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Efficacy of Intraarterial Transplantation of Mesenchymal Multipotent Stem Cells in Patients with Liver Cirrhosis and Portal Hipertension
Jafarli R.E.
Azerbaijan Medical University, Baku, Azerbaijan;
International center for biotechnology and cell cultivation "Biostem", University of emergency and reconstructive surgery named. V.K.Gusak, Donetsk, Ukraine
Summary
The article is devoted to the results of clinical trials studying the effectiveness of autologous transplantation of mesenchymal multipotent stem cells to patients with liver cirrhosis and portal hipertension. In the transplantation of obtained cell lines, we used the method of intraarterial administration. Efficiency has been studied by morphological analysis (index of histological activity ) and immunohistochemical staining (detection of proliferative activity, expression of activated myofibroblasts and sinusoidal capillarization).
During intra-arterial administration of autologous of mesenchymal stem cells observed higher quality of structural changes in the liver in the cirrhotic on its defeat. These abovementioned circumstances were shown a significant reduction in the proliferation of hepatocytes, reduction in the number of myofibroblasts in the parenchyma, which leads to faster resolution of the zone of perisinusoidal fibrosis and restoration of the normal structure of sinusoidal. The obtained results allow to conclude about the prospect of autologous transplantation of mesenchymal multipotent stem cells in the treatment of patients with liver cirrhosis.
Keywords: liver cirrhosis, autologous mesenchymal multipotential stem cells, histomorphological study.
Introduction
Increasing the number of predisposing factors, the prevalence among young adults, as well as unfavorable outcomes indicate the importance of social and medical solutions to this problem [1, 2].
In the treatment of cirrhosis of the liver depending on its severity and the reasons illness caused takes a conservative and surgical methods of correction [2]. However, despite the reached achievement results of treatment are often overshadowed by poor results mainly related to disease progression, complications and high mortality rate [2].
Achievements of the last decades of Cell Biology and conducted experimental researches have shown the possibility of cell transplantation to recover and activate regenerative capacity of the liver parenchyma with its various lesions [3].
Among the currently used treatment methods CPU with stem cells can be distinguished the following main directions, which have been intensively developed in the experiment and find their practical application:
Direction I is based on using of embryonic stem cells [4]. Direction II based on local or intravenous transplantation of differentiated fetal tissues in the form of islets or the cell
suspension which are obtained from abortive material during 9 - 12 week of pregnancy [4].
Direction III based on harnessing the potential of transdermal of differentiation "adult" SC [4]. Transplantation of these cells (hematopoietic, mesenchymal, neural stem cells) produced after or without pre-dates their transdifferentiation in the treatment of cellular elements
In experiments on animals has been shown opportunity to stimulate liver regeneration with autologous bone marrow stem cells [5].The first positive results of treatment of liver diseases in humans installed high-performance application of autologous mesenchymal multipotent stem cells in the treatment of virus hepatitises was obtained [6]. Moreover, stem cell with a phenotype CD133 + obtained from the blood of patients and entered into the liver through the portal vein in 2.5 times accelerated the process of liver regeneration after resection [7]. The positive dynamics of liver function test was noted within the first phase of clinical studies when patients were with chronic hepatitis [8] Thus, by the beginning of our work there were theoretical, experimental and clinical prerequisites for clinical trials of usage of transplantation in the treatment of AMMSK CPU.
The purpose of the study
Assess the effectiveness of intra-arterial transplantation of autologous multipotent mesenchymal stem cells on the morphofunctional condition of the liver in the cirrhotic on its defeat.
Materials and methods of the studies
The present study is based on study of the effectiveness of transplantation of autologous mesenchymal multipotent stem cells (AMMSK) in the treatment of 7 patients with HC (6 (85.7%) men and 1 (14.3%) women) with the presence of portal hypertension (PH). The reason of CPU in 5 (71.4%) cases was alcoholic and in 2 (28.6%) patients with virus. The age of the patients was from 32 to 65 years
( average age 43.6 + 8.2 years). Duration of disease from diagnosis was from 1 to 8 years and constituted on average 3,7 ± 2,4 years.
The severity of disease was evaluated according to the criteria of the Child-Pugh [8]. Distrubution of patients according to the criteria of the Child-Pugh initially as follows - the functional class A was observed in 4 (57.1%) patients and class B - in 3 (42.9%) patients. Among the study, patients with functional class C were not observed.
