Distribution and Migration of Human Placental Mesenchymal Stromal Cells in the Brain of Healthy Rats after Stereotaxic or Intra-Arterial Transplantation.

Human placenta mesenchymal stromal cells were injected to healthy rats either stereotaxically into the striatum or intra-arterially through the internal carotid artery. Some cells injected into the brain migrated along the corpus callosum both medially and laterally or concentrated around small blood vessels. A small fraction of MSC injected intra-arterially adhered to the endothelium and stayed inside blood vessels for up to 48 hours mostly in the basin of the middle cerebral artery. Neither stereotaxic, nor intra-arterial transplantation of mesenchymal stromal cells modulated the proliferation of neural stem cells in the subventricular zone of the brain, but stereotaxic transplantation suppressed activation of their proliferation in response to traumatization with the needle.

Individual Peculiarities of the Development and Differentiation of Embryonic Neocortex Transplants in Intact Adult Mouse Brain.

We studied individual peculiarities of the development and differentiation of allogeneic transplants of neocortical cells isolated from embryos at different stages of development in intact brain of adult mice. Despite standard transplantation technique, intraparenchymal grafts considerably varied in size, morphology, and structural organization. The cells in the transplants developing inside the brain ventricles of the recipient formed histotypical structures resembling organoids. Transplants of each age group (12.5, 14.5, and 19.5 days) demonstrated individual peculiarities of cell migration, differentiation, and fiber growth. Only from cells of 12.5-day transplants formed spiny pyramidal neurons typical of V layer of the cerebral cortex. Differentiation of catecholaminergic neurons untypical of brain cortex was observed only in 14.5-day transplants. In few transplants of each age group, extensive cell migration from the transplant was observed. In some transplants, dense astrocyte accumulation was seen. In all cases (n=52), the response of the recipient's glia to the transplant was observed, but formation of an extensive glial barrier was noted only in one case. Our findings suggest that the entire range of the results determined by individual peculiarities of the transplant growth and recipient's response should be thoroughly realized when introducing the methods of neurotransplantation into regenerative medicine.

Reprogramming of Human Retinal Pigment Epithelial Cells under the Effect of bFGF In Vitro.

We studied the effect of bFGF on human retinal pigment epithelial cells in vitro In ARPE-19 cells, enhanced expression of KLF4 mRNA and reduced expression of PAX6, MITF, and OTX2 mRNA specific for retinal pigment epithelium were observed after bFGF application. The expression of KLF4 mRNA peaked in 72 h after bFGF application and then sharply decreased, which was accompanied by a 3-fold increase in TUBB3 mRNA expression (neuronal marker). Immunocytochemical analysis showed that in the presence of bFGF, some cells retained epithelial properties and showed positive staining for connexin-43, while others had long axon-like processes and demonstrated positive staining for ßIII-tubulin, which attests to their neuronal transdifferentiation. Despite the prevalence of the epithelial properties, ARPE-19 cells under the influence of bFGF can show proneuronal properties.

Heterogeneity of Retinal Pigment Epithelial Cells from Adult Human Eye in Different Culturing Systems.

We studied the behavior of retinal pigment epithelial cells from adult human eye derived from different donors in different culturing systems: on plastic, in collagen gel, and on decellularized neural retina substrate. The cells diverge into two subpopulations similar by their morphology and behavior: one subpopulation migrated to the surface of the dense substrate and the other formed spheroid structures consisting of aggregated cells. This fact confirms the data on genetically-predetermined phenotypic heterogeneity of retinal pigment epithelium cells that should be taken into account when using these cells in tissue engineering.

Analysis of Neural Stem Cells from Human Cortical Brain Structures In Vitro.

Comparative immunohistochemical analysis of the neocortex from human fetuses showed that neural stem and progenitor cells are present in the brain throughout the gestation period, at least from week 8 through 26. At the same time, neural stem cells from the first and second trimester fetuses differed by the distribution, morphology, growth, and quantity. Immunocytochemical analysis of neural stem cells derived from fetuses at different gestation terms and cultured under different conditions showed their differentiation capacity. Detailed analysis of neural stem cell populations derived from fetuses on gestation weeks 8-9, 18-20, and 26 expressing Lex/SSEA1 was performed.

Differentiation and Cell-Cell Interactions of Neural Progenitor Cells Transplanted into Intact Adult Brain.

We studied the behavior and cell-cell interactions of embryonic brain cell from GFP-reporter mice after their transplantation into the intact adult brain. Fragments or cell suspensions of fetal neocortical cells at different stages of development were transplanted into the neocortex and striatum of adult recipients. Even in intact brain, the processes of transplanted neurons formed extensive networks in the striatum and neocortical layers I and V-VI. Processes of transplanted cells at different stages of development attained the rostral areas of the frontal cortex and some of them reached the internal capsule. However, the cells transplanted in suspension had lower process growth potency than cells from tissue fragments. Tyrosine hydroxylase fibers penetrated from the recipient brain into grafts at both early and late stages of development. Our experiments demonstrated the formation of extensive reciprocal networks between the transplanted fetal neural cells and recipient brain neurons even in intact brain.

