Biological Properties of the Stem Cells and their Effect on Restoring Animal Myocardium after Experimental Ischemic Infarction

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURE

Abstract

Introduction

Materials and research methods

Research results

Conclusion

Bibliographical references

LIST OF TABLES

Table 1 Surface marker expression in the cells population that were derived from rat’s myocardium from I to IV passage

Table 2 Expression of surface markers in stem cells population derived from a rat’s red bone marrow

Table 3 The results of cytogenetic analysis of the I-VI passages stem cells culture obtained from the rat’s myocardium

Table 4 The results of micronuclear test of the rat’s myocardium stem cells culture of І–VI passages

Table 5 The results of cytogenetic analysis of the stem cells population, derived from the red bone marrow of a rat

Table 6 Proliferative activity of the stem cells culture of rat’s myocardium during the study of cytotoxic activity of the lymphocytes and blood serum of intact and sensibilized animals

Table 7 The percentage ratio of the necrotized myocardium tissue to the total cut section are after transplanting the studied cell cultures with the consideration of the experimental rat’s ischemia infarction

Table 8 The number of the cat’s myocardium stem cells in the monolayer depending on the initial material processing method

Table 9 Comparing different methods of obtaining the cat’s adipose tissue stem cells culture

Table 10 Proliferative activity of the stem cells obtained from different sources, depending on the concentration of IGF-1 in the culture medium

Table 11 Proliferative activity of the stem cells obtained from different sources, depending on the concentration of FGF-2 in the culture medium

Table 12 Proliferative activity of the stem cells obtained from different sources, depending on the rhGH concentration in the culture medium

Table 13 Proliferative activity of the stem cells culture of different origin after adding to the Biolaminin 521 LN medium

Table 14 The results of cytogenetic analysis of the cat’s stem cells under growth factors effects

Table 15 The results of the cytogenetic analysis of the cat cells culture under rhGH and LN521 effect

LIST OF FIGURE

Fig. 1. The microphotos of monolayer of the rat’s myocardium stem cells culture, 0 passage

Fig. 2. The microphotos of monolayer of a rat’s myocardium stem cells culture

Fig. 3. The level of expression of the surface markers in the rat’s myocardium stem cells, IV passage

Fig. 4. The microphotos of monolayer of red bone marrow cells culture, III passage

Fig. 5. Microphotos of rat metaphase plates (І passage)

Fig. 6. Microphotos of the cells with the nucleus changes (IV passage)

Fig. 7. Rat myocardium, I test group: 1) connective tissue fibers; 2) cardiomyocytes

Fig. 8. Rat myocardia, I test group: 1) hyperemic vessel; 2) dystrophic changes of cardiomyocytes; 3) connective tissue; 4) cardiomyocytes

Fig. 9. Rat myocardia, II test group: 1) cardiomyocytes with disrupted structure; 2) cardiomyocytes; 3) vessel

Fig. 10. Rat myocardium, the 7th day after the ligation of the branch of the left coronary artery: 1) signs of coagulation necrosis (outlines of dead cardiomyocytes) 2) erythrocytes; 3) cardiomyocytes

Fig. 11. Rat myocardium, the 7th day after the ligation of the branch of the left coronary artery: 1) stroma cell proliferation; 2) nuclei; 3) cardiomyocytes

Fig. 12. Rat myocardia, the 12th day after the ligation of the branch of the left coronary artery: 1) accretion of collagen; 2) cardiomyocytes

Fig. 13. Rat myocardium, the 12th day after the ligation of the branch of the left coronary artery: 1) isles of necrotized cardiomyocytes; 2) cardiomyocytes; 3) рericardium

Fig. 14. Rat myocardium, the 17th day after the ligation of the branch of the left coronary artery: 1) formed collagen fibers; 2) cardiomyocyte

Fig. 15. Rat myocardium, the 25th day after the ligation of the branch of the left coronary artery: 1) collagen fibers; 2) cardiomyocytes; 3) granulation tissue

Fig. 16. Rat myocardium, the 25th day after the ligation of the branch of the left coronary artery: 1) connective tissue scar; 2) cardiomyocytes

Fig. 17. Coculturing the rat’s myocardium stem cells of the I passage with: a) lymphocytes; b) blood serum of intact animals

Fig. 18. Coculturing the rat’s myocardium stem cells of the I passage with addition of: а) leucocytes; б) blood serum of sensibilized animals

Fig. 19. Detecting the intramyocardially administered cells marked by Hoechst in the cardiac muscle

