[THE AGING OF MICROVASCULAR NETWORK FORMED IN CORTEX FOLLOWING INTRACEREBRAL TRANSPLANTATION OF MESENCHYMAL STEM CELLS].

Using a TV device to study microcirculation in brain we found that intracerebral transplantation of mesenchymal stem cells to 12-months old rats led to a significant increase (circa 1,5-fold times) of microvascular density in pia tissue and to increased constriction reactions of pia arterioles in response to noradrenalin application on a brain surface. Both microvascular density and pia arterioles reactivity was completely preserved in aging until 22-24 months.

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit.

The present study focuses on the involvement of reactive oxygen species (ROS) in the process of mesenchymal stem cells "waking up" and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs), we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol) blocked G1-S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

[Age-related changes in blood microcirculation functional state of the brain cortex of rats].

Male Wistar-Kyoto rats, aged 22-24 months, were intracerebrally transplanted syngeneic mesenchymal stem cells (MSC). Cognitive functions of these animals were tested in 3 weeks. The density of microvascular bed, the tissue blood flow, saturation of mixed blood in cortex microvessels were measured under the standard conditions, under the impact of noradrenaline and in the context of global ischemia. The control groups consisted of rats aged 2-3 months and 22-24 months. MSC transplantation have a positive impact on the microcirculation in brain cortex of old animals: reduction of vascular density was prevented, level of saturation in the blood vessels was increased, which enabled animals to endure easier extreme impacts, but did not correct age-related deterioration of cognitive functions.

[Efficacy of mesenchymal stem cells intracerebral transplantation for the correction of age-related cerebral microcirculation alterations in rats].

Using a television-based vital microscopy method and immunohystochemical analysis, we have assessed the effect of intracerebral transplantation of syngeneic mesenchymal stem cells (MSC) on the brain cortex structure and the microcirculation in the pia mater of old rats. Using "open field" system, we have studied the effect of MSC transplantation on position-finding and discovery behavior of older animals. We have found that density of microvascular network of the pia mater increased ca. 1.9-fold in MSC recipients, compared to age-matched intact animals. Density of the arteriolar area of microvascular network of the pia mater increased ca. 2-fold. Reactivity of the newly formed arterioles was nearly equal to that of native microvessels. Intracerebral transplantation procedure itself was traumatic for brain cortex of rats, but it had no effect on the microcirculation in the contralateral hemisphere. Intracerebral transplantation of MSC did not improve locomotor behavi- or and emotional stage of old rats, did not increase their position-finding and discovery activity.

[BDNF secretion in human mesenchymal stem cells isolated from bone marrow, endometrium and adipose tissue].

The ability of mesenchymal stem cells (MSCs) to differentiate into neuronal lineage determines the potential of these cells as a substrate for a cell replacement therapy. In this paper we compare the neurogenic potential of MSCs isolated from bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was found that the native eMCSs, BMSCs and AD MSCs express neuronal marker ß-III-tubulin with a frequency of 90, 50 and 14%, respectively. We also showned that eMSCs have a high endogenous level of brain-derived neurotrophic factor (BDNF), whereas the BMSCs and the AD MSCs are characterized by low basal BDNF levels. As induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMF caused changes in the cells morphology, the increased expression of ß-III-tubulin, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. BDNF secretion during differentiation was significantly enhanced in the BMSCs and decreased in the eMSCs cultures. However, no correlation between the basal and induced levels of the neuronal markers expression and BDNF secretion in the studied MSCs has been established.

Effect of intracerebral transplantation of mesenchymal stem cells on reactivity of pial arterioles in old rats.

Using a TV device for studying microcirculation (×160), we analyzed the responses of arterioles in the pia mater of the sensorimotor cortex in young (2-3 months) and old (22-24 months) rats after local application of a vasoconstrictor (norepinephrine, 10(-6) M) or vasodilator (acetylcholine, 10(-6) M). The responses of the arterioles were evaluated by changes in their diameter and by the number of responding vessels in the field of view. The constrictor responses of the pial arteries to norepinephrine did not significantly differ in intact young and old rats. The number and degree of dilatory responses to acetylcholine in old rats were lower than in young animals by 14 and 30%, respectively. Intracerebral transplantation of mesenchymal stem cells to old rats had practically no effect on reactivity of pial arterioles to acetylcholine, while the number of constricted vessels in response to norepinephrine increased by ~20%.

[Recellularization of tissue engineered vascular grafts in perfusion bioreactor].

Small diameter tissue engineered vascular grafts could be a potential solution to the shortage of vascular substitutes in reconstructive cardiovascular surgery. Previously, we have developed a decellularization method for human umbilical arteries, which could be used as a scaffold in vascular tissue engineering. Objective of the study was to optimize the recellelularization of decellularized scaffolds with mesenchymal stem cells. In the study, the possibility of cell growth on decellularized vessel has been shown. We also has proved that the use of perfusion-bioreactor improves the results of recellularization.