Effect of hrWnt7a on Human Retinal Pigment Epithelial Cells In Vitro.

Human recombinant protein Wnt7a (hrWnt7a) inhibits cell proliferative activity and triggers cell polarization. Although cell polarization process was maintained only over a short time, probably via microenvironmental stimuli, hrWnt7a is involved in the transformation of the retinal pigment epithelium. Analysis of Wnt signaling pathway and its regulation will help to understand the processes in retinal pigment epithelial cells under pathological conditions, which can be useful in developing new generation drugs.

[Stem Cells in the Brain of Mammals and Human: Fundamental and Applied Aspects].

Brain stem cells represent an extremely intriguing phenomenon. The aim of our review is to present an integrity vision of their role in the brain of mammals and humans, and their clinical perspectives. Over last two decades, investigations of biology of the neural stem cells produced significant changes in general knowledge about the processes of development and functioning of the brain. Researches on the cellular and molecular mechanisms of NSC differentiation and behavior led to new understanding of their involvement in learning and memory. In the regenerative medicine, original therapeutic approaches to neurodegenerative brain diseases have been elaborated due to fundamental achievements in this field. They are based on specific regenerative potential of neural stem cells and progenitor cells, which possess the ability to replace dead cells and express crucially significant biologically active factors that are missing in the pathological brain. For the needs of cell substitution therapy in the neural diseases, adequate methods of maintaining stem cells in culture and their differentiation into different types of neurons and glial cells, have been developed currently. The success of modern cellular technologies has significantly expanded the range of cells used for cell therapy. The near future may bring new perspective and distinct progress in brain cell therapy due to optimizing the cells types most promising for medical needs.

Effect of transplants of retinal pigment epithelial cells from adult human eye on degenerative processes in the brain of rats with experimental acute hypoxia.

Stimulation of cell regeneration in the brain and eye retina in various degenerative processes is a pressing problem in neurobiology. A promising approach is transplantation of somatic cells reprogrammed towards neural lineage. We studied the effect of transplantation of retinal pigment epithelial cells from adult human eye transdifferentiated in culture on degenerative processes in the brain of rats subjected to acute hypoxia. Immunohistochemical and molecular genetic analysis suggests that retinal pigment epithelial cells transdifferentiate in vitro and express markers of low-differentiated neural cells. The cells transplanted into rat brain survive for at least 20 days. During this period, they stimulate compensatory and reparative processes that protected cortical neurons in the recipients from hypoxia-induced degeneration.

[Experimental models of human retinal degenerations: genetic models].

Existing approaches in experimental animal modeling of human retinal degenerations, genetic models in particular, that allow to study the pathogenesis are reviewed.

[Experimental models of human retinal degenerations: induced models].

Existing approaches in experimental animal modeling of human retinal degenerations are reviewed, in particular models, in which pathological processes in animals were induced by chemical, biological and physical exposure.

Characteristics of induced human pluripotent stem cells using DNA microarray technology.

We performed transcriptome analysis of some human induced pluripotent stem cells, embryonic stem cells, and human somatic cells using DNA microarrays. PluriTest bioinformatic system was used for evaluation of cell pluripotency. Changes in the genome structure and status of X-chromosome gene expression was analyzed using microarray technology.

Comparative analysis of the expression of neural stem cell-associated genes during neocortex and retina development in human.

We compared the expression of Sox2, Oct4, Nanog, Pax6, Prox1 genes associated with plasticity of neural stem and progenitor cells during human neocortex and retina development and in cell cultures. At the analyzed stages of neurogenesis, Pax6 gene is expressed in the neocortex and retina at constant levels, the expression is by one order of magnitude higher in the retina. The dynamics of Sox2 and Pax6 expression in the neocortex was similar. The expression of Oct4 and Nanog genes during neurogenesis in the neocortex and human fetal retina reflects the existence of a high-plasticity cell pool. The dynamics of ßIII-tubulin expression indicates that the retina develops more rapidly than the neocortex. Our experiments showed that genetically determined cell potencies typical of native cells are realized in primary cultures without specific stimulation.

Expression of multipotent and retinal markers in pigment epithelium of adult human in vitro.

Immunoperoxidase and molecular genetic analysis showed that retinal pigment epithelial cells from adult human eye undergo morphogenetic changes in vitro. They lose expression of tissue-specific protein RPE65 and start to express stem cell markers: Oct4 (POU5F1), Nanog, Prox1, Musashi 1, and Pax6, which indicates their differentiation. Expression of Musashi 1 and Pax6 attest to neural differentiation, which is also confirmed by the expression of ßIII-tubulin, a neuroblast marker, and markers of differentiated neuronal cells, tyrosine hydroxylase and neurofilament proteins. These findings attest to the capacity of retinal pigment epithelium from adult human eye to transdifferentiation into neural lineage cells, which makes them an interesting object for cell therapy in neurodegeneration.