Fig. 20. Cat’s myocardium stem cells monolayer under effect of different tissue treating methods (the 3rd day of culturing, the 0 passage)

Fig. 21. The cat’s myocardium stem cells culture monolayer under different methods of obtaining (the 5th day of culturing, 0 passage)

Fig. 22. Micro photo of the cat’s adipose tissue stem cells culture (0 passage, 7th day of culturing)

Fig. 23. The microphotos of the red bone marrow stem cells under different mitogens effect

Fig. 24. Microphotos of the adipose tissue stem cells under the effect of various mitogens

Fig. 25. Microphotos of the myocardium stem cells culture under the FGF-2 effect

Fig. 26. Microphotos of the bone marrow stem cells culture under the rhGH effect

Fig. 27. Microphotos of myocardium stem cells culture under the LN521 effect

Fig. 28. Microphotos of the metaphase cell plates of a cat

ABSTRACT

The monograph deals with the current issues of veterinary medicine, in particular, experimental solution of the scientific issue of the effect of stem cells derived from myocardium, bone marrow, and adipose tissue on the restoration of myocardium after experimental pale infarction.

Study were focused on the myocardium-derived cultures of stem cells as an alternative source of cellular material to treat heart diseases.

It is important to emphasize that in the course of in vitro cultivation, cells in the culture tended to change their phenotypic characteristics: morphology, expression of CD markers, and karyotype.

The primary culture of myocardium-derived stem cells of a rat reached 90–100 % confluence in 8 days average. In the process of subcultivation, 70–80 % confluence was reached in 3 days. In the process of cultivation, the number of fusiform cells tended to increase with each passage. Thus, in the process of culture of myocardium-derived stem cells cultivation, heterogeneous culture became more homogeneous

Having analyzed the data of the immunophenotypic profile of the rat myocardium cells, it is possible to make a conclusion that is heterogeneous in terms of cellular composition that additionally changes in the process of cultivation. Thus, at the beginning of the study, the culture had more cells with the signs of cardiomyocytes, in particular, high levels of CD10+ and troponin Ihigh, CD95+, low level of CD34low, CD38low, CD45low, СD326low, and the absence of expression of CD48-, CD54- CD56-, СD227-. It is important to emphasize that in the process of cultivation it appeared that cells had markers peculiar for committed cells: CD34+, CD45low and epithelial cells: pan-keratinlow, СD326+, СD227low. The cytogenetic analysis cultures showed a gradual percent increase in cells with aneuploidy, which correlated with a percent increase in cells with micronuclei. Directed differentiation in the cardiomyogenic direction causes bone marrow-derived stem cells to have cardiomyocytes-like peculiarities. This is marked by an increase in the level of expression of CD10 and troponin I and a decrease in the levels of CD45, CD56, CD227, CD326, and pan-keratin.

In the course of the study, neither lymphocytes nor blood serum of intact animals showed in vitro cytotoxic action to the culture of myocardium-derived stem cells rat, explained by the absence of preliminary sensitization by the antigens of the studied culture of these animals.

However, a sensitized animals experiment showed a significant cytotoxic effect of lymphocytes - marked by a great decrease in the proliferation index of studied cells – and blood serum.

In order to study the ability of stem cells to migrate, the area of myocardial damage was treated by 0.5 million of fluorochrome-labeled stem cells of the bone marrow culture by intracardiac, intramyocardial and intravenous administration. In case of intramyocardial administration, we detected labeled cells in the administration channel on the second day after transplantation. The analysis of cardiac samples after intracardiac and intravenous transplantation showed no labeled stem cells in the cardiac muscle structure, neither on the second nor on the eighth day.

The final stage in the course of the research of rat stem cell cultures is the study of their effect on experimental infarction. The study showed that stem cells helped to decrease the area of necrotic tissue of animal myocardium. The study revealed that culture of myocardium-derived stem cells appeared to be the most effective: it managed to make the area of necrosis 1.53 times lesser if compared with the control group.

Since the therapeutic effect of the studied cultures on the development of experimental myocardial infarction had been proven, we were to determine the best conditions for isolation from different sources and cultivation of cultures of cat stem cells for their further putting into veterinary practice.

In order to obtain the cat myocardium stem cells culture, there were compared the method of explants and 5 variants of enzymatic treatment of the cardiac tissue. In order to determine the optimal method for obtaining adipose tissue stem cells culture, there were compared the method of explants and 6 combinations of enzymes.

