Neural stem cells

Experimental evidence of the possibility of regeneration of CNS cells was obtained much earlier than the discovery of embryonic stem cells in studies that showed the presence of cells in the neocortex, hippocampus and olfactory bulbs of the brain of adult rats that capture 3H-thymidine, i.e., are capable of protein synthesis and division. Back in the 60s of the last century, it was assumed that these cells are precursors of neurons and are directly involved in the processes of learning and memory. A little later, the presence of synapses on neurons formed de novo was revealed and the first works on the use of embryonic stem cells for the purpose of inducing neurogenesis in vitro appeared. At the end of the 20th century, experiments with the directed differentiation of ESCs into neural progenitor cells, dopaminergic and serotonergic neurons led to a revision of the classical ideas about the ability of mammalian nerve cells to regenerate. The results of numerous studies have convincingly proven both the reality of the restructuring of neural networks and the presence of neurogenesis throughout the entire period of postnatal life of the mammalian organism.

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Sources of neural stem cells

Human neural stem cells are isolated during operations on the subventricular region of the lateral ventricles and the dentate gyrus of the hippocampus, the cells of which form neurospheres (neural spheres) in culture, and after dispersion and preformation of the latter - all the main cell types of the central nervous system or, in a special medium, new microspheres. In suspension cultures of dissociated tissue isolated from the periventricular regions of the embryonic brain, neurospheres also arise.

Markers of immature brain cells include nestin, beta-tubulin III (neuronal lineage marker), vimentin, GFAP, and NCAM, which are identified immunocytochemically using monoclonal antibodies. Nestin (intermediate neurofilament protein type IV) is expressed by multipotent neuroectodermal cells. This protein is used to identify and isolate multipotent neuroepithelial progenitor cells from the CNS using monoclonal antibodies Rat-401, which can detect up to 95% of neural tube cells in rat embryos on the eleventh day of gestation. Nestin is not expressed on differentiated descendants of neural stem cells, but is present in early neural progenitor cells, postmitotic neurons, and early neuroblasts. This marker has been used to identify neuroepithelial progenitor cells and to prove the existence of stem cells in the CNS. Vimentin (intermediate neurofilament protein type III) is expressed by neural and glial progenitor cells, as well as neurons, fibroblasts, and smooth muscle cells. Therefore, both immunocytochemical markers lack the specificity required to separately identify neural stem and progenitor cells. Beta-tubulin III establishes the neuronal direction of stem cell differentiation, whereas type I astrocytes are identified by GFAP expression, and oligodendrocytes specifically express galactocerebroside (Ga!C).

FGF2 and EGF serve as mitogens for neural progenitor cells, supporting the proliferation of undifferentiated progenitor cells in culture with the formation of neurospheres. The rate of neural stem cell division increases significantly under the influence of FGF2, as well as with the use of a combination of FGF2 + EGF. The proliferative effects of FGF2 are mediated by FGF2-R1 receptors. Heparin increases the affinity of FGF2 receptor binding and dramatically enhances its mitogenic effect on neuroepithelial cells. In the early stages of embryogenesis, FGF2 receptors are expressed in the rat telencephalon, while in later stages their localization is limited to the ventricular zone. The peak of FGF2-R1 expression by postmitotic cells is observed upon completion of the early neurogenesis period. The initial period of telencephalon development is characterized by a low level of EGF receptor expression, mainly in the cells of the ventral region. At later stages of embryogenesis, EGF-R expression increases in the dorsal direction. In the rodent brain, EGF has a high affinity for the transforming growth factor beta receptor (TGF-beta-R), which it preferentially binds to. Indirect evidence for the functional role of EGF-R is provided by data on cortical dysgenesis of the forebrain that occurs in the late period of embryogenesis and postnatal ontogenesis, decreased forebrain function, cortical cell death, and hippocampal ectopia in EGF receptor gene knockout mice. In addition, the presence of TGF-a in the nutrient medium is absolutely necessary for the formation of neurospheres. After the removal of growth factors from the conditioned medium, the cells stop dividing and undergo spontaneous differentiation with the formation of neurons, astrocytes, and oligodendroblasts.

Taking this into account, reaggregation of dissociated stem cells and cultivation of neurospheres are carried out in nutrient media containing EGF and basic FGF or FGF2, but without adding serum. It has been shown that EGF induces proliferation of stem cells of the subependymal zone of the lateral ventricles, and basic FGF promotes proliferation of stem cells of the striatum, hippocampus, neocortex and optic nerve of the mature brain. The combination of EGF and basic FGF is absolutely necessary for active proliferation of stem cells isolated from the ependyma of the third and fourth ventricles of the forebrain, as well as from the spinal canal of the thoracic and lumbar spinal cord.

After dissociation, the suspension of neural stem cells is cultured in plastic dishes or multi-well plates without an adhesive substrate to increase the size of the new neurospheres formed, which usually takes about 3 weeks. The method of multiple dispersion and reproduction of neurospheres allows obtaining a sufficient number of linear clones of multipotent stem cells for intracerebral transplantation. This principle is also the basis for creating a bank of stem cells isolated from the human embryonic brain. Their long-term (over several years) cloning makes it possible to obtain stable lines of neural stem cells, from which catecholaminergic neurons are formed during induced differentiation.

If neurospheres are not dispersed and grown on adhesive substrates in media lacking growth factors, proliferating stem cells begin to spontaneously differentiate to form neuronal and glial precursor cells expressing markers of all types of nerve cells: MAP2, Tau-1, NSE, NeuN, beta-tubulin III (neurons), GFAP (astrocytes) and CalC, 04 (oligodendrocytes). Unlike mouse and rat cells, neurons account for more than 40% of all differentiated cells in human neural stem cell cultures (from 1 to 5% in rodents), but significantly fewer oligodendrocytes are formed, which is very important from the point of view of cell therapy of demyelinating diseases. The problem is solved by adding B104 culture medium, which stimulates the formation of myelin-producing cells.

