Effect of Transplantation of Mesenchymal Stem Cells on the Density of Pial Microvascular Network in Spontaneously Hypertensive Rats of Different Age.

Using a TV device for studying microcirculation (×40), we analyzed the density of the whole microvascular network and the density of arterioles in the pia mater of the sensorimotor cortex in SHR rats of different ages (3-4 and 12 months) after intracerebral transplantation of human mesenchymal stem cells. We found that the density of pial microvascular network in SHR rats receiving transplantation of human mesenchymal stem cells increased to a level observed in young Wistar-Kyoto rats.

EFFICACY OF MESENCHYMAL STEM CELLS USED FOR THE IMPROVEMENT CEREBRAL MICROCIRCULATION IN SPONTANEOUSLY HYPERTENSIVE RATS.

Elaboration of new methods of correction of microcirculatory disorder in the brain caused by persistent high blood pressure is a topical task both for medicine and for biology. We studied influence of intracerebral transplantation of human mesenchymal stem cells (MSCh) to cerebral microcirculation in young (4 months) and aged (12 months) spontaneously hypertensive rats (SHR). It was shown that transplantation MSCh promoted the rise of the density of microvascular network of young SHR ca. 1.6-fold; density of the arteriolar area of microvascular network of the pia mater increased ca. 1.9-fold. The density of microvascular network of aged SHR increased ca. 1.4­1.5-fold after transplantation MSCh. The perfusion and tissue saturation of sensorimotor cortex of young SHR increased to the level of young normotensive rats, and in aged SHR the perfusion and tissue saturation of sensorimotor cortex was not increased. Conclusion: the intracerebral transplantation MSCh almost completely leveled the pathological changes of the microcirculation in the sensorimotor cortex of the brain of young SHR and improved unimportantly microcirculation in aged SHR.

Morphological changes in rat hippocampus after brain injury.

We performed morphological analysis of the structure of rat hippocampus after ablation of the left sensorimotor cortex. Four experimental groups were formed: two control groups (intravenous and intracerebral injections of the culture medium) and two experimental groups (intravenous and intracerebral transplantation of MSC). Ten weeks after surgery, disturbed cytoarchitectonics and great number of dead neurons were found in all zones of the hippocampus in animals of the control groups. In animals receiving cell therapy, no pathological changes in the structure of the hippocampus were found: hyperchromatic neurons were absent and the cells had regular shape and closely adjoined to each other.

[Transplantation of mesenchymal stem cells in multiple sclerosis].

To assess safety and tolerability of treatment with autologic multipotent mesenchymal stem cells (MSC) in multiple sclerosis (MS), we have obtained autologic red bone marrow-derived MSC from 8 patients. Proliferation, immunophenotype and caryotype of MSC, their sterility, the absence of hemopoetic cells, chromosomal aberrations and signs of aging were controlled during the cell growth. The inverse injection of MSC in patient's blood was conducted in accordance to the elaborated protocol in a short intravenous infusion in dose 2.0 x 10(6)/kg of body mass once in 30 days. The duration of treatment was from 4 to 8 months. The efficacy of treatment was assessed after 4, 8 and 12 months. All patients tolerated repeated intravenous infusions of autologic MSC well with no significant side-effects as in the early as well in the remote periods of treatment. The distinct positive effect was seen in some cases 2 months after the beginning of treatment. The improvement of 0.5 point on EDSS was seen in 5/8 patients after 4 months. After 12 months, the improvement of 0.5-1 point on EDSS was seen in 6/8, stabilization in 1/8, progression in 1/8. These results revealed the safety of the elaborated protocol of treatment and the moderate clinical efficacy of treatment in non-curable patients or those with poor response to treatment that suggested continuing the study and enrollment of new patients.

Mesenchymal stem cells restore orientation and exploratory behavior of rats after brain injury.

