Y, Urata Y, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006; 24:2292–2298.  Back to cited text no. 7
    
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Whitehouse T, Wendon J. Acute liver failure. Best Pract Res Clin Gastroenterol 2013; 27:757–769.  Back to cited text no. 8
    
9.
Kang X, Zang W, Song T, Xu X, Li D, Meng K, et al. Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro. World J Gastroenterol 2005; 11:3479–3484.  Back to cited text no. 9
    
10.
Baldo G, Giugliani R, Uribe C, Belardinelli MC, Duarte ME, Meurer L, et al. Bone marrow mononuclear cell transplantation improves survival and induces hepatocyte proliferation in rats after CCL4 acute liver damage. Dig Dis Sci 2010; 55:3384–3392.  Back to cited text no. 10
    
11.
Shizhu J, Xiangwei M, Xun S, Mingzi H, Bingrong L, Dexia K, et al. Bone marrow mononuclear cell transplant therapy in mice with CCl4-induced acute liver failure. Turk J Gastroenterol 2012; 4:344–352.  Back to cited text no. 11
    
12.
Fujimoto J, Iimuro Y. Carbon tetrachloride-induced hepatotoxicity. World J Gastroenterol 2010; 9:251–272.  Back to cited text no. 12
    
13.
Carvalho A, Quintanilha L, Dias J, Paredes B, Mannheimer E, Carvalho F, et al. Bone marrow multipotent mesenchymal stromal cells do not reduce fibrosis or improve function in a rat model of severe chronic liver injury. Stem Cells 2008; 26:1307–1314.  Back to cited text no. 13
    
14.
Yang J, Cao H, Pan Q, Yu J, Li J, Li L. Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice. Hepatobiliary Pancreat Dis Int 2015; 14:186–193.  Back to cited text no. 14
    
15.
Cao B, Lin J, Zhong Y, Huang S, Lin N, Tang Z, et al. Contribution of mononuclear bone marrow cells to carbon tetrachloride-induced liver fibrosis in rats. World J Gastroenterol 2007; 13:1851–184.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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