When culturing neural progenitor cells from the brain of human embryos in a medium containing EGF, basic FGF and LIF, the number of neural lineage precursor cells increases 10 million-fold. Cells expanded in vitro retain the ability to migrate and differentiate into neural and glial elements after transplantation into the brain of mature rats. However, in vivo the number of divisions of multipotent precursor cells is limited. It has been repeatedly noted that the Hayflick limit for an “adult” neural stem cell (about 50 mitoses) is still unattainable even in an experiment - cells in the form of neurospheres retain their properties for only 7 months and only after 8 passages. It is believed that this is due to the peculiarities of their dispersion methods during passaging (trypsinization or mechanical action), which sharply reduces the proliferative activity of cells due to disruption of intercellular contacts. Indeed, if instead of dispersion the method of dividing neurospheres into 4 parts is used, the viability of cells during passaging increases significantly. This method allows human neural stem cells to be cultivated for 300 days. However, after this period the cells lose mitotic activity and undergo degeneration or enter the stage of spontaneous differentiation with the formation of neurons and astrocytes. On this basis, the author believes that 30 mitoses is the maximum number of divisions for cultured neural stem cells.

When human neural stem cells are cultured in vitro, predominantly GABAergic neurons are formed. Without special conditions, neural progenitor cells give rise to dopaminergic neurons (necessary for cell therapy of Parkinson's disease) only in the first passages, after which all neurons in the culture consist exclusively of GABAergic cells. In rodents, IL-1 and IL-11, as well as fragments of nerve cell membranes, LIF and GDNF, cause the induction of dopaminergic neurons in vitro. However, this methodological approach has proven unsuccessful in humans. Nevertheless, when GABAergic neurons are transplanted intracerebrally in vivo, under the influence of microenvironmental factors, nerve cells with different mediator phenotypes arise.

Search for combinations of neurotrophic factors showed that FGF2 and IL-1 induce the formation of dopaminergic neuroblasts, which, however, are not capable of producing dopaminergic neurons. Differentiation of hippocampal stem cells into excitatory glutamatergic and inhibitory GABA-ergic neurons occurs under the influence of neurotrophins, and EGF and IGF1 induce the formation of glutamatergic and GABA-ergic neurons from neural progenitor cells of human embryos. Sequential addition of retinoic acid and neurotrophin 3 (NT3) to the culture significantly increases the differentiation of mature brain hippocampal stem cells into neurons of various mediator nature, while a combination of brain-derived neurotrophic factor (BNDF), NT3 and GDNF can produce pyramidal neurons in hippocampal and neocortical cultures.

Thus, the results of numerous studies indicate that, firstly, stem cells from different brain structures under the influence of local specific tissue factors are capable of differentiating in vivo into neuronal phenotypes inherent to these structures. Secondly, targeted induced differentiation of neural stem cells in vitro using cloning of progenitor cells makes it possible to obtain nerve and glial cells with specified phenotypic characteristics for intracerebral transplantation in various forms of brain pathology.

There is no doubt that pluripotent stem cells isolated from embryos or the adult CNS can be considered as a source of new neurons and used in the clinic for the treatment of neurological pathology. However, the main obstacle to the development of practical cellular neurotransplantation is the fact that most neural stem cells do not differentiate into neurons after implantation into non-neurogenic zones of the mature CNS. To circumvent this obstacle, a very original innovative method is proposed that allows in vitro obtaining of a pure population of neurons from human fetal neural stem cells after transplantation into the CNS of a mature rat. The authors prove that the differentiation of cells implanted by this method ends with the formation of neurons of the cholinergic phenotype, which is due to the influence of factors of the surrounding microenvironment. The proposed technology is of interest from the point of view of developing new types of stem cell-based therapy and replacing neurons damaged due to injury or neurodegenerative disease, since cholinergic neurons play a leading role in the development of motor, memory and learning functions. In particular, cholinergic neurons isolated from human stem cells can be used to replace motor neurons lost in amyotrophic lateral sclerosis or spinal cord injuries. Currently, there is no information on methods for producing a significant number of cholinergic neurons from a population of mitogen-preformed stem cells. The authors propose a fairly simple but effective method for stimulating mitogen-preformed primary human embryonic neural stem cells to develop into virtually pure neurons after implantation in both non-neurogenic and neurogenic zones of the CNS of a mature rat. The most important result of their work is the conversion of a sufficiently large number of transplanted cells into cholinergic neurons when implanted into the middle membrane and spinal cord.