We studied the effects of intravenous and intracerebral transplantation of MSC on restoration of orientation and exploratory behavior of Wistar-Kyoto rats after removal of the left motor cortex. Removal of the motor cortex led to a significant reduction of the number of behavioral acts in the open field test. Two weeks after removal of the motor cortex and intravenous transplantation, the animals were as inhibited as the controls, but during the next 10 weeks, the behavioral status of these rats remained unchanged, while controls exhibited further behavioral degradation. After injection of MSC into the brain, the behavior of rats with trauma did not change in comparison with intact rats over 10 weeks.

Effect of transplantation of mesenchymal stem cells on neuronal survival and formation of a glial scar in the brain of rats with severe traumatic brain injury.

We studied the effect of various methods of transplantation of mesenchymal stem cells on neuronal survival in rat brain 1 and 6 weeks after severe traumatic brain injury. It was found that intracerebral and systemic transplantation of mesenchymal stem cells improves neuronal survival in the piriform cortex of the contralateral hemisphere without affecting neuronal survival in the marginal zone of the traumatic cavity and amygdaloid nuclei. Intracerebral transplantation of mesenchymal stem cells increases the content of the astroglial component of the scar in the borderline zone of the traumatic cavity.

Alterations of cytological and karyological profile of human mesenchymal stem cells during in vitro culturing.

Transplantation of human bone marrow mesenchymal stem cells is considered as a promising therapeutic approach to the therapy of many diseases. However, the problem of possible alterations of the properties of mesenchymal stem cells during their expansion in in vitro cultures before transplantation is not solved. In our study, one of two hundred examined cultures of mesenchymal stem cell cultures derived from donors without bone marrow pathologies and developed under standard culturing conditions demonstrated spontaneous disturbances in morphology, proliferation, and karyotype at early passages. The cells of this abnormal culture retained immunophenotype characteristic of normal mesenchymal stem cells, but some of them (15-25%) had numerous numerical and structural chromosome aberrations.

[Stem cells from adult differentiated tissues].

The review thoroughly describes all types of adult stem cells which have been isolated by the present time - hematopoietic, muscle, neural, skin, endothelial, intestinal and mesenchymal stem cells. Their isolation, in vitro cultivation, and possible application in cell therapy of various diseases are discussed. Mesenchymal stem cells (MSC) are of great concern now because they are, in the authors' opinion, the most promising material for application in the therapy of humans.

[Cell therapy for myocardial infarction].

The review discusses cell therapy; one of the most promising approaches to myocardial infarction treatment. The possibility to use cell material of various origins is analyzed. The review sums up data on the application of fetal and neonatal cardiomyocytes, myoblasts, bone marrow mononuclear fraction, hematopoietic and mesenchymal stem cells (MSX) as cell therapy agents. The conclusion is made that MSC are promising cell material for myocardial infarction therapy. MSC are able to migrate to the injured area, differentiate into myocardial lineage. They produce a wide range of factors that stimulate angiogenesis and increase viability of cells, including cardiomyocytes.

[Methods for mathematical data processing in enzyme immunoassay. I. Theoretical bases].

The paper deals with the mathematical processing of enzyme immunoassay (EIA) data. The paper comprises two parts. Part 1 contains a brief comparative account of the basic ways of presenting experimental data and constructing a standard curve, as well as practical guidelines for choosing the mathematical methods for calculation of concentrations. The recommendations proposed in Part II are based on the comparative study of photometric equipment and software that is of common use in EIA diagnosis at the Russian clinical laboratories. The experiment imitates competitive analysis illustrates a relationship of the obtained clinical result to the choice of either mode of constructing the standard graph on the basis of the optic densities of calibration samples. The findings indicate that an incorrectly chosen method for data processing may make both a false rejection of the whole analysis on the basis of the erroneous calculation of the concentration of a test agent in the control serum and to underdiagnosis on the basis of the erroneous calculation of the concentration of a test agent in a patient's sample near the point of taking a medical decision.

Distribution of mesenchymal stem cells in the area of tissue inflammation after transplantation of the cell material via different routes.

