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11THE EFFECTIVENESS OF THE DEVELOPED BIOENGINEERING STRUCTURE FOR THERAPY
OF LACK OF FUNCTIONS OF THE LIVER
Kostyukova S.
Candidate of Biological Sciences, Associate Professor of the Department of Medical Biology and Genetics
Ural State Medical University, Yekaterinburg, Russia
Korotkov A.
Candidate of Medical Sciences, Associate Professor of the Department of Medical Biology and Genetics
Ural State Medical University, Yekaterinburg, Russia
Sichkar D.
Assistant at Department of medical biology and genetics, Ural State Medical University, Yekaterinburg,
Russia Satonkina O.
Candidate of Biological Sciences, Associate Professor of the Department of Medical Biology and Genetics
Ural State Medical University, Yekaterinburg, Russia
Makeev O.
Professor, Doctor of Medical Sciences Head of the Department of Medical Biology and Genetics of Ural
State Medical University, Yekaterinburg, Russia,
Head of the Laboratory of Gene and Cellular Technologies of Institute of Medical Cell Technology, Yekaterinburg, Russia
Abstract
The goal of the study is to obtain convincing data on the effectiveness of the use of multipotent mesenchymal stromal cells (MMSCs) transfected with a plasmid vector with the hepatocyte growth factor gene (HGF), as well as spheroids from MMSCs transfected with the same vector (SFMSC).
Morphological and morphometric studies were performed on Wistar rats (n = 54) 30 and 90 days after transplantation of a suspension of transfected MMSCs and cell spheroids. The results obtained were statistically processed.
Positive changes of the studied parameters in the dynamics of observation are shown. Thus, therapy with the suspension of transfected MMSCs and SFMSCs by the number of binuclear cells and the thickness of connective tissue septa significantly differed from the corresponding indices in the control group.
Keywords: cirrhosis, multipotent mesenchymal stromal cells, transdifferentiation, gene therapy, hepatocyte growth factor
INTRODUCTION
Liver failure and cirrhosis is a widespread pathology. So, in Russia, every fourth patient in a hospital (regardless of profile) has one or another degree of severity of liver failure. Every year up to one and a half thousand people are diagnosed with cirrhosis of the liver. In other countries, the incidence of this pathology is close to domestic Russian, and the mortality from liver cirrhosis is about 30 cases per 100,000 population [1].
An uncontested method of treating patients with the final stage of liver cirrhosis is liver transplantation. At the same time, the lack of donor organs remains a global problem. Thus, only one out of ten patients on the waiting list receives such assistance. And the number of those awaiting transplantation is steadily increasing [2]. In turn, the difficulties with the survivability of the transplanted organ and the duration of its delivery determine the graft survival rate within 65%. Therefore, a significant number of research teams are involved in the development of non-transplant methods of treatment for both failure of functions and cirrhosis of the liver, as an extreme degree of development of this pathology [3, 4]. Taking into account the physiological features of liver tissue regeneration, the lack of proven information about the existence of hepatic stem cells, as well as the involvement of almost all functionally preserved hepatocytes in the process of restoring the integrity of this organ, the possibility of stem cell transplantation attracts much attention. It is assumed
that the cells injected will differentiate or transform (using stem cells from another germ layer) into cells of the liver tissue. Meanwhile, the proportion of transplanted cells undergoing reprogramming under natural conditions is extremely small and barely reaches tenths of a percent. Nevertheless, this approach is considered promising for the treatment of liver cirrhosis [5].
Thus, the aim of the work was to confirm the effectiveness of the developed tool, which provides for the use of reprogrammed multipotent mesenchymal stromal cells (MMSC) transfected with a plasmid vector with the hepatocyte growth factor (HGF) gene, as well as spheroids from MMSC transfected with the same vector (SMMSC).