[Mesenchymal stem sell donor age effect on the cerebral cortex microvascular net density in old age rats-recipients of transplant].

Male Wistar-Kyoto rats aged 22-24 months were intracerebrally transplanted with syngenic bone marrow mesenchymal stem cells (BM MSC) established from the donor aged 3-4 months and 20-22 months, respectively. Using a TV device to study microcirculation in vivo, we have established that transplantation of BM MSC from young donors increased a density of the microvascular network in the pia mater of the sensorimotor cortex in old rats approximately 1.9-fold, comparing to age-matched controls, while a density of the arteriolar compartment increased approximately 2.1-fold. Transplantation of BM MSC from old donors did not lead to the significant increase in the density of the microvascular network in the pia mater, while a density of the arteriolar compartment increased approximately 1.5-fold.

[Neurogenic potential of human mesenchymal stem cells isolated from bone marrow, adipose tissue and endometrium: a comparative study].

Mesenchymal stem cells (MSCs) can be isolated from many adult tissue sources. These cells are a valuable substrate in cell therapy for many diseases and injuries. Different types of MSCs vary in plasticity. We performed a comparative study of the neurogenic potential of three types of human MSCs derived from bone marrow (BMSCs), subcutaneous adipose tissue (ADSCs) and endometrium (isolated from the menstrual blood) (eMSCs). It was shown that all three types of MSC cultures demonstrate multipotent plasticity and predisposition to neurogenesis, based on the expression of pluripotency markers SSEA-4 and neuronal precursors' markers nestin and beta-III-tubulin. Further analysis revealed the transcription of the neuronal marker MAP2 and neurotrophin-3 in undifferentiated BMSCs and ADSCs. Additionally, a significant basal level of synthesis of brain-derived neurotrophic factor (BDNF) in eMSC culture was also observed. Stimulation of neural induction with such agents as 5-azacytidine, recombinant human basic fibroblast growth factor (bFGF), recombinant human epidermal growth factor (EGF), a recombinant human fibroblast growth factor 8 (FGF8), morphogen SHH (sonic hedgehog), retinoic acid (RA) and isobutyl-methyl-xanthine (IBMX), showed further differences in the neurogenic potential of the MSCs. The components of the extracellular matrix, such as Matrigel and laminin, were also the important inducers of differentiation. The most effective neural induction in BMSCs proceeded without the RA participation while the cells pretreated with 5-azacytidine. In contrary, in the case of eMSCs RA was a necessary agent of neural differentiation as it stimulated the transcription of neurotrophin-4 and the elevation of secretion level of BDNF. The use of laminin as the substrate in eMSCs appeared to be critical, though an incubation of the cells with 5-azacytidine was optional. As far as ADSCs, RA in combination with 5-azacytidine caused the elevation of expression of MAP2, but reduced the secretion of BDNF. Thus, the effect of RA on neural differentiation of ADSCs in ambiguous and, together with the study of its signaling pathways in the MSCs, requires further research. The therapeutic effect of transplanted MSCs is commonly explained by their paracrine activity. The high basal level of BDNF synthesis in the eMSCs, along with their high proliferative rate, non-invasive extraction and neural predisposition, is a powerful argument for the use of the intact eMSCs as a substrate in cell therapy to repair nerve tissue.

Effect of transplantation of mesenchymal stem cells on the density of pial microvascular network in rats of different age.

Using a TV device for studying microcirculation (×40), we studied the density of the whole microvascular network and arteriolar its compartment in the pia mater of the sensorimotor cortex in rats of different age (2-3, 12, and 24 months) after intracerebral transplantation of mesenchymal stem cells or nutrient medium (control). The density of the microvascular network in the pia mater remained practically unchanged until 1 year, but then decreased by 1.8 times with adding (up to 2 years). MSC transplantation 1.5-1.8-fold increased the density of the pial microvessels in animals of all age groups in comparison with intact and control rats; the density of the arteriolar compartment increased by 2.1-2.4 times. Intracerebral injection of MSC to 1-year-old animals prevented pathological decrease in the density of microvascular network during the next year of life.

[The effect of intracerebral transplantation of mesenchymal stem cells on orientation and exploratory behavior of aged animals].

Male Wistar-Kyoto rats (12 and 22-24 months old) were intracerebrally transplantated with syngeneic mesenchymal stem cells (MSC). Three weeks or one year later, orientation and exploratory behavior of the same animals (not 2 years old) was assessed using an «open field¼ test. Older rats (22-24 months old) were inhibited in the basic behavioral acts in comparison to younger animals (2-3 months old). Transplantation of MSC did not cause improvement of an orientation and exploratory behavior of aged rats. Morphological and immunohistochemical analysis suggests intracerebral MSC transplantation led to the injury of the cortex of the ipsilateral hemisphere.