In experiments on male Wistar-Kyoto rats we studied the distribution of mesenchymal stem cells in intact body and in the presence of a focus of acute tissue inflammation. In healthy animals mesenchymal stem cells were transplanted intravenously. In the second case we used various routes of transplantation of mesenchymal stem cells: intravenous, into tissue adjacent to the inflammation focus, and into intact lobe of the damaged organ (prostate gland). Three weeks after transplantation, mesenchymal stem cells labeled with a fluorescent dye were detected in the bone marrow and intestine of intact animals. In case of inflammation focus, mesenchymal stem cells after transplantation migrated into the bone marrow, intestine, and prostate gland. After injection into the adjacent zone, these cells formed a compact agglomerate at the site of injection. After transplantation into the intact lobe of the prostate gland the cells migrated towards the inflammation focus. Thus, transplantation of mesenchymal stem cells into the venous blood is less traumatic and led to more uniform distribution of cells in the damaged tissue.

[Mesenchymal stem cells-based therapy of brain ischemic stroke in rat].

Mesenchymal stem cells (MSCs)-based therapy is a promising modern attempt to improve the recovery after stroke. Experiments were carried out on inbred Wistar-Kyoto rats. MSCs were isolated, expanded in cultute and labeled with vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO), followed by injection of 5 x 10(6) rat MSCs into the tail vein 3 days after MCAO. Control group animals received PBS injection (negative control). Therapy results were estimated by the following parameters: behavioral and neurological testing, the brain injure area, the state of damaged region "border" zone and the vessels quantity in the "borden" area. It was shown that control group animals (PBS injection) did not restore their initial behavioral and neurological state, while the experimental group animals (MSCs injection) showed the same parameters as intact rats at 2-3 weeks after MCAO. The size of the damaged region in the control group was approximately 1.5 as large as in the experimental group. The damage in the experimental group was limited to neocortex; caudate nucleus, capsula externa and piriform cortex remained uninjured. Small vessels quantity in the "border" regions was twine higher compared to control group and was approximately equal to an intact brain vessel number. Moreover, it was shown for the first time that after MSCs transplantation the vessels quantity in the neocortex and caudate putamen of contralateral hemisphere was twice as much as in control. We demonstrated that the MSCs transplantation definitely exerted a positive influence upon the brain tissue reparation after stroke.

[Blood sampling systems and interference in immunoassay].

Immunoassay interference caused by reagents (additives, excipients) incorporated in the blood sampling systems is undetectable by the conventional methods for intralaboratory quality control, by using reference substances. A large number of manufacturers and a great physicochemical variety of additives in the current vacuum and non-vacuum systems require that additional measures be made to monitor systemic errors in immunochemical studies. The review considers the mechanisms of this type of interference and the problems of its monitoring and prevention.

[Mesenchymal stem cells transplantation influences upon dynamics of morphological changes in rat brain after stroke].

Study of dynamic morphological changes if the brain after ischemic stroke is an important phase of pre-clinical trial of mesenchymal stem cell (MSC) therapy for this widespread disease. Experoments were carried out in inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture and labeled with vital fluorescent dye PKH26. Animals were subjected to middle cerebral artery occlusion (MCAO) followed by injection of 5 x 10(6) rat MSCs into the tail vein on the day of MCAO. Control group of animals received PBS injection (negative control). Animals were sacrificed in 1, 2, 3 and 5 days and in 1, 2, 4 and 6 weeks after operation. MSCs were revealed in the brain on the third day transplantation. They distributed around brain vessels in both the ipsilateral and contralateral hemispheres. This pattern of distribution remained unchanged during 6 weeks of observation. It was demonstrated that inflammation process and scar formation in the experimental group progressed 25-30 % faster than in the control group. MSC transplantation stimulated endogenous stem cell proliferation on the subependimal zone of lateral ventricles (subventrecular zone). What is more, MSC injection showed neuroprotective effect: almost all penumbra neurons in animals treated with cell therapy retained their normal structure, whereas in animals of control group penumbra neurons died or had signs of serious damage.