Materials and methods
Linear laboratory rats (Wistar) n = 54 were used to evaluate the effect of transplantation of pre-differen-tiated MMSCs, as well as spheroids of these cells in cirrhotic liver injury. The work was carried out on the basis of approval of the study design by the local ethical committee of the Ural State Medical University of the Ministry of Health of the Russian Federation, as well as being guided by the European Convention for the Protection of Vertebrate Animals (Strasbourg, 03/18/1986) and the Order of the Ministry of Health of the Russian Federation (199n, 04/01/2016).
To simulate liver damage in laboratory animals, a well-known technique was used that combines two ap-
proaches to the pathology of this organ. Rats were injected intraperitoneal^ with a 50% solution of carbon tetrachloride in vegetable oil at the rate of 0.25 ml of pure CCl4 2 times a week. In addition to carbon tetrachloride, a solution of thiocytamide 150 mg / kg per week was used. The latter provides an improved supply of carbon tetrachloride to hepatocytes. This method makes it possible to achieve a stable output of liver cirrhosis in almost 100% of experimental animals.
According to the formation of liver cirrhosis, the administration was stopped and the animals were divided into 3 groups: two experimental and a control. To perform the cell therapy procedure, we used samples of adipose tissue from the inguinal girdle of intact rats due to the presence of the least differentiated and functionally active mesenchymal stem cells in this tissue [6]. The adipose tissue was collected under intravenous anesthesia.
Isolation and cultivation of MMSC from adipose tissue was carried out according to the standard technique [7].
We used pBABE-puro (Addgene plasmid 176) as a vector base. Synthesis of DNA encoding HGF was
carried out by reverse transcription using the RTS kit (Promega). PCR primers were designed using data and programs located on the NCBI website. Cells were removed from vials using 0.2% and 0.25% trypsin-Versene solution, respectively (Sigma Aldrich). The harvested cells were placed in 25 cm mattresses and cultured in an incubator at 5% CO2 in DMEM / F12 medium with fetal bovine serum from 3 to 12%, which was determined by the degree of differentiation of the cell culture. At the initial stage, the cells were cultured with the addition of penicillin, streptomycin, and tylo-sin at "culture" concentrations. The medium was changed every 3 days.
Upon reaching 70% confluency, cells were trans-fected with pBABE-puro HGF (Addgene) using the Lipofectamine ™ 2000 kit (Invitrogen) according to the manufacturer's protocol. After 24 hours, cells were washed from free plasmid vector and transfection medium.
Transfection made it possible to obtain cells with phenotypic characteristics of hepatocytes from multipotent mesenchymal stromal cells of adipose tissue.
Table 1
Study design
Experimental group Group characteristics
Group 1 experimental group No. 1 (n = 18) Upon completion of the modeling of liver cirrhosis, the rats continued intraperitoneal administration of physiological saline in an amount equivalent to the control for 6 days. On the 7th day, a single intraportal injection of a suspension of transfected MMSCs was carried out at the rate of 5 * 106 / kg of animal weight (106 cells for a 200-gram rat) in 0.5 ml of saline.
Group 2 experimental group No. 2 (n = 18) Upon completion of the modeling of liver cirrhosis, the rats continued intraperitoneal administration of physiological saline in an amount equivalent to the control for 6 days. On day 7, a single intraportal injection of a suspension of spheroids from transfected MMSCs was carried out at the rate of 5 * 106 / kg of animal weight (106 cells for a 200-gram rat) in 0.5 ml of saline.
Group 3 control group (n = 18) In animals, cirrhosis of the liver was simulated and saline solution (0.2 ml) was injected daily 6 times intraperitoneally and the seventh (0.5 ml) - intraportally. The animals did not receive any therapeutic effects. The results of the study of this group were used as an object of comparison with the animals of the experimental groups.
At the end of 30 and 90 days after the introduction of cells (or saline solution to a control group of animals), the animals were euthanized (by air embolism), which made it possible to study the relatively long-term results of therapy. To study the isolated liver, microscopic examination of the affected tissue [8] with he-matoxylin-eosin staining was performed.