In addition, we were study the optimal concentration of insulin-like growth factor-1, growth factor of fibroblasts-2, growth hormone and Biolaminin 521 LN for proliferation index growth of stem cells of bone marrow, adipose tissue and myocardium, and accounted for the number of genetic errors in studied cultures.

INTRODUCTION

Myocardial ischemia is a heart dysfunction caused by insufficient blood supply to its muscle tissue. This supply decrease can be related to the narrowing of coronary arteries, coronary thrombosis, or diffusive narrowing of arterioles and other small-sized vessels of the heart. Stopping the blood supply to the myocardium may lead to cardiac muscle necrosis (myocardial infarction) 72.

Coronary arteries perfuse the heart and help transpor the nutrients and Oxygen to metabolically active myocardium 95. Nowadays, myocardial ischemia, which is associated with atherosclerosis and coronary arteries obstruction, is the leading cause of death for both men and women, and according to the predictions it will remain so until 2030 64. Myocardial ischemia and myocardial infarction, which is associated with coronary arteries diseases, can also happen to small animals like cats and dogs [35, 95], which may lead to a sudden death or death while under anesthesia 95.

Because the heart of adult mammals has extremely limited regeneration ability 90 and the lost cells are replaced with fibrous scars 103, there is a need for search for treatment methods that are aimed at restoring the structure of cardiac muscle after the ischemia.

Cell technologies are a promising treatment method for animals with myocardial infarction that restores its structure and contractile function [111, 42, 57, 102, 54]. In the past 50 years there have been a significant number of experimental researches that deal with the cell technologies issues. There are hundreds of published papers that indicate the positive results of using the stem cells (SC) to treat different diseases found in humans and animals [22, 46]. As a result of the research, we understand that stem cells and their derivatives may lay foundation for developing the innovative cell technologies, at any stage of histogenesis. The spectrum of clinical issues that this kind of treatment methods is expected to help with is astounding 15.

However, the introduction of cell technologies to the clinical practice requires a thorough guide for protocols on how to obtain the stem cells, cell culture and usage, deep study of recipient organism and the transplanted cells interaction, close control of phenotypic and genetic changes in order to provide high quality and safety of the used cell materials in restorative clinical therapy of post myocardial infarction changes in the myocardium.

The research mainly focuses on the culture of myocardium-derived stem cells as an alternative source of the cell materials for treating patients with heart diseases 65.

MATERIALS AND RESEARCH METHODS

All manipulations with animals were conducted in compliance with the Law of Ukraine "On the Protection of Animals from Cruelty" (No. 3447 - IV, dated 02.21.2006) (Law of Ukraine 2006).

The research used the cell culture of bone marrow, pancreas (that were obtained from the bone marrow of 12 days old rat`s tubular bones and hearts), and adipose tissue (obtained from 4-5 months old rats). The obtained cell mass was cultured in a standard medium: 80 % − Dulbecco's modified Eagle medium; 20 % − fetal bovine serum; 10 mcL/cm3 – antibiotic-antifungal agent “Sigma”, USA): in СО2 incubator at 37 ºС and 5% concentration of СО2 50, up to 90-100% confluence.

The cells were detached according to a standard method (0.25% solution of trypsin/EDTA) 3. Further passaging was executed in a 1:3 dilution. For microscopic analysis and culture evaluation the inverted microscope Axiovert 40 (Carl Zeiss) was used.

Phenotype change control was made by detecting CD-markers (СD10, CD38, CD34, CD45, CD48, CD54, CD56, CD66e, CD96, CD227, CD326б, СD pan-keratin) The preparations were prepared according to the standard method) 50.

The results analysis was conducted based on the number of cells with expression (green light of cells) and evaluated with the H-Score classical method: S = 1×A + 2×B + 3×C, where S is the “H-Score” indicator, and its measure ranges from 0 (protein is not expressing) to 300 (strong expression in 100% of cells); А – cells with a weak expression; В – percentage of cells with a moderate protein expression; С – cell percentage with strong expression. The level of expression is considered negative if the score is in 0-50 range; 51-100 is considered low; 101-200 is considered moderate; 201 and more is considered high. The research was conducted with the help of a Leica DMR fluorescent microscope (Germany) 68.