In addition, for the preformation of neural stem cells from the 8-week human embryonic cerebral cortex into cholinergic neurons in vitro, it is proposed to use various combinations of the following trophic factors and chemical elements: recombinant basic FGF, EGF, LIF, mouse amino-terminal sound peptide (Shh-N), trans-retinoic acid, NGF, BDNF, NT3, NT4, natural laminin and mouse heparin. The original line of human neural stem cells (K048) was maintained in vitro for two years and withstood 85 passages without changes in proliferative and differentiating properties while maintaining a normal diploid karyotype. Undispersed neurospheres of passages 19–55 (weeks 38–52) were plated on poly-d-lysine and laminin and then treated with the above-mentioned factors in different concentrations, combinations, and sequences. The combination of basic FGF, heparin, and laminin (abbreviated as FHL) gave a unique effect. After one day of culturing embryonic neural stem cells in FHL medium with or without Shh-N (the combination of Shh-N + FHL in the abbreviation SFHL), a rapid proliferation of large planar cells was observed. All other one-day protocols (such as basic FGF + laminin), on the contrary, led to a limited radial spread of spindle-shaped cells, and these cells did not leave the core of the neurospheres. After 6 days of activation and subsequent 10 days of differentiation in B27-containing medium, large multipolar neuron-like cells were detected at the edge of FHL-activated spheres. In other protocol groups, most neuron-like cells remained small and bipolar or unipolar. Immunocytochemical analysis showed that small (< 20 μm) bipolar or unipolar cells were either GABAergic or glutamatergic, whereas most large multipolar cells localized at the edge of FHL-activated neurospheres were cholinergic, expressing markers characteristic of cholinergic neurons (Islet-1 and ChAT). Some of these neurons simultaneously expressed synapsin 1. As a result of five series of independent experiments, the authors found that the total population of cells in the single-layer zones differentiated into TuJ1+ neurons by 45.5%, while cholinergic (ChAT^) neurons constituted only 27.8% of the cells of the same population. After 10 days of additional differentiation in vitro, in addition to cholinergic neurons, a significant number of small neurons were found in the FHL-activated neurospheres - glutamatergic (6.3%), GABA-ergic (11.3%), as well as astrocytes (35.2%) and nestin-positive cells (18.9%). When using other combinations of growth factors, cholinergic neurons were absent, and the marginal cells of the neurospheres formed either astrocytes or small glutamatergic and GABA-ergic neurons.Monitoring of reserve and active potentials using the whole-cell patch clamp technique showed that after seven days of FHL activation, most large polypolar cells had a resting potential of -29.0±2.0 mV in the absence of action potential. After 2 weeks, the resting potential increased to -63.6±3.0 mV, and action potentials were observed at the moment of induction of depolarizing currents and were blocked by 1 M tetrodotoxin, indicating the functional activity of cholinergic immature neurons.

The authors further established that FHL or SFHL activation in vitro per se does not result in the formation of mature neurons and attempted to establish whether FHL- or SFHL-preformed stem cells are capable of differentiating into cholinergic neurons when transplanted into the CNS of mature rats. For this purpose, activated cells were injected into the neurogenic zone (hippocampus) and into several non-neurogenic zones, including the prefrontal cortex, middle membrane, and spinal cord of adult rats. The implanted cells were tracked using the CAO-^^p vector. OCP is known to label both cellular ultrastructure and cellular processes (molecular level) without leakage and can be directly visualized. In addition, OCP-labeled neural stem cells maintain a profile of neuronal and glial differentiation identical to that of non-transformed stem cells of the embryonic brain.

One to two weeks after implantation of 5 x 10 ⁴ activated and labeled neural stem cells, they were found in the spinal cord or brain of rats, with OCD+ cells located mainly near the injection site. Migration and integration processes were observed as early as one month after transplantation. The migration limits varied depending on the injection site: when injected into the prefrontal cortex, OCD+ cells were located 0.4-2 mm from the injection site, while in the case of implantation into the middle membrane, hippocampus or spinal cord, the cells migrated much longer distances - up to 1-2 cm. The transplanted cells were localized in highly organized CNS structures, including the frontal cortex, middle membrane, hippocampus and spinal cord. OCD-labeled neuronal elements were visible as early as the first week after transplantation, with their number significantly increasing one month after the operation. Stereological analysis showed a higher survival rate of implanted cells in various structures of the brain, compared to the spinal cord.

It is known that in most tissues of the adult mammalian organism, a population of regional stem cells is preserved, the transformation of which into mature cells is regulated by specific tissue factors. The proliferation of stem cells, differentiation of progenitor cells and the formation of neuronal phenotypes specific to a given brain structure in vivo are expressed to a much greater extent in the embryonic brain, which is determined by the presence of high concentrations of morphogenetic factors of the local microenvironment - neurotrophins BDNF, NGF, NT3, NT4/5 and growth factors FGF2, TGF-a, IGF1, GNDF, PDGF.

Where are neural stem cells located?

It has been established that neural stem cells express glial acidic fibrillary protein, which among mature cells of the neural lineage is retained only on astrocytes. Therefore, astrocytic cells may be the stem reserve in the mature CNS. Indeed, neurons originating from GFAP-positive precursors were identified in the olfactory bulbs and dentate gyrus, which contradicts traditional ideas about the progenitor role of radial glia, which does not express GFAP in the dentate gyrus in adulthood. It is possible that there are two populations of stem cells in the CNS.

The question of the localization of stem cells in the subventricular zone also remains unclear. According to some authors, ependymal cells form spherical clones in culture that are not true neurospheres (like clones of subependymal cells), since they are capable of differentiating only into astrocytes. On the other hand, after fluorescent or viral labeling of ependymal cells, the marker is detected in the cells of the subependymal layer and olfactory bulbs. Such labeled cells in vitro form neurospheres and differentiate into neurons, astrocytes, and oligodendrocytes. In addition, it has been shown that about 5% of cells in the ependyma express stem markers - nestin, Notch-1, and Mussashi-1. It is assumed that the mechanism of asymmetric mitosis is associated with the uneven distribution of the membrane receptor Notch-1, as a result of which the latter remains on the membrane of the daughter cell localized in the ependymal zone, while the mother cell migrating to the subependymal layer is deprived of this receptor. From this point of view, the subependymal zone can be considered as a collector of progenitor precursors of neurons and glia formed from stem cells of the ependymal layer. According to other authors, only glial cells are formed in the caudal parts of the subventricular zone, and the source of neurogenesis are the cells of the rostral-lateral part. In the third variant, the anterior and posterior parts of the subventricular zone of the lateral ventricles are given equivalent neurogenic potential.