Morphometry was performed on an Olympus CX-41 microscope using a package of special programs in 10 fields of view with a magnification from 100 to 300 [9]. The number of two or more nuclear cells, the degree of dystrophic changes and the thickness of the connective tissue interlobar septa were investigated [10].
The results were processed using the Mann-Whitney, Kruskal-Wallis and Pearson xi-square tests. The calculations were performed using the STATISTICA 8.0 package. The results were considered significant at p <0.05.
Results
The introduction of carbon tetrachloride and thio-acetamide was accompanied by the occurrence of liver
cirrhosis. The organ was visually enlarged, compacted, with rounded edges and tuberosity.
Among the typical signs observed in the liver with cirrhosis, there was the appearance of a rounded shape of unfilled cavities, edema, pigmentation, hyperemia of blood vessels, lysis of erythrocytes, as well as an increase in the number of binuclear cells with signs of necrosis and dystrophy.
An important sign observed during microscopy is fibrosis of the liver tissue with the formation of false lobules due to the proliferation of connective tissue septa.
The average value of the thickness of the connective tissue septa in the 3rd group did not exceed 35.12 ^m, in the 1st group - no more than 34.48 ^m and in the 2nd group - 33.60 ^m. No significant differences were found between the indicators of individual groups.
The average value of cellularity (binucleated / multinucleated cells, number in the field of view) in the 3rd control group was 12.0 (11.7 - 12.5), in the 1st group - 11.8 (11.3 - 12, 3) and in group 2 - 11.6 (11.4 -
11.8), which also did not allow finding statistical differences.
The assessment of the degree of development of signs of dystrophy after the reproduction of the cir-rhotic liver model in all three studied groups also did not reveal significant statistical differences.
Histological analysis of liver samples 30 days after administration of transfected MMSCs in the experimental groups revealed statistically significant differences from the control group.
Figure 1. Histological section of the liver 30 days after the injection of the MMSC suspension, group 1, hema-
toxylin-eosin stain, magnification x 100
So, in the control group (group No. 3), the uniform arrangement of binuclear / multinucleated cells, the presence of a fibrosing process of stromal tissue, degenerative changes in hepatocytes were determined.
In group 1, histological studies revealed a number of two or more nuclear cells, proliferation of connective tissue formations, and the presence of false lobules (Figure 1).
In group 2 (with the introduction of SMMSC), af- intercellular and interlacunar connective tissue septa ter 30 days, a smaller number of two- or more nuclear were found in the control group (Figure 2). cells, areas of moderate dystrophy and thinning of the
Figure 2. Histological section of the liver 30 days after the injection of SMMSK, group 2, hematoxylin-eosin
stain, magnification x 100
30 days after the administration of transfected MMSC / SMSCC, the mean value of the thickness of connective tissue septa in group 3 was 29.62 (28.8 -30.7) ^m, in group 1 (with the introduction of a suspension of transfected MMSC) - 22.18 (21, 56 - 23.6) microns and in the 2nd group 19.9 (17.8 - 20.5).
In the control group, the significance of the differences with the experimental groups was confirmed (pr0.05). Also, differences attributed to statistically significant were noted between the experimental groups.
Hepatocytic dystrophy in the 3rd (control) group was expressed in 4 animals and moderately expressed in 2; in the 1st group (with the introduction of a suspension of transfected MMSC) and slightly expressed in 3 rats; and in the 2nd group (with the introduction of
SMMSC) dystrophy was observed in 2 rats and weakly in 4 rats.
Comparison of the control and experimental groups demonstrated the statistical significance of the differences. No other significant differences were found.
In the morphological and morphometric analysis of liver samples 90 days after the injection of trans-fected MMSC in the form of a suspension of cells or spheroids, the changes were identified as significant compared to the control.
So, the 3rd group was characterized by the presence of mild processes of liver tissue repair. This was manifested in the form of preservation of degenerative changes, fibrosis of structures and a decrease in the number of binucleated cells.