Cytogenetic analysis was conducted on 30 metaphase plates of cell cultures from every passage. The modified standard cytogenetic method was used to obtain the chromosome preparation 50. The obtained preparations were colored with coloring tools (Leukodif 200) as per manufacturer`s instructions. The metaphase plates were analyzed with Leica DMR (Germany), ×400, ×1000 magnification. The mentioned preparations contained quantity chromosome disorder - aneuploidy (A), polyploidy (PP), the binucleated cells (BC), micro-nuclei cells (MC), mitiotic index (the percentage of the cell at the separation stage against the total number of the analyzed cells (MI) 2, apoptotic index (the percentage of the cells with the signs of apoptosis against the total number of the analyzed cells (AI) 9. The frequency of BC, MC, AP occurrence was calculated per 500 cells (%).

To determine the cytotoxic activity of lymphocytes in animals sensibilized to culture cells, on the 15th day after administrating the cells, 1.5 ml of blood was taken and produced lymphocytes according the Boyum A. method 24. After that 3×104 of the researched cells (that the animals were sensibilized to) were placed in 96-well flat bottomed plates (Sarstedt, Germany) with 0,1 ml of standard culture medium and then placed in a СO2-incubator for 1 hour in order to glue the cells to the bottom of the culture plastic. After the selected time had passed, 1×104 of lymphocytes (sensibilized and intact animals depending on the group) in 0.1 ml of standard culture medium (target cell:lymphocytes ratio 1:3) were placed into the walls with the researched cells. After that, the plates were placed in a СO2-incubator and cultured for 18 hours with 5% of СO2, 100% humidity and at t=37°С 7.

The cytotoxic activity of the blood serum to stem cells cultures was determined on the 15th day according to H.V. Didenko method 4 that was modified by the department employees. To obtain the blood serum taken from the researched animals, it was stored until a full clotting of the fibrinogen. Then the blood serum was taken with a Pasteur pipette and centrifuged for 5-7 minutes at 3000 r/m. Simultaneously, 3×104 of the researched cells were placed in 96-well flat bottomed plates with 0.1 ml of standard culture medium (that animals were sensebilized to) and added 0.1 ml of researched animals’ serum. The cells were incubated for 40 minutes at a room temperature. After the time had passed, 0,1 mcL of complement (1:4 dilution) was added and incubated in standard conditions for 18 hours.

After the time had passed the wells were washed with PBS in order to remove non-adhesive cells and lymphocytes (if there were any). To detach the adhesive cells from the culture plastic 100 mcL of 0.25% trypsin/EDTA (t=37 °С) was placed into every wall with cells, and 1-2 minutes later the detached cells were pipetted. In order to count their number, Goryaev chamber was used with a 200 magnification in all squares and was calculated by the following formula:

Abbildung in dieser Leseprobe nicht enthalten

where:

Х is number of cells per 1 cm3;

А is number of cells in all squares;

1000 is number of mm3 in cm3;

0,9 is the volume of Goryaev chamber in mm3.

The cytotoxic effect on transplanted cells was proved by the absence of their proliferative activity.

The vital nuclear stain Hoechst 33258 (Sigma, USA) was used to mark the stem cells. The red bone marrow stem cell culture with 60-80% confluence was stained. Before administrating Hoechst 33258 the adhesive cells were washed with PBS twice and a new standard culture medium was added that had been heated to t=37 ºС, adding 2 mcL/mL of fluorochrome and incubated in a СО2-incubator for 30 minutes.

After the time had passed the incubated cells were washed with PBS once and the culture medium was changed. After marking, the cells fluorescent with a bright green color.

The III passage cells were used to induce the differentiation of the culture of bone marrow-derived stem cells into cardiomyocytes. CBMDSC were cultured in standard conditions until a monolayer appeared at 50-60%. Then 10 μm 5-azacytidine (SIGMA, USA) was added to the culture medium. To prevent the cells from dying due to 5-azacytidine long activity, the adhesive cells were washed with PBS 3 times and a new portion of a standard culture medium was added after differentiation induction during 24 hours. The cells had been cultivated for 3 weeks, changing the half of culture medium for a new one every 3 days for a full differentiation 84. The stem cell culture was studied on the 6th day after the differentiation induction in order to track the phenotypical changes, genetic changes, and cytotoxic effect of leucocyte and blood serum.

To determine an optimal method of myocardial infarction modeling two options were used. In the first test group (n=3) myocardial infarction was modeled by ligating the left coronary artery branch

In the second group (n=3) myocardial infarction was modeled by coagulating the artery in the above-mentioned area with a hot needle. 0.5 million stem cells in 0,05 cm3 of Igla culture medium, modified by Dulbecco (DMEM) (Sigma, USA), were transplanted intramyocardially with an insulin syringe (Chirana, Slovakia) immediately after ligating the left coronary artery, 1 mm lower from the area of ligating. The cells were administered gradually in order to prevent an extreme cardiac muscle extension. The control group of animals had 0,05 cm3 of DMEM administered.

