[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.

[CHARACTERISTIC OF ENDOMETRIAL MESENCHYMAL STEM CELLS IN CULTURE OBTAINED FROM PATIENT WITH ADENOMYOSIS].

Adenomyosis is form of endometriosis, common diseases of female reproductive system, which can lead to infertility in women. in this study we are obtained and characterized cell line endometrial mesenchymal stem cells from a patient with adenomyosis, and compare obtained cells with the cell line of healthy donor. Aim of this study was to assesses the extent of differences between cells from donor with adenomyosis and cells from healthy donor. Was established that compared lines had morphology like fibroblasts, were differentiated in adipocytes, were expressed mesenchymal markers and didn't expressed haematopoietic markers. Cytogenetic analysis of differentially stained metaphase chromosomes on G-banding (passage 6-7) showed that healthy donor's cells had predominantly normal karyotype. The cellular line from a patient with diagnosis of "adenomyosis" had a lot of cells with changes in karyotype's structure. These changes were related with aneuploidy of cellular population and the presence non-random chromosomal breaks, often in chromosomes 7 and 11. Analysis of this data allows the cells from adenomyosis characterized physiological stability in culture and karyotypic instability with non-random involvement certain chromosomal set. The cellular line obtained from donor with adenomyosis showed signs destabilization of he genome, typical for cell transformation. Division of adenomyosis cells to the 26th passage is stopped and these cells entered into a phase of replicative aging. Based on this, we can conclude that founded karyotype's hanges do not lead to transformation and immortalization of cells in vitro.

[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.

[Optional protocols of hematopoietic stem cell expansion in vitro].

Hematopoietic stem cells (HSC) effectively and continuously renew a full spectrum of blood cell populations. Bone marrow and umbilical cord blood stem cells transplantation (SCT) restore hematopoietsis, when used in various hematological and oncohematological disorders in adults and children. However, wider clinical application of effective SCT-based approaches is limited by the low number of the primitive HSC in the available biospecimens. Development of effective protocols for HSC expansion in vitro is therefore necessary. In this review, a concept of bone marrow hematopoiesis niche being a complex cellular system is discussed; a comparative analysis of various methods for HSC expansion in vitro is provided. The review is illustrated by the authors own data supporting application of various feeder cell types for human HSC expansion in vitro.

[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 intracerebral transplantation of mesenchymal stem cells on pial microcirculation in rats.

We studied the effect of intracerebral transplantation of bone marrow mesenchymal stem cells on microcirculation (density of microvascular network and reactivity of arterioles) in the pia mater of 2-3-month-old rats. It was found that after transplantation of mesenchymal stem cells, the density of pial microcirculatory network in the contralateral hemisphere significantly increased (by 1.7 times; p<0.05) in comparison with both intact animals and controls. The number of arterioles in the studied area increased most markedly (by ≈2.5 times; p<0.05) in comparison with other groups. Intracerebral transplantation of mesenchymal stem cells or conditioned culture medium (α-MEM) had no effect on reactivity of pial arterioles.

[First experience of the study "Intramyocardial Multiple Precision Administration of Mononuclear Bone Marrow Cells in the Treatment of Myocardial Ischemia"].

Study aim - to elucidate possibilities of the use of precision administration of mononuclear bone marrow cells (MBMC) for the treatment of myocardial ischemia and heart failure. "Intramyocardial Multiple Precision Administration of Mononuclear Bone Marrow Cells in the Treatment of Myocardial Ischemia" was a double blind randomized placebo controlled study in which we included patients more or equal 6 months after Q-wave myocardial infarction with systolic myocardial dysfunction (ejection fraction <35%), not requiring myocardial revascularization, receiving stable optimal medical therapy for more or equal 8 weeks, and with implanted cardioverter-defibrillator. Transplantation of MBMC was guided by fluoroscopy and tridimensional NOGA XP Cardiac Navigation System. For assessment of efficacy of the method we used surrogate end points: decrease of number of fixed perfusion defects according to SPECT data and improvement of regional myocardial contractility according to data of echocardiography. Results of dynamic observation of the first experience of MBMC administration are presented in this paper.