The fourth variant of the organization of the stem reserve in the central nervous system seems preferable, according to which three main types of neural progenitor cells are distinguished in the subventricular zone - A, B and C. A-cells express early neuronal markers (PSA-NCAM, TuJl) and are surrounded by B-cells, which are identified as astrocytes by the expression of antigens. C-cells, having no antigenic characteristics of neurons or glia, have high proliferative activity. The author has convincingly proven that B-cells are precursors of A-cells and de novo neurons of the olfactory bulbs. During migration, A-cells are surrounded by strands of neural progenitor cells, which differs significantly from the migration mechanism of postmitotic neuroblasts along the radial glia in the embryonic brain. Migration ends in the olfactory bulbs with mitotic division of both A and B cells, the derivatives of which are incorporated into the granular cell layers and into the glomerular layer of the olfactory zone of the brain.

The developing embryonic brain lacks differentiated ependymal cells, and the ventricular walls contain proliferating stem cells of the ventricular germinal and subventricular zones, where primary neuro- and glioblasts migrate. Based on this, some authors believe that the subependymal region of the mature brain contains reduced embryonic germinal neural tissue consisting of astrocytes, neuroblasts, and unidentified cells. True neural stem cells constitute less than 1% of the cells in the germinal zone of the lateral ventricular wall. Partly for this reason, and also in connection with the data that astrocytes of the subependymal zone are precursors of neural stem cells, the possibility of transdifferentiation of astrocytic glial elements with the acquisition of neuronal phenotypic characteristics is not excluded.

The main obstacle to a final solution to the problem of neural stem cell localization in vivo is the lack of specific markers for these cells. Nevertheless, very interesting from a practical point of view are the reports that neural stem cells were isolated from CNS regions that do not contain subependymal zones - the third and fourth ventricles of the forebrain, the spinal canal of the thoracic and lumbar regions of the spinal cord. Of particular importance is the fact that spinal cord injury increases the proliferation of ependymal stem cells of the central canal with the formation of progenitor cells migrating and differentiating into astrocytes of the gliomesodermal scar. In addition, precursor cells of astro- and oligodendrocytes were also found in the uninjured spinal cord of adult rats.

Thus, the literature data convincingly demonstrates the presence in the CNS of adult mammals, including humans, of a regional stem reserve, the regenerative-plastic capacity of which, unfortunately, is capable of providing only the processes of physiological regeneration with the formation of new neuronal networks, but does not meet the needs of reparative regeneration. This poses the task of searching for opportunities to increase the stem resources of the CNS by exogenous means, which is unsolvable without a clear understanding of the mechanisms of CNS formation in the embryonic period.

Today we know that during embryonic development, neural tube stem cells are the source of three cell types - neurons, astrocytes and oligodendrocytes, i.e. neurons and neuroglia originate from a single precursor cell. Differentiation of the ectoderm into clusters of neural progenitor cells begins under the influence of the products of the proneural genes of the bHLH family and is blocked by the expression of receptor transmembrane protein derivatives of the Notch family genes, which limit the determination and early differentiation of neural precursor cells. In turn, the ligands of the Notch receptors are the transmembrane Delta proteins of neighboring cells, due to the extracellular domain of which direct intercellular contacts with inductive interaction between stem cells are carried out.

The further implementation of the embryonic neurogenesis program is no less complex and, it would seem, should be species-specific. However, the results of neuroxenotransplantation studies indicate that stem cells have a pronounced evolutionary conservatism, due to which human neural stem cells are able to migrate and develop when transplanted into the rat brain.

It is known that the mammalian CNS has an extremely low capacity for reparative regeneration, which is characterized by the absence of any signs of the emergence of new cellular elements in the mature brain to replace neurons that died as a result of injury. However, in the case of neuroblast transplantation, the latter not only engraft, proliferate and differentiate, but are also able to integrate into brain structures and functionally replace lost neurons. When transplanting committed neuronal progenitor cells, the therapeutic effect was significantly weaker. Such cells have been shown to have a low capacity for migration. In addition, neuronal progenitor cells do not reproduce the architecture of neural networks and are not functionally integrated into the recipient's brain. In this regard, issues of reparative-plastic regeneration during transplantation of non-preformed multipotent neural stem cells are being actively studied.

In the study by M. Aleksandrova et al. (2001), in the first version of the experiments, the recipients were sexually mature female rats, and the donors were 15-day-old embryos. A section of the occipital cortex of the brain was removed from the recipients, and mechanically suspended tissue of the presumptive embryonic cortex containing multipotent stem cells of the ventricular and subventricular regions was transplanted into the cavity. In the second version of the experiments, neural stem cells of a 9-week human embryo were transplanted into the brain of sexually mature rats. The authors isolated pieces of tissue from the periventricular region of the embryonic brain, placed them in an F-12 nutrient medium, and obtained a cell suspension by repeated pipetting, and then cultured them in a special NPBM medium with the addition of growth factors - FGF, EGF and NGF. The cells were grown in a suspension culture until neurospheres were formed, which were dispersed and again planted in the culture. After 4 passages with a total cultivation period of 12-16 days, the cells were used for transplantation. The recipients were ten-day-old rat pups and sexually mature two-month-old Wistar rats, to which 4 μl of the human neural stem cell suspension were injected into the lateral ventricle of the brain without immunosuppression. The results of the work showed that dissociated cells of the ventricular and subventricular zone of the embryonic anlage of the rat cerebral cortex continued their development during allotransplantation into the mature brain, i.e., the factors of the microenvironment of the differentiated recipient brain did not block the growth and differentiation of the neural stem cells of the embryo. In the early stages after transplantation, multipotent cells continued mitotic division and actively migrated from the transplantation area to the recipient's brain tissue. Transplanted embryonic cells with a huge migration potential were found in almost all layers of the recipient's cerebral cortex along the transplantation track and in the white matter. The length of the migration tract of nerve cells was always significantly shorter (up to 680 μm) than that of glial elements (up to 3 mm). Blood vessels and fiber structures of the brain served as structural vectors for astrocyte migration, which was also noted in other studies.