In vitro and in vivo differentiation of mesenchymal stem cells in the cardiomyocyte direction.

The possibility of mesenchymal stem cell differentiation in the cardiomyocyte direction was studied on Wistar-Kyoto rats with myocardial infarction induced by ligation of the left coronary artery. In vitro treatment of mesenchymal stem cells with 5-azacitidine led to spontaneous contractions of about 15% cells in culture. Analysis of the expression of matrix RNA showed expression of fetal and functional markers of the myocardium in this cell culture. In vivo on day 21 after myocardial infarction and intravenous transplantation of mesenchymal stem cells into the periinfarction area, myocardial cells carrying donor label were detected. Immunohistochemical analysis showed that these cells were cardiomyocytes integrated into the myocardium. These cells can be a result of differentiation of transplanted mesenchymal stem cells or fusion of endogenous cardiomyocytes with exogenous mesenchymal stem cells.

Effect of mesenchymal stem cell transplantation on cognitive functions in rats with ischemic stroke.

The effect of intravenous transplantation of mesenchymal stem cells on the recovery of cognitive functions was studied in Wistar-Kyoto rats after brain stroke induced by occlusion of the middle cerebral artery in the left hemisphere. Analysis 2 and 5 weeks after stroke showed that transplantation of mesenchymal stem cells 3 days after middle cerebral artery occlusion reduced the area of cerebral injury, preserved cognitive functions, and decreased mortality in experimental animals.

Effect of mesenchymal stem cells on rejection of xenogenic bone transplant.

We studied the effects of mesenchymal stem cells on rejection of xenogenic bone transplant. Experiments were carried out on Wistar-Kyoto rats transplanted chicken demineralized bone matrix populated or not populated with mesenchymal stem cells to the site of parietal bone defect. Histological analysis showed complete resorption of the xenogenic transplant not populated with mesenchymal stem cells by day 119. In experimental animals chicken matrix without signs of tissue inflammation was in fact completely retained over the entire period of observation. Numerous new vessels, mineralization fields, and areas of bone tissue formation were seen in the transplant.

[The influence of mesenchymal stem cell transplantation time on myocardial reparation in rat experimental heart failure].

Mesenchymal stem cells (MSCs) have an ability to migrate in the organism to injured tissue to exert influence on inflammation and reparation in these regions. The aim of this study was to determine the optimal time of MSCs transplantation for myocardial reparation in rat experimental heart failure. The experiments were carried out on inbred line Wistar-Kyoto rats. Myocardial experimental infarction (EI) was induced by ligation of the left descending coronary artery. MSCs were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the time of MSCs transplantation exerted an essential influence on angiogenesis in a damaged myocardium, on ventricular dilatation and morphological structure of the scar. The best time for MSCs transplantation was determined within two days before EI, and seven days after EI. As a result, the overload of the border zone of infarct region decreased, and no features of infarction relapse were shown in the border zone.

[The influence of mesenchymal stem cells on bone tissue regeneration upon implantation of demineralized bone matrix].

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC are able to differentiate into chondroblasts, adipocytes, neurons, glia, cardiomyocytes, or osteoblasts. The problem of MSC usage in cell therapy of bone defects is widely discussed at present. The experiments were carried out using rats of inbred line Wistar-Kyoto. MSC were isolated from bone marrow and cultivated in vitro. Demineralized bone matrices (DBM) were obtained from parietal bones of rats and hens. Part of DBM was loaded with MSC. Bone defects were made in cranium parietal regions. DBM with or without MSC or metal plates were transplanted in these regions. It was shown that the application of MSC increased angiogenesis and osteogenesis in the damaged bone. The implantation of rat's DBM with MSC led to the formation of a full value bone. MSC suppressed inflammation, when transplantation of hen's DBM was carried out. The application of MSC always improved bone tissue regeneration.