[Adult human retinal pigment epithelial cells - a potential source of cells for regeneration retina].

Retinal pigment epithelium (RPE) arises from neuroectoderm and plays a key role in support of photoreceptor functions. Several degenerative eye diseases, such as macular degeneration or retinitis pigmentosa, are associated with impaired RPE function that may lead to photoreceptor loss and blindness. RPE cell culture derived from adult human eyes autopsy could be an important source for transplantation to cure such retinal degenerative diseases. RPE cells subsequent isolation and maintenance in culture are described. Besides the results of immunocytochemical analysis that characterizes dedifferentiated state of cultured adult human RPE cells are given. Our findings demonstrate that mature human RPE cells have the capacity to express neural markers in response to conditions that promote dedifferentiation.

[Effects of human cultural neuronal and mesenchymal stem cells on the rat learning and brain state after acute hypoxia].

The aim of the study was to compare the effects of neurotransplantation of cultural neural stem cells (NSC) and mesenchymal stem cells (MSC) on the rat behaviour and brain state after acute hypoxia. It was shown that development of two-way avoidance defensive conditioning in a shuttle box improved in rats-recipients with NSC, but not MSC as compared to control. Both the transplants of NSC and transplants of MSC exert neuroprotective influence on the rat brain. NSC both in vitro (before transplantation) and in vivo (on day 27 after transplantation) gave rise to all neural cell types: stem/progenitor cells, precursors of neurons and glia, neurons and glial cells. MSC population in vitro and in vivo (on day 10 after transplantation) consisted of fibroblast-like cells which were eliminated by day 20 after transplantation and were surrounded by reactive glia. We suggest that effects of NSC may be connected with their good survival and potential to differentiate into neurons and with trophic influence on the brain of recipient, whereas MSC only have possible positive trophic effect at early stages after transplantation.

Phenotypic plasticity of retinal pigment epithelial cells from adult human eye in vitro.

Phenotypic plasticity of retinal pigment epithelial cells from adult human eye was studied by immunohistochemical methods under different culturing conditions. It was found that retinal pigment epithelium in adult human eye is a heterogeneous population of cells demonstrating different behavior in vitro. Some cells retain epithelial morphology for a long time in culture, while others are rapidly transformed into fibroblast-like cells and synthesize proteins typical of proneural, neural, glial, and photoreceptor cells. However, irrespective of initial morphological features differentiation of retinal pigment cells can be modulated by varying culturing conditions.

[Immunohistochemical analysis of development of suspension and tissue neurotransplants].

Engraftment and development of suspension and whole tissue allografts from the mouse brain embryonal tissue, in which cells of the green fluorescent protein (GFP) are synthesized, have been studied. The transgenic mouse cells with synthesized GFP can be used for neurotransplantation. Whole tissue and suspension transplants are able to survive more than 30 days without rejection in the brain of adult mice. It was found that the cells with synthesized GFP are able to differentiate in the neuronal and glial directions in both whole tissue and suspension transplants. The results of immunohistochemical analysis showed the reciprocal ingrowth of fibers between the cells of donor and recipient in both cases. A study of proliferation and differentiation of the cells showed the higher ability of tissue transplants for development.

[Effect of tririum on growth and bioluminescence of P. phosphoreum bacteria].

Effect of tritium labeled amino acid valine (0.3-1.0 MBq/ml) on luminous bacteria P. Phosphoreum was studied. The amino acid was used as a nutrient medium for the bacteria. Tritium was found to suppress bacterial growth, but stimulate luminescence: luminescence intensity, quantum yield and time of light-emitting were increased. Activation of the luminescent function is explained by redistribution of electronic density at beta-decay, and affecting biochemical processes in the bacterial media. Effects of alpha- and beta-radiation on luminous bacteria are compared.

In vitro phenotypic modification of pigmented epithelium cells from human eye at early stages of development.

Multipotent characteristics of human fetal (9-11.5 weeks) pigmented epithelial retinal cells and capacity to transdifferentiation in neuronal direction were studied in vitro under different culturing conditions. The cultures were analyzed using a wide spectrum of antibodies. It was found that pigmented epithelium of human eye is a heterogeneous cell population with three subtypes differing by adhesion characteristics, migration, transdifferentiation potential, and reaction to microenvironmental factors. Subtype 1 cells steadily retain their epithelial characteristics, subtype 2 cells change their morphotype and produce neuroblast and photoreceptor cell proteins, and subtype 3 cells form free floating spheres and are capable to multipotent differentiation.