In group 1, histological examination revealed thinning of the intercellular and interlacunar connective tissue septa with insignificant compaction of the cells of
the hepatic lobules and mild cellular dystrophy (Figure
3)
Figure 3. Histological section of the liver 90 days after the introduction of a suspension of transfected MMSC,
group 1, hematoxylin-eosin stain, magnification x 100
Histological sections of rat liver in group 2 were closest to normal values. Over a larger area of sections, residual fibrosis, thinning of the connective tissue
septa, mild dystrophy in some areas are revealed. The number of bi- and multinucleated cells did not differ
from intact values (Figure 4).
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Figure 4. Histological section of the liver 90 days after the injection of SMMSK, group 2, hematoxylin-eosin
staining, magnification x 100
The average value of the thickness of the connective tissue septa in the 3rd group was 25.5 (24.68 - 26.11) microns; in the 1 st group (with the introduction of a suspension of transfected MMSC) - 16.76 (15.34 -17.87); in the 2nd group (with the introduction of SMMSC) - 12.89 (11.76 - 13.85) microns. In the 3rd group, the thickness of the connective tissue septa significantly exceeded the corresponding indicator of the experimental groups. At the same time, differences were also revealed between the experimental groups (1st and 2nd).
Signs of hepatic tissue dystrophy in group 3 were observed in 2 animals and were poorly expressed in 4 animals. In the 1st group, they were weakly expressed in the 1st rat and were practically not observed in 5 animals. In group 2, there were practically no signs of hepatic dystrophy.
In group 3, the average value of two or more nuclear cells in one visual field did not exceed 11.5 (10.8 - 11.9), in group 1 - 9.0 (8.7 - 10.1), and in the 2nd group - 8.2 (7.9 - 8.7). Found statistical differences between the indicators of the 1st and 2nd groups (p = 0.02).
Discussion
Studies of the histological parameters of animals after a two-month modeling of liver cirrhosis by the introduction of solutions of carbon tetrachloride and thi-oacetamide demonstrated the reproducibility of this method in all 54 of the 54 studied animals, conventionally divided into 3 groups - experimental and control. The lack of statistical significance of morphological parameters under the condition of a stochastic division of groups indicates the stability of this model, which gives rise to a study of drugs for the treatment of liver cirrhosis.
30 days after the reconstruction of the hepatotoxic model, as well as the introduction of a suspension of transfected MMSC (group 1) and spheroids from transfected MMSC (group 2) in comparison with a group of animals without cell therapy (group 3, control), some improvements in morphohistological parameters were revealed, which found expression in the form of a lesser thickness of connective tissue interlacunar and intercellular septa, as well as signs of dystrophy and the number of two- or more nuclear hepatocytes.
After 90 days, more pronounced positive changes were observed in the groups with cell therapy compared
to both the baseline values (in all groups) and the corresponding indicators of the control group.
It seems important that in laboratory rats that were injected with cell spheroids, the morphohistological parameters were better than in rats that were treated with a suspension of transfected MMSC. This is manifested in a decrease in the number of binucleated cells, as well as in the thickness of the connective tissue septa.
The use of the formed spheroids, apparently, to a greater extent corresponds to the structural and functional features of the liver tissue under physiological conditions, when the layer-by-layer structure of cells differs in activity, depending on the distance from the lacunae.
Conclusion
The administration of MMSC transfected with a plasmid vector (carrying a useful hepatocyte growth factor gene) as monotherapy, as well as spheroids from MMSC transfected with the same vector, is accompanied by an improvement in the morphohistological parameters of the damaged liver in dynamics for 90 days after MMSC transplantation.
The introduction of SMMSC was accompanied by slightly better indicators than those in rats with the intraduction of a suspension of MMSC.
Thus, the forms of the bioengineering agent selected for the study (a suspension of transfected MMSC and SMMSC) confirmed the efficacy in the treatment of liver cirrhosis in preclinical studies. At the same time, cell spheroids are apparently more promising, since they differ from other groups in accelerated repair of liver tissue damage.
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