The area of damaged myocardium was determined on the 25th day after ligating the left coronary artery by staining the heart section with 2,3,5- triphenyl tetrazolium chloride (TTCh) (Sigma USA) 99.

To determine the area of the necrotized area of the myocardium, the heart was dissected with a blade into 3 segments of an equal width. The sections were then put into a Petri dish with a 1% TTCh solution on phosphate buffer solution (Sigma, USA) and incubated for a day in a СО2-incubator. The TTCh staining method allows for separating the inevitably damaged myocardium from a vital tissue on a macroscopic level. The vital myocardium with preserved NAD-dependent enzymes activity is stained in a dark red color, while the necrotized tissues remain pale-pink. The stained sections where shot on photo and were digitally processed in Adobe Photoshop. The total area of the cicatricial tissue was determined by 3 sections and shown in a percentage ratio to the total area of the section.

The heart samples for histological research when studying the pale infarction course were taken on the 7th, 12th, 17th and 25th days. The samples of tissues were taken, and histological studies were conducted according to the standard methods after euthanizing the test animals. Dewaxing and section staining according to Van Gieson method, Carrazzi's hematoxylin, and eosin was done on an HMS 70 (MICROM, Germany) linear type tissues staining device.

RESEARCH RESULTS

When culturing the culture cells in vitro, they may change their phenotypical properties: morphology, expression of CD markers, karyotype). And the initial culture will have the most heterogeneity which is the result of all the original tissue cells being present that have the adhesive properties depending on the source where they were obtained from. During culturing, they are removed and the cells that are able to divide are proliferated. It also worth mentioning that every cell population has its own speed of division that may change the phenotypical peculiarities of the culture in the future.

At this moment, the cell cultures obtained from the bone barrow and the adipose tissue are well-studied. At the same time, the culture of myocardium-derived stem cells (CMDSC) as an alternative source of stem cells for heart diseases treatment is poorly studied. Knowing this, the first stage of our research was to study the phenotypical properties of the culture of rat’s myocardium stem cells in the culturing process. The obtained data shows the phenotypical and genetic stability of the culture with a safe usage in the future.

When studying the morphology of the rat’s myocardium stem cell culture we discovered that explanation method causes the initial culture to start growth from the pieces of tissue that was on the very bottom of the cultural plastic (Fig. 1a). The further cultivation has shown that the initial culture of adhesive myocardium stem cell of a rat is morphologically heterogenic and there were a moderate number of spindle cells among the dominant epithelioid cells (Fig. 1b).

These results can be explained by the variety of cell pool in the material that CMDSC was obtained from. According to Urbanek K. et.al. 107, Oh H. et.al. 78 the cardiac muscle contains three populations of proliferating cells: stem cells are located in the myocardium, as well as the endothelial cells and progenitor cells of smooth-muscle tissue, orderly placed in coronary vessels.

The initial CMDSC of the rat reached the 90-100% confluence on average in 8 days (Fig. 1b). Reaching the 70-80% confluence took 3 days during the subculturing.

Abbildung in dieser Leseprobe nicht enthalten

Fig. 1. The microphotos of monolayer of the rat’s myocardium stem cells culture, 0 passage: a) 4th day; b) 8th day of culturing. Native preparations; Magnification: ×50. (Author’s own work)

Abbildung in dieser Leseprobe nicht enthalten

Fig. 2. The microphotos of monolayer of a rat’s myocardium stem cells culture: a) I passage; b) IV passage. Native preparations; Magnification: ×50.

During the subculturing the stem cell culture obtained from the rat’s myocardium was visually getting more homogenic due to fibroblast-like cells that came to be because of their proliferating activity (Fig. 2b). Meanwhile the epithelioid-like morphology cells were less active.

It should be noted that immunophenotyping of the population of stem cell cultures obtained from a rat’s myocardium also revealed a change in the expression of the studied surface markers with passages (Table 1).

Thus, the intensity of CD45, CD227 and pan-keratin was getting higher in the culturing process within the “absence of expression” level with 3,3±3,9, 0±0 and 0±0 points on the I passage up to 23,7±6,8, 22,7±8,9 and 41,3±9,1 points on IV passage respectively.

Abbildung in dieser Leseprobe nicht enthalten

Fig. 3. The level of expression of the surface markers in the rat’s myocardium stem cells, IV passage. A) CD66e; b) pancytocreatine. Fluorescent microscoping; Magnification: ×1000. (Author’s own work).