Introduction of autologous mesenchymal multipotent stem cells (AMMSK) in common hepatic artery performed by means
First-generation' stem cells
lilac crest aspirate
Endothelial progenitor cells
iDoS©'!
Mesenchymal stem cells
Figure 1. Approaches to isolation and preparation of stem cells
of endovascular angiography in the operating room once.
Observed patients before embarking on a AMMSK transplantation previously informed about the planned manipulation, possible side effects, complications and applied technologies. Subsequently, consent of the patient confirmed by consent in writing.
DHBs (20 - 40 million units in 2.0 - 5 ml) used for the transplantation which was pre-allocated out of the bone marrow of the patient [6].
AMMSK human culture previously isolated from bone marrow obtained at a puncture of the iliac wing (2 month prior to transplantation AMMSK) (figure 1).
Aspirate of bone marrow was diluted with Hanks' solution («Biolot», Russia) in a ratio of 1: 5.2. In a centrifuge tube in
Figure 2. Photomicrography of culture of goat bone marrow adherent cells. (A) Cells after 24 hours in culture. Note the non-adherent mononuclear cell cluster and the presence of cells already bonded to the culture plate, in a spindle shape. (B) Cells after the first wash. Note the decrease in the amount suspended cells and the higher definition of the adherent mononuclear cells. (C) Cells after eight days culture presenting 60% confluence. Note the small amount of suspended non-adherent cells. (D) The 14th culture day and (E) on the 17th culture day, both with 80% confluence. Note the spindle shape pattern and the radial growth adopted during this particular phase. Rod: A-E 100im.
the capacity of 50 ml was poured a density gradient Histopaque 1077 (Sigma, USA) at the rate of 1 ml gradient per 1 ml of bone marrow.
Carefully was layered diluted bone marrow on a gradient. Then was centrifuged tube under room temperature in the mode of 1800-2000 rev / min for 30-40 minutes. They collected interphase cells containing MSCs in a centrifuge tube in the volume of 15 ml with small amounts of Hanks' solution and resuspended slurry. They centrifuged tube under 800-1000 r / min for 8-10 minutes
They poured supernatant resuspended precipitate in a solution of Hanks and twice repeated centrifugation. Then the precipitate is mixed with a
growth medium, which is containing the DMEM / F12 (Sigma, USA), 20% FBS («Biolot», Russia), and mitogens. After that the slurry were plated on plastic bottles in a area of 75 cm2 (Nuclon, USA) in a density of 1-2 x 105 / cm2 and placed them in carbon dioxide incubator for 3 nights (370C, 5% CO2). Then changed their environment simultaneously removing not adhered (hematopoietic) cells. Subsequently replacement of environment was carried out every 2 days (figure 2).
In order to identify and characterize cell lines of mesenchymal stem cells take into account the basic criteria, recommended by the International Society for Cell Technology (ISCT) in 2006, which were used in our study [5].
Among them: a) the ability of stem cells to adhesion to the plastic under standard culturing conditions; b) the presence more than 95% of the cellular elements of positive markers SD105 (endoglin), CD73 (ecto-5'-nucleotidase) and CD90 (thymocyte antigen 1); c) presence of no more than 2% of the cells of negative markers CD45, CD34, CD14 and CD11b, CD79? or CD19, HLA-DR; g) the ability of the cell lines in vitro to differentiation of osteoblasts and adipocytes. Term of cultivation AMMSK was 42 days.
It should be noted that all stduied patients was implemented CPU transplantation AMMSK addition to the usual basic curative interventions (hepatoprotectors, vitamin therapy, antiviral therapy, infusion, if necessary hormone therapy and. etc..).
In all patients in the immediate and distant periods subjected to comprehensive clinical examination, comprising the general laboratory and instrumental (X-ray, ultrasound, endoscopy) methods. Special biochemical studies have been performed to assess the functional state of the liver.
In order to study the effect of different methods of transplantation AMMSK on morphological status of the liver we before and one month after administration of cell cultures carried out a puncture biopsy of the liver (under stationary conditions).
Biposy avascular zone of the liver was performed under ultrasound local anesthesia. They used a puncture type mono-jekt ABC (Sherwood Medical).