Previously, it was believed that the accumulation of labeled astrocytes in the area of damage to the recipient's cerebral cortex may be associated with the formation of a glial barrier between the tissues of the transplant and recipient. However, a study of the structure of compactly located cell transplants showed that their cytoarchitecture is characterized by chaos, without any layered distribution of transplanted cells. The degree of orderliness of transplanted neurons approached that of normal cerebral cortex cells only in the absence of a glial barrier between the tissues of the donor and recipient. Otherwise, the structure of the transplant cells was atypical, and the neurons themselves were subject to hypertrophy. Using neuroimmunochemical typing of transplanted cells, inhibitory GABA-ergic neurons were found in the transplants and expression of PARV, CALB, and NPY proteins was detected. Consequently, the mature brain retains microenvironmental factors capable of supporting the proliferation, migration, and specific differentiation of neural multipotent cells.

In the culture of human stem cells isolated from the periventricular region of the brain of 9-week embryos, M. Aleksandrova et al. (2001) found a large number of nestin-positive multipotent cells in the fourth passage, some of which had already undergone in vitro differentiation and were developing according to the neuronal type, which corresponded to the results of studies by other authors. After transplantation into the brain of adult rats, the cultured human stem cells mitotically divided and migrated into the tissue of the xenogeneic recipient brain. In the cell transplants, the authors observed two populations of cells - small and larger. The latter migrated both in the parenchyma and along the fiber structures of the recipient brain over insignificant distances - within 300 μm. The greatest extent of the migration path (up to 3 mm) was characteristic of small cells, some of which differentiated into astrocytes, which was established using monoclonal antibodies to GFAP. Both cell types were found in the wall of the lateral ventricle, indicating that the transplanted cells entered the rostral migration tract. Astrocytic derivatives of neural stem cells from both humans and rats migrated predominantly through the blood capillaries and fiber structures of the recipient brain, which coincides with the data of other authors.

Analysis of human stem cell differentiation in vivo using monoclonal antibodies to GFAP, CALB, and VIM revealed the formation of both astrocytes and neurons. Unlike the cells in rat transplants, many human stem cells were vimentin-positive. Consequently, some of the human multipotent cells did not undergo differentiation. The same authors subsequently showed that human neural stem cells transplanted without immunosuppression survive in the rat brain for 20 days after transplantation, with no signs of immune aggression from the glial elements of the mature brain.

It has been established that even neural stem cells of Drosophila engraft and undergo differentiation in the brain of a taxon as distant from insects as the rat. The correctness of the authors' experiment is beyond doubt: transgenic Drosophila lines contained genes for human neurotrophic factors NGF, GDNF, BDNF, inserted into the CaSper vector under the Drosophila heat-shock promoter, so that the body temperature of mammals automatically evoked their expression. The authors identified Drosophila cells by the product of the bacterial galactosidase gene using histochemical X-Gal staining. In addition, it turned out that Drosophila neural stem cells specifically respond to neurotrophic factors encoded by human genes: when xenotransplanting cells of a transgenic Drosophila line containing the gdnf gene, the synthesis of tyrosine hydroxylase in its differentiating neural stem cells increased sharply, and cells with the ngf gene actively produced acetylcholinesterase. The xenotransplant induced similar gene-dependent reactions in the allotransplant of embryonic neural tissue transplanted together with it.

Does this mean that specific differentiation of neural stem cells is induced by species-non-specific neurotrophic factors? According to the authors' results, the xenograft producing neurotrophic factors had a specific effect on the fate of allografts, which in this case developed more intensively and were 2-3 times larger in size than allografts introduced into the brain without the addition of xenografts. Consequently, xenograft cells containing neurotrophin genes, in particular the gene encoding human glial cell-derived neurotrophic factor (GDNF), have a species-non-specific effect on allograft development similar to the action of the corresponding neurotrophin. GDNF is known to increase the survival of dopaminergic neurons in the rat embryonic midbrain and enhance dopamine metabolism by these cells, and induce the differentiation of tyrosine hydroxylase-positive cells, enhancing axon growth and increasing the size of the neuronal cell body. Similar effects are also observed in cultured rat midbrain dopaminergic neurons.

Active migration of human neural stem cells is observed after xenotransplantation into the brain of mature rats. It is known that the process of migration and differentiation of neural stem cells is controlled by a set of special genes. The initiating migration signal to the precursor cell to begin differentiation is given by the protein product of the c-ret protooncogene together with GDNF. The next signal comes from the mash-1 gene, which controls the choice of the cell development path. In addition, the specific reaction of differentiating cells also depends on the a-receptor of the ciliary neurotrophic factor. Thus, given the completely different genetic constitution of xenogenic human neural stem cells and the recipient rat brain cells, it is necessary to recognize not only the species-nonspecificity of neurotrophic factors, but also the highest evolutionary conservatism of the genes responsible for the specific differentiation of neural stem elements.

The future will show whether xenotransplantation of embryonic neuromaterial will be possible in neurosurgical practice of treating neurodegenerative pathological processes caused by disruption of myelin synthesis by oligodendrocytes. In the meantime, the most intensively addressed issues of neurotransplantation are those related to obtaining allogeneic neural stem cells from the embryonic or mature brain in culture with their subsequent directed differentiation into neuroblasts or specialized neurons.