Table 1

Surface marker expression in the cells population that were derived from rat’s myocardium from I to IV passage, M±m, n=3

Abbildung in dieser Leseprobe nicht enthalten

Note: *р<0.05; **р<0.01, compared to control (I passage served as the control)

During the entire study we did not detect CD48, CD54 and CD56 expression (0±0 points). The CD38 expression level kept decreasing within this level from 22.7±4.5 at the І passage to 18.7±5.0 points at the ІV. The CD10 and troponin I expression kept decreasing from a high level (235.7±35.6 and 257.0±23.2 points respectively) on the I passage to a moderate level (170.3±27.7 and 181±9.9 points respectively) on the IV level. The CD34 intensity grew from “absence of expression” (8.3±5.6 points) on the I passage to moderate (170.0±19.7 points) on the IV, and CD326 grew from “absence of expression” (6.3±3.7 points) of I passage to low (54.0±8.7 points) on the IV.

The CD66e expression level grew from moderate (185.3±35.8 points) on the I passage to high (287.0±5.8 points) level on the IV (Fig. 3, b). Meanwhile the CD95 intensity was lowering during the cultivation within the moderate level, from 164.0±13.4 points on the I passage to 108.3±8.9 points on the IV.

As we know, 5-azacytidine induces the differentiation of the stem cells in cardiomyogenic direction that goes with changes in the morphology of the cultured cells and the heart marker expression, such as GATA-4, Nkx2.5, troponin І [84, 30]. However, there is a lack of data regarding the marker expression that are normal for weakly differentiated (stem) cells in the culture that was affected by 5-azacytidine. It should be noted that the information about the condition of the genetic apparatus of the mentioned cells in the available literature is simply absent. Our next task was to study the above-mentioned issues. We noted the changes in cell morphology from spindle-shaped to epithelioid (Fig. 4) on the 6-7th day of culturing since the start of differentiation of the cultures of red bone marrow stem cells.

Abbildung in dieser Leseprobe nicht enthalten

Fig. 4. The microphotos of monolayer of red bone marrow cells culture, III passage: a) control; b) on the 6th day after adding 5-azacytidin. Native preparations. Magnification: ×50. (Author’s own work).

The decrease of their proliferating activity was also noted. On the 10-22nd day of culturing the studied cell cultures started having occasional shortages of certain areas in the cell monolayer which further proves their differentiation in the cardiomyogenic direction.

In the process of immunophenotyping the culture of red bone marrow stem cells of a rat that was affected by 5-azacytidin, we noted the change in expression of the studied surface markers towards what is more natural for the culture derived from myocardium (table 1 and 2).

Table.2

Expression of surface markers in stem cells population derived from a rat’s red bone marrow, M±m, n=3

Abbildung in dieser Leseprobe nicht enthalten

Note: *p<0.05; **p<0.01; ***p<0.001 compared to control

Thus, the CD10 expression level has grown compared to control and scored 107±19 points. СD34, СD38 and СD95 intensity has grown by 21, 18 and 22 points respectively. СD48, СD54 and СD66е expression levels remained within the same level, and the culture affected by a directed differentiation scored 83±10, 0±0 and 110±11 points respectively. The СD45, СD56, СD227 and СD326 intensity level changed from low to “absence of expression” and scored 0±0, 0±0, 0±0 and 10±6 respectively.

The level of pan-keratin expression decreased from high (253 ± 19 points) to moderate (77 ± 14 points), while the degree of troponin I intensity in culture subjected to targeted differentiation with 5-azacitidine, increased significantly from the level of "absence of expression" (17 ± 10 points) to moderate 105 ± 9 (points).

When immunophenotyping the bone marrow cell cultures under 5-azacitidine effect and those derived from myocardium, we noted curious patterns in the studied CD-markers expressions.

We noted that during culturing, the CD10 expression level (neutral peptidase 24.11 or NEP) in rat’s myocardial stem cells decreased from high level to moderate. It’s worth mentioning that rat’s CBMDSC expression of this marker has grown to moderate, it is the one that was subjected to a direct differentiation in a cardiomyogenic direction.

NEP has different functions that depend on the cells type. Broccolini A. et.al. 25 state that CD10 directly or indirectly via insulin-like growth factor I may have a crucial role in muscle cells differentiation. More so, Fielitz J. et.al. 36 state that the NEP has the ability to degrade bradykinin and natriuretic peptides in the cardiac muscle. So, discovering CD10 expression in cells of the studied cells was predicted. Furthermore, the fact that CD10 was present on rat’s myocardium cells is proved by the research of Piedimonte G. et.al. 82.