The effectiveness of different methods of transplantation AMMSK as assessed by morphological and immunohisto-chemical methods of evaluation: 1) study of the proliferative activity of cells of the liver [on expression nuclear antigen of proliferating cells Proliferation Cell Nuclear Antigen, PCNA; 2] the activation of myofibroblasts [on the expression of alpha-smooth muscle actin, a-SMA]; 3] of the sinusoids capillariza-tion [on the expression of CD34].
It should be noted that in order to estimate the proliferation was a study of expression of hepatocyte regenerative response for damage.
a -GMA - is one of the proteins of the contractile smooth
muscle which in healthy liver detected only in muscle cells in blood vessels of the liver. When CPU occurs transdifferentiation of perisinusoidal liver cells in myofibroblasts, which intensively synthesize collagen that leads to the development of fibrosis.
The marker of myofibroblasts is a -GMA. The appearance of a-GMA- positive myofibroblasts such a way leads to the deposition of new collagen and fibrosis.
CD34 - is a glycoprotein which is located on the outside membrane surface of endothelial cells and hematopoietic progenitor cells. In particular, it is used in isolation of hematopoietic stem cells as their marker. when morphological study of biopsy specimens CD34 interest us as a marker of endothelial of the sinusoids of the liver.
In order to perform of morphological analysis of liver biopsy material fixed 10% neutral formalin and poured into paraffin according to standard methods.
Immunohistochemical staining of liver sections was performed with a streptavidin-biotin method [3] by using a commercial monoclonal antibody against PCNA, a-SMA, CD34.
Results and discussions
Observation of patients for 8 weeks after the introduction of stem cells showed improvement in overall health, increasing efficiency, disappearance of weakness and discomfort in the right upper quadrant.
In order to asses objectively the comparative effectiveness of intra-arterial and ontraportal transplantation AMMSK during the treatment we conducted a study of the functioanal state of the liver during the treatment (Table 1).
In 2 months after the introduction of stem cells in all patients decreased performance of cytolysis (compare the levels of ALT, AST), increased prothrombin index, there was an improvement of albumin-producing ability of the liver (2 cases to the norm).
Table 1. The results of biochemical analysis on functional state of the liver in the studied patients with liver cirrhosis (n = 7)
Indicators Before transplantation Within 8 weeks after transplantation Norm
Albumin (g/L) 31.4±4.2* 37.4±3.6** 3В-41
Total bilirubin (|jmol/L) 3б.В±14.2 23.9±12.6 <1В.В
ALAT (U/L) Вб^^.б 41.1±15.4 <37
ASAT (U/L) 93.2±19.б 43.6±13.5* <39
PI (%) б7.3±9.4 76.5±8.3 7В-120
'-Significant differences in comprasion with initial data, p<0.05, ** p<0.01
As can be seen from Table 1 the overall dynamics of the studied parameters in the observed patients was positive. However, there were significant differences. In 2 months after transplantation noted a clear improvement over the morphofunctional state of the liver in comparison with the original data.
Thus, albumin synthesizing capacity of the liver before transplantation AMMSK was reduced in all patients observed by 21.5% from the lower limits of normal (r<0,05). In 2 months after transplantation AMMSK level of albumin in the blood increased and reached to 37,4 ± 3,6 g / L. The indicator is exceeded the original data by 19.1% and almost corresponded to indicator of the norm.
As shown our research, indicators of ALT and AST before transplantation AMMSK were higher than the average
rates on 133.5 and 138.97%, respectively (r<0,05). Intraarterial transplantation AMMSK accompanied by a decrease phenomena of cytolysis. In 8 weeks after transplantation performance levels of ALT and AST were lower than baseline on 52.4% and 53.2% (r<0,05).
A similar positive dynamics of intra arterial transplantation into the liver DHBs was marked and in the study of changes of the prothrombin index in dynamics of treatment also characterizing the functional state of the liver (Table 1).
According to the protocol all patients was performed puncture biopsy of the liver before and one month after transplantation AMMSK. Comparative analysis of histological activity index (HAI) is presented in Table 2.