Neural stem cell transplantation

To stimulate the proliferation and differentiation of neural stem cells of an adult organism, embryonic nervous tissue can be transplanted. It is possible that the stem cells of the embryonic nervous tissue brought in with the allograft can themselves undergo proliferation and differentiation. It is known that after a spinal injury, regeneration of nerve conductors occurs through the elongation of damaged axons and collateral sprouting of axons of undamaged processes of motor neurons. The main factors that prevent spinal cord regeneration are the formation of a connective tissue scar in the area of damage, dystrophic and degenerative changes in central neurons, NGF deficiency, and the presence of myelin breakdown products in the area of damage. It has been shown that transplantation of different cell types into the damaged spinal cord - fragments of the sciatic nerve of adult animals, embryonic occipital cortex, hippocampus, spinal cord, Schwann cells, astrocytes, microglia, macrophages, fibroblasts - promotes regeneration of damaged axons by sprouting and allows newly formed axons to grow through the spinal cord injury zone. It has been experimentally proven that transplantation of embryonic nervous tissue into the spinal cord injury area, through the action of neurotrophic factors, accelerates the growth of damaged axons, prevents the formation of glial scar and the development of dystrophic and degenerative processes in central neurons, while the cells of the transplanted embryonic nervous tissue survive in the spinal cord, integrate with adjacent tissues and promote axon growth through the injury area with the formation of dendritic synapses on spinal neurons.

This area of regenerative-plastic medicine has received the greatest development in Ukraine thanks to the work of the scientific team led by V. I. Tsymbalyuk. First of all, these are experimental studies of the effectiveness of embryonic nerve tissue transplantation in spinal cord injuries. During autotransplantation of the peripheral nerve, the authors observed the most pronounced destructive changes in the distal suture zone, where on the 30th day after the operation they were combined with reparative processes. During allotransplantation, the morphofunctional state of the implanted nerve on the 30th day was characterized by pronounced destruction with fatty degeneration and amyloidosis against the background of focal inflammatory lymphoid cell infiltration with predominant atrophy of Schwann cells. Transplantation of embryonic nerve tissue contributed to the restoration of spinal cord conductivity to a greater extent, especially in animals that underwent surgery during the first 24 hours after injury: against the background of a decrease in the intensity of inflammatory and destructive processes, hypertrophy and hyperplasia of protein-synthesizing and energy-producing ultrastructural elements of spinal neurons, hypertrophy and hyperplasia of oligodendrocytes were observed, the amplitude of the muscle action potential was restored by 50% and the impulse conduction velocity by 90%. When assessing the effectiveness of transplantation of embryonic nerve tissue depending on the transplantation zone, it was found that the best results were observed when the graft was introduced directly into the spinal cord injury zone. With complete transection of the spinal cord, transplantation of embryonic nerve tissue was ineffective. Dynamic studies have shown that the optimal time for performing embryonic nerve tissue transplantation is the first 24 hours after spinal cord injury, while performing surgery during the period of pronounced secondary ischemic-inflammatory changes that occur on the 2nd-9th day after injury should be considered inappropriate.

It is known that severe traumatic brain injury provokes powerful and prolonged activation of lipid peroxidation at the initial and intermediate stages of the post-traumatic period both in the damaged brain tissue and in the body as a whole, and also disrupts the processes of energy metabolism in the injured brain. Under these conditions, transplantation of embryonic nervous tissue into the area of traumatic injury promotes stabilization of lipid peroxidation processes and increases the potential of the antioxidant system of the brain and the body as a whole, enhances its antiradical protection on the 35-60th day of the post-traumatic period. In the same period after transplantation of embryonic nervous tissue, energy metabolism and oxidative phosphorylation processes in the brain are normalized. In addition, it has been shown that on the first day after experimental traumatic brain injury, the impedance of the tissue of the injured hemisphere decreases by 30-37%, the contralateral - by 20%, which indicates the development of generalized cerebral edema. In animals that underwent transplantation of embryonic nerve tissue, edema involution occurred significantly faster - already on the seventh day, the average impedance value of the tissues of the injured hemisphere reached 97.8% of the control level. Moreover, complete restoration of impedance values on the 30th day was noted only in animals that received transplantation of embryonic nerve tissue.

The death of some neurons in the brain after a severe craniocerebral injury is one of the main causes of post-traumatic complications. Neurons of the integrating dopaminergic and noradrenergic systems of the midbrain and medulla oblongata are especially sensitive to injury. A decrease in the dopamine level in the striopallidal complex and the cerebral cortex significantly increases the risk of developing motor disorders and mental disorders, epileptiform states, and a decrease in dopamine production in the hypothalamus can be the cause of numerous vegetative and somatic disorders observed in the late post-traumatic period. The results of studies conducted in experimental craniocerebral injury indicate that transplantation of embryonic nerve tissue helps restore dopamine levels in the injured cerebral hemisphere, dopamine and norepinephrine in the hypothalamus, and increase norepinephrine and dopamine levels in the midbrain and medulla oblongata. In addition, as a result of transplantation of embryonic nervous tissue in the injured hemisphere of the brain of experimental animals, the percentage ratio of phospholipids is normalized and the content of fatty acids increases (C16:0, C17:0, C17:1, C18:0, C18:1 + C18:2, C20:3 + C20:4, C20:5).

These data confirm the stimulation of regenerative-plastic processes by transplanted embryonic nervous tissue and indicate the reparative-trophic effect of the transplant on the recipient's brain as a whole.