CD34 expression on culture cells that are derived from rat’s myocardium also changed depending on the passage. Its levels went from “absence of expression” to moderate. CD34 intensity growth to moderate level was also noted in the culture of red bone marrow stem cells that was treated with 5-azacitidine.

CD34 is a marker that can be seen on most of the progenitor cells 100. Even though its function is not clear, we know that the cells with high expression of CD34 have a well-developed ability to form colonies as well as a long-term proliferating activity [85, 109, 32]. However, there is controversial data regarding the expression of this particular marker in cell culture that is derived from myocardia. Darryl R. et.al. 32 explain the expression of this marker in the initial culture obtained from myocardium by the presence of CD34-positive endothelial cells. They claim that the cardiac muscle progenitor cells are CD34-negative. Zhou Q et.al. 117 state that the initial myocardium culture contains telocytes (a type of interstitial cells that usually have a small body with a few long branching-telopodes) that express this particular marker and are able to divide [117, 116].

CD38 expression remained within “absence of expression” level while 5-azacitidine use on CBMDSC led to increase within low level.

СВ38 functions as a NAD (P)(H) cell regulator in heart, however, it activates only during an oxidative stress (regular for ischemia) [108, 23]. The expression of this marker is different in different heart cells accordingly. According to Boslett J. et.al. 23 endothelial heart cells have a high level of CD38 expression, while fibroblasts have a low level, and cardiomyocytes can have only traces of that.

CD45 expression in stem cells that are derived from rat’s myocardium also had a “absence of expression” level during the entire research. But CBMDSC direct differentiation on rat in the cardiomyogenic direction led to lower expressions of this particular marker from low to above-mentioned level.

As of today, we do not know for sure the role of CD45 in the life of stem cells 98. However, there is data about incubation effect of this marker on the differentiation and proliferation of the cells by affecting the JAK/STAT 52 signal route. It should be mentioned that the data about CD45 expression in heart cells is also debatable. According to Sandstedt J. et.al. 93, the cardiac muscle has both CD45-positive and CD45-negative cells. They also described CD45- cells as endothelial progenitor cells, while CD45+ were categorized as mast cells. In their further studies 92, the scientists point CD45-negative stem cells in the cardiac muscle that additionally express the cardiac markers.

CD48 expression on stem cells that are derived from the rat’s myocardium was not marked during the entire period of culturing. The level of cell culture that was affected by 5-azacitidine was identical to the control group.

CD48 functions as immunity regulator and it is a hematogenic cell 67 that explains why it is not in the culture of stem cells derived from the rat’s myocardium.

CD54 expression (ICAM-1) was not found in the cultures throughout the entire duration of study. ICAM-1 is constitutively placed on endothelial cells, but its expression is heightened upon inflammatory cytokines activity 59, as it participates in changing the permeability of the vessel wall during an inflammation 38. CD54 expression can be found on cardiomyocytes under an acute myocardial infarction [77, 21]. The fact that there is no expression of this particular marker in the studied cultures can be explained with the absence of the conditions for an inflammation reaction when culturing the cells in in vitro system.

No expression of CD56 (NCAM) in the cultures was detected throughout the entire research. CD56 mediates the cell adhesion and signalization in the nervous system 37. However, according to Nagao K et.al. 73, NCAM functions as a cardioprotection that can be seen in cardiomyocytes under a metabolic stress. The scientists claim that the CD56 expression is clearly visible in the remaining heart myocytes in the infarction area. The absence of CD56 in the researched cultures is explained by the absence of the metabolic stress for the cells that are cultured in in vitro system.

The CD66e (CEACAM5, СЕА) expression level in the culture of a rat myocardium cells during culturing increased from moderate to high.

It’s worth noting that CEA has various functions in in vitro system, including the cell adhesion that helps binding collagen and activating ecto-ATPases 106.

However, the expression of this particular marker sampled from healthy heart is usually not noted 27. That is why the discovered high CD66e expression level in the culture of a rat myocardium cells indicates the need for the further study of this particular marker functions.

CD95 (Fas) expression level in the culture of a rat myocardium cells decreased within the moderate level, while the expression level in rat CMBDSC under the effect of 5-azacitidine increased within the said level.

According to Woller K. C. et.al. 114 Fac is constitutively expressed in myocardium and cardiomyocytes, and the expression level depends on the stress conditions in in vivo system, so increased CD95 level does not trigger these cells’ reaction that led to apoptosis. Thus, this particular marker expression in the researched cultures can be explained by the presence of progenitor cardiomyocytes and the cells in the state of apoptosis.