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.

[Risks of the xenogenic origin in stem cells applications].

Stem cells can be applied as the substrate of cell therapy of a variety of diseases, including those previously considered incurable. Furthermore, studying the fundamental mechanisms underlying proliferation and differentiation of stem and progenitor cells is most important to uncover the principles of growth, development, adaptation and regeneration, which could be altered in pathology. However, many steps of stem cell studies (including cell isolation, expansion and differentiation in vitro) are associated with factors of the xenogenic origin. Human stem cell exposure to the xenogenic factors over the various steps of work may complicate an interpretation of the proteomics, genomics, transcriptomics and metabolomics data. It is also associated with the risk of immune response and zenozoonoses development in cell transplantation recipients. In the review, key issue related to xenogenic factors in stem cell applications are discussed, as well as the possible means to lower the risks of the xenogenic origin.

[Cell technologies and the development of skin substitutes].

Large skin lesions might be of different cause and sometime resistant to the conventional and surgical treatment. For many years, skin grafting used to serve one of the most important methods to treat large and deep skin lesions. However, a limited availability of the substrate for transplantation restricts wider application of the approach. Utilization of wound covers and skin replacements of various types (including synthetic, biological and biosynthetic ones) provides an alternative. Skin substitutes--biosynthetic covers of the complex structure--constitute the most important niche in a wide spectrum of wound covers developed and actively utilized by today. Cell substrates used in the former include dermal fibroblasts, neonatal foreskin fibroblasts and keratinocytes. In the current review, key properties of the modern skin substitutes, structure features of the most widespread types. and principle of the work with the particular cell types are analyzed. Comparative analysis of the efficiency of wound covers is provided.

[Cell therapy in treating age-related intervertebral disc pathology].

Pathology of the spine significantly affects the quality of life and is associated with a high rate of disability. Prevalence of the pathology of the spine increases with advanced age. It may have various causes and is associated with a wide spectrum of significant and variable symptoms. Low back pain (back pain) is the most common syndrome. It is mostly caused by the degeneration of the intervertebral discs, associated with unspecific and irreversible structural alterations in the latter. Surgical treatment of degenerative disc disease (DDD) is symptomatic, thus requiring a development of alternative approaches. In the current review, the principles of regenerative cellular therapy and cell replacement therapy of DDD are analyzed. Various cell types utilized as cellular therapy substrates are compared.

[Generation of dopamine neurons from human embryonic stem cells in vitro].

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

[Cell therapy for Parkinson's disease: III. Neonatal, fetal and embryonic stem cell-based applications].

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

[Cell therapy for Parkinson's disease: II. Somatic stem cell-based applications].

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.

[Cell therapy for Parkinson's disease: IV. Risks and future trends].

Motor dysfunctions in Parkinson's disease are believed to be primarily due to the degeneration of dopaminergic neurons located in the substantia nigra pars compacta. Numerous cell replacement therapy approaches have been developed and tested, including these based on donor cell transplantation (embryonic and adult tissue-derived), adult mesenchymal stem cells (hMSCs)-, neural stem cells (hNSCs)- and finally human embryonic stem cells (hESCs)-based. Despite the progress achieved, numerous difficulties prevent wider practical application of stem cell-based therapy approaches for the treatment of Parkinson's disease. Among the latter, ethical, safety and technical issues stand out. Current series of reviews (Cell therapy for Parkinson's disease: I. Embryonic and adult donor tissue-based applications; II. Adult stem cell-based applications; III. Neonatal, fetal and embryonic stem cell-based applications; IV. Risks and future trends) aims providing a balanced and updated view on various issues associated with cell types (including stem cells) in regards to their potential in the treatment of Parkinson's disease. Essential features of the individual cell subtypes, principles of available cell handling protocols, transplantation, and safety issues are discussed extensively.