Table 2. Indicators of histological activity index in patients with liver cirrhosis observed during the treatment
Indicator of histological activity index (HAI) The obtained results (number of points)
Before transplantations AMMSK 12
After transplantation AMMSK 8
Note: HAI of 1-3 - chronic hepatitis with minimal activity, 4-8 - poorly articulated activity, 9-12 - moderate activity of chronic hepatitis, 1318 decompensated cirrhosis.
Table 2 shown changes in histological activity index (HAI) observed cirrhotic patients in dynamics of treatment. HAI was calculated taking into account the presence of intralobular degeneration, periportal necrosis and portal inflammation as well as fibrosis withdrawn in the biopsy of the liver tissue.
As can be seen from Table 2 in all observed patients before treatment with stem cells HAI was 12, indicating the presence of moderate activity of following hepatitis. In 8 weeks after transplantation AMMSK HAI decreased by 4 points. We consider that this dynamic primarily related reduction of intralobular necrosis area of degeneration of the parenchyma and portal inflammation. At the specified time severity of necrosis around the portal tracts has not changed and in biopsies were marked porto-dockside bridging necrosis. These necrosis always were localized in the area of portodockside of connective tissue septa due to which patients were diagnosed with fibrosis.
Obviously, 2 months it is not enough time for permit fibrosis. At the same time, reduction HAI due to reduction of intralobular degeneration and portal inflammation reflects positive changes in the condition of the liver after transplantation. Moreover, favorable morphological changes in the liver parenchyma explain positive dynamics of biochemical parameters.
The most dramatic morphological change, which we found was significant reduction zone of germination connecting with tissue from the space of Disse. Prior to transplantation, all patients were identified perisinusoidal fibrosis and a month later after stem cell transplantation we did not found deposition of collagen fibers between the endothelium and hepatocytes.
According to the protocol, bioptates were stained with antibodies against PCNA, a-SMA and CD34.
High proliferative activity in hepatocytes before transplantation (Fig. 3B) indicates on the strength of the regenerative response. This kind of intensity always observed in case of damage of hepatocytes and is an response to reduce the cell mass or hepatocyte damage. In 2 months after transplantation AMMSK number of proliferating hepatocytes was within 1-4% which is an indicator of close to the norm (Figure 3C).
Interesting changes occurred after a stem cell transplant
Figure 3 - expression of proliferating cell nuclear antigen (PCNA) in liver (8 weeks posttransplantation) (x200 magnification) (A) Control group; before transplantation (B); 8 weeks after transplantations via hepatic artery (C).
with sinusoidal endothelium. In all patients before transplantation indicated capillarization of sinusoids - endothelium cells express CD34). This type of change of the phenotype endothelium in the sinusoids of the liver indicate that the fenestrated endothelium became less permeable. In 2 months after transplantation situation radically changed - in the sinusoids showed only isolated CD34 + endothelium cell.
Certain changes were observed in the expression of aSMA. Prior to transplantation, all patients within of connective tissue septa present myofibroblasts that indicated the ongoing synthesis components of connective tissue and progression of fibrosis. In one patient myofibroblasts were found in the sinusoids which is a adverse prognostic factor plan on further fibrosing of the liver parenchyma.
After the transplantation number of myofibroblasts decreased markedly and few a-SMA cells detected only in the range already formed of connective tissue septa, basically same a-SMA was present in smooth muscle cells of blood vessels. As exactly myofibroblasts are the main source of connecting tissue of regenerating liver decrease in their number explains as the restoration of the normal structure of sinusoidal and the disappearance of perisinusoidal fibrosis.
The results of this study allow us to conclude that transplantation AMMSK in common hepatic artery with cirrhotic liver disease in a safe and effective procedure. After 2 months transplantation AMMSK improves the general condition of the patient and liver function tests, positive changes in the morphology of the liver (reduced histological activity index by reducing the severity of portal inflammation and intralobular necrosis).
The most important result of transplantation AMMSK is that liver for qualitative structural changes - is greatly reduced proliferation of hepatocytes decreases the number of myofibroblasts in the parenchyma that leads to the solution
of perisinusoidal fibrosis and restoration of the normal structure of sinusoidal.
Thus, findings allow to conclude the prospects of this method in the treatment of CPU and of highly informative immunohistochemical methods in the assessment of the effectiveness of transplantation AMMSK. Obtained positive results show topicality and prospectivity use of stem cells in the treatment of cirrhosis of the liver.
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