The clinical experience of the staff of the A.P. Romodanov Institute of Neurosurgery of the Academy of Medical Sciences of Ukraine in transplantation of embryonic nerve tissue in cerebral palsy, an extremely complex pathology with severe motor dysfunction, deserves special attention. Clinical forms of cerebral palsy depend on the level of damage to the integral structures responsible for the regulation of muscle tone and the formation of motor stereotypes. Currently, there is sufficient evidence to support the fact that pathological changes in the striopallidal-thalamocortical motor control system play an important role in motor function and muscle tone disorders. The striopallidal link of this system carries out the control function through nigrostriatal dopamine production. The direct pathway for the implementation of thalamocortical control begins from the neurons of the putamen, is mediated by gamma-aminobutyric acid (GABA) and substance P and is projected directly into the motor zone of the internal segment of the globus pallidus and the substantia nigra. The indirect pathway, the effect of which is realized with the participation of GABA and enkephalin, originates from neurons of the putamen and affects the nuclei of the basal ganglia through a sequence of connections including the external segment of the globus pallidus and the subthalamic nucleus. Disturbances in the conductivity of the direct pathway cause hypokinesia, while a decrease in the conductivity of the structures of the indirect pathway leads to hyperkinesia with corresponding changes in muscle tone. The integrity of GABAergic conduction pathways at different levels in the motor control system and the integration of dopaminergic connections at the level of the putamen are essential for the regulation of thalamocortical interactions. The most common manifestation of motor pathology in various forms of cerebral palsy is a violation of muscle tone and a closely related change in reflex muscle activity.

Transplantation of embryonic nerve tissue in cerebral palsy requires a thorough analysis of the nature of damage to brain structures. Based on the determination of dopamine and GABA levels in the subarachnoid cerebrospinal fluid, the authors detailed the level of disruption of the integration of functional brain structures, which made it possible to objectify the results of surgical intervention and correct repeated neurotransplantations. Embryonic nerve tissue (abortion material of a 9-week embryo) was transplanted into the parenchyma of the cortex of the precentral convolutions of the cerebral hemispheres depending on the severity of atrophic changes. No complications or deterioration in the condition of patients were observed in the postoperative period. Positive dynamics was noted in 63% of patients with spastic forms, in 82% of children with an atonic-esthetic form, and only in 24% of patients with a mixed form of the disease. A negative effect of a high level of neurosensitization with the presence of autoantibodies to neurospecific proteins on the results of the operation was established. Transplantation of embryonic nerve tissue was found to be ineffective in patients aged 8-10 years and older, as well as in cases of severe hyperkinetic syndrome and epilepsy. Clinically, the effectiveness of transplantation of embryonic nerve tissue in patients with spastic forms of cerebral palsy was manifested by the formation of new statomotor skills and voluntary movements with correction of the pathological motor stereotype and a decrease in the degree of spasticity, pathological postures and attitudes. The authors believe that the positive effect of transplantation of embryonic nerve tissue is the result of the normalizing effect on the functional activity of the supraspinal structures involved in the regulation of postural tone and voluntary movements. At the same time, the positive clinical effects of transplantation of embryonic nerve tissue are accompanied by a decrease in the content of neurotransmitters in the subarachnoid cerebrospinal fluid, which indicates the restoration of integral interactions of the affected brain structures.