CD227 (MUC1) expression in the studied cultures ranged within “absence of expression” level. It should be known that 5-azacitidine effect on CMBDSC caused the expression of this particular marker in cells to disappear.

MUC1 is usually found on epithelial and tumorous cells. It is able to interact with growth factors 96 and inhibit own way of apoptosis [86, 113]. While maintaining the balance between the growth and apoptosis. According to Thie H. et.al. 105, CD227 expression will not be found in a healthy heart cells which is also true according to our data.

CD326 (EpCAM) expression level in cells obtained from the rat myocardium increased from “absence of expression” to low. In the meantime, 5-azacitidine effect on the culture of red bone marrow stem cells had an opposite result.

CD326 has an important role in intercellular interaction 48. We know that this marker sustains the “stem” features in somatic cells [62, 80]. Sarrach S. et.al.94 claim that EpCAM high level of expression is natural for proliferating cells, but it is low for differentiated cells.

The above-mentioned data may explain the increase of the marker in CMDSC in the process of culturing by increasing the cells quantity during the division. While 5- azacytidine effect on CBMDSC lowered the CD326 expression that indicates the cells differentiation.

Pan-keratin (pancytokeratin) expression in cells derived from the rat’s myocardium increased within the “absence of expression” level, while 5- azacytidine effect on CBMDSC lowered its expression.

Cytokeratin is a group of intermediate filaments found in epithelial cells of all types, so they are considered to be specific markers for epithelial lineage cells. Pan-keratin can be found on the early stage of the cell terminal differentiation 81. Furthermore, the research by Gown A.M. 44 in vitro and in vivo systems show the presence of cytokeratines in smooth-muscle tissue as well as in the cardiac muscle.

The appearance of pan-keratin expression in rat at the CMDSC III passage with its subsequent growth can be explained by the appearance of differentiated cells in the culture. But the lower level of this particular marker in cells under the effect of 5- azacytidine proves their differentiation in cardiomyogenic direction.

Troponin I or cardiotroponin I is the troponin isoform that can be found only in the heart and is a cardiomyocytes marker 16. In their research Saito T. et.al. 91 and Wang H. S. et.al. 110 state the appearance of this marker expression in stem cells under differentiation in a cardiomyogenic direction.

During our research, we noted the decrease of troponin I expression in cell cultures taken from the rat’s myocardium that can indicate the lower the passage, the lower the number of cells with cardiomyocytes properties.

The directed CBMDSC differentiation of a rat by using the 5-azacytidine lead to a certain increase of troponin I expression level that is also true according to the data gathered by other scientists.

After analyzing the immunophenotypic profile of the cells obtained from the rat’s myocardium we can conclude that the CMDSC is heterogeneous in its cell composition that further changes during the process of culturing. So, at the beginning of the research the following cells with cardiomyocytes were dominant: CD10+, CD34low, CD38low, CD45low, CD48-, CD54- CD56-, CD95+, СD227-, СD326low, troponin Іhigh. During culturing we begin to discover markers that are natural for epithelial cells: pan-keratinlow, СD326+, СD227low; increased marker expression that is inherent to committed cells: CD34+, СD45low. The data informs us about the differentiated cells that can no longer divide (cardiomyocytes), because of increased number of the progenitor and epithelial cells, being removed from the culture.

Differentiation in the cardiomyogenic direction leads the stem cells of the red bone marrow to obtain features inherent to cardiomyocytes which is further proved by the presence of epithelial-like cells that express markers usually found in cardiomyocytes (CD10+, CD45-, CD95+, СD326low, troponin І+).

Studying the stability of cells karyotype in culture is an important condition to use the cell material for transplantation, because the chromosome mutations play a vital part in carcinogenesis. Subsequently, the next stage of our research was studying the genetic stability of cells in the culture during the process of culturing by determining the percentage composition of cells with micronucleus, aneuploid and polyploid sets of chromosomes, binucleated cells, and mitotic index.

We analyzed the chromosome variation of I-VI passage cells (table 3) in order to study the genetic stability of the stem cells derived from the rat’s myocardium.

Table 3

The results of cytogenetic analysis of the I-VI passages stem cells culture obtained from the rat’s myocardium, M±m, n=3

Abbildung in dieser Leseprobe nicht enthalten

Note: *р<0.05; ***р<0.001, compared to control (passage I served as the control)

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