There is another severe form of neurological pathology - apallic syndrome, the problem of treatment of which, unfortunately, is far from being solved. Apallic syndrome is a polyetiological subacute or chronic condition that occurs as a result of severe organic lesions of the central nervous system (mainly the cerebral cortex), and is characterized by the development of panapraxia and panagnosia with relatively preserved function of the segmental-stem sections and formations of the limbic-reticular complex of the brain. Follow-up studies (from 1 year to 3 years) have shown that apallic syndrome is not a final diagnosis of persistent damage to the nervous system in children, but is transformed either into organic dementia or into a chronic vegetative state. In the Department of Restorative Neurosurgery of the A.P. Romodanov Institute of Neurosurgery of the Academy of Medical Sciences of Ukraine, 21 patients with the consequences of apallic syndrome underwent transplantation of embryonic nerve tissue. Under general anesthesia, a crown burr was used to make a burr hole over the area of the most pronounced atrophic changes revealed by computed tomography or magnetic resonance imaging, and in the presence of diffuse atrophy of the gray or white matter, the transplant was introduced into the precentral and central gyri of the brain. After opening the dura mater, pieces of tissue from the sensorimotor cortex of 8-9-week embryos were implanted intracortically using a special device. The number of implanted tissue samples ranged from 4 to 10, which was determined by the size of the burr hole and the size of local changes in the brain matter. Unlike other types of pathology, in apallic syndrome the authors sought to implant as much embryonic tissue as possible into the most accessible areas of the brain. The dura mater was sutured, and plastic surgery of the skull defect was performed. During the operation, all patients showed significant changes in both the cortex (atrophy, absence of convolutions, change in color and pulsation of the brain matter) and the meninges (thickening of the dura mater, significant thickening of the arachnoid membrane with the presence of its own blood vessels, fusion of the membranes with the underlying brain matter). These changes were more pronounced in patients with a history of inflammatory brain lesions. In patients who had undergone CNS hypoxia, diffuse atrophic changes in the brain matter, especially in the cortex, with an increase in the subarachnoid space, predominated, without significant changes in the meninges. Half of the patients had increased bleeding of soft tissues, bones, and brain matter. After the operations, within six months to three years, the condition improved in 16 patients, and remained unchanged in five patients. Positive dynamics were observed in both the motor and mental spheres. Muscle tone decreased in ten patients,In 11 patients, motor activity increased (paresis decreased, coordination of movements improved), in five children, the manipulative ability of the upper limbs significantly increased. In four patients, the frequency and severity of epileptic seizures decreased, and in one child there were no seizures at all during the entire observation period after the operation. Aggression decreased in two children, in two patients with severe bulbar disorders, the act of swallowing improved, two children were able to chew independently already 2 weeks after the operation. A decrease in the severity of mental disorders was noted, nine children became calmer after the operation, sleep and attention improved in seven patients. Three patients with the consequences of apallic syndrome began to recognize their parents, one - to follow instructions, two - to pronounce words, in three, the degree of dysarthria decreased. The authors note that a noticeable improvement in the condition of patients begins 2 months after the operation, reaches a maximum by 5-6 months, then the rate of improvement slows down and by the end of the year the process stabilizes in 50% of patients. The positive effect of neurotransplantation served as the basis for a repeated operation in six patients with the consequences of apallic syndrome, but on the other hemisphere of the brain. The technique and methods of the second transplantation were identical to those in the first operation, but the clinical effect of the second operation was lower, although no serious complications arose after either the first or the second surgical intervention. According to the authors, the mechanism of the therapeutic effect of neurotransplantation is associated with the neurotrophic effect of the transplanted embryonic nerve tissue, which contains a large number of growth, hormonal and other biologically active substances that stimulate the reparation of damaged neurons and plastic reorganization of the recipient's brain tissue. An activating effect on the activity of nerve cells that were previously morphologically preserved, but lost their functional activity due to the disease, is also possible. It is the rapid neurotrophic effect that can explain the improvement of bulbar functions in some children already at the end of the first or second week after the operation. It is assumed that, in addition to this, by the third or fourth month, morphofunctional connections are established between the transplant and the host brain, through which the neurotransplant replaces the functions of dead brain cells, which is the substrate for improving both the motor and mental functions of patients.Two children were able to chew independently already 2 weeks after the operation. A decrease in the severity of mental disorders was noted, nine children became calmer after the operation, sleep and attention improved in seven patients. Three patients with the consequences of apallic syndrome began to recognize their parents, one - to follow instructions, two - to pronounce words, in three the degree of dysarthria decreased. The authors note that a noticeable improvement in the condition of patients begins 2 months after the operation, reaches a maximum by 5-6 months, then the rate of improvement slows down and by the end of the year the process stabilizes in 50% of patients. The positive effect of neurotransplantation served as the basis for a repeated operation in six patients with the consequences of apallic syndrome, but on the other hemisphere of the brain. The technique and method of the second transplantation were identical to those in the first operation, but the clinical effect of the second operation was lower, although there were no serious complications after either the first or the second surgical intervention. According to the authors, the mechanism of the therapeutic effect of neurotransplantation is associated with the neurotrophic effect of the transplanted embryonic nervous tissue, which contains a large number of growth, hormonal and other biologically active substances that stimulate the reparation of damaged neurons and plastic reorganization of the recipient's brain tissue. An activating effect on the activity of nerve cells that were previously morphologically preserved, but lost their functional activity due to the disease, is also possible. It is precisely the rapid neurotrophic effect that can explain the improvement of bulbar functions in some children already at the end of the first or second week after surgery. It is assumed that, along with this, by the third or fourth month, morphofunctional connections are established between the transplant and the host brain, through which the neurotransplant replaces the functions of dead brain cells, which is the substrate for improving both motor and mental functions of patients.Two children were able to chew independently already 2 weeks after the operation. A decrease in the severity of mental disorders was noted, nine children became calmer after the operation, sleep and attention improved in seven patients. Three patients with the consequences of apallic syndrome began to recognize their parents, one - to follow instructions, two - to pronounce words, in three the degree of dysarthria decreased. The authors note that a noticeable improvement in the condition of patients begins 2 months after the operation, reaches a maximum by 5-6 months, then the rate of improvement slows down and by the end of the year the process stabilizes in 50% of patients. The positive effect of neurotransplantation served as the basis for a repeated operation in six patients with the consequences of apallic syndrome, but on the other hemisphere of the brain. The technique and method of the second transplantation were identical to those in the first operation, but the clinical effect of the second operation was lower, although there were no serious complications after either the first or the second surgical intervention. According to the authors, the mechanism of the therapeutic effect of neurotransplantation is associated with the neurotrophic effect of the transplanted embryonic nervous tissue, which contains a large number of growth, hormonal and other biologically active substances that stimulate the reparation of damaged neurons and plastic reorganization of the recipient's brain tissue. An activating effect on the activity of nerve cells that were previously morphologically preserved, but lost their functional activity due to the disease, is also possible. It is precisely the rapid neurotrophic effect that can explain the improvement of bulbar functions in some children already at the end of the first or second week after surgery. It is assumed that, along with this, by the third or fourth month, morphofunctional connections are established between the transplant and the host brain, through which the neurotransplant replaces the functions of dead brain cells, which is the substrate for improving both motor and mental functions of patients.although no serious complications arose after either the first or the second surgical intervention. According to the authors, the mechanism of the therapeutic effect of neurotransplantation is associated with the neurotrophic effect of the transplanted embryonic nervous tissue, which contains a large number of growth, hormonal and other biologically active substances that stimulate the reparation of damaged neurons and the plastic reorganization of the recipient's brain tissue. An activating effect on the activity of nerve cells that were previously morphologically preserved, but lost their functional activity due to the disease, is also possible. It is precisely the rapid neurotrophic effect that can explain the improvement of bulbar functions in some children already at the end of the first or second week after surgery. It is assumed that, along with this, by the third or fourth month, morphofunctional connections are established between the transplant and the host brain, through which the neurotransplant replaces the functions of dead brain cells, which is the substrate for improving both the motor and mental functions of patients.although no serious complications arose after either the first or the second surgical intervention. According to the authors, the mechanism of the therapeutic effect of neurotransplantation is associated with the neurotrophic effect of the transplanted embryonic nervous tissue, which contains a large number of growth, hormonal and other biologically active substances that stimulate the reparation of damaged neurons and the plastic reorganization of the recipient's brain tissue. An activating effect on the activity of nerve cells that were previously morphologically preserved, but lost their functional activity due to the disease, is also possible. It is precisely the rapid neurotrophic effect that can explain the improvement of bulbar functions in some children already at the end of the first or second week after surgery. It is assumed that, along with this, by the third or fourth month, morphofunctional connections are established between the transplant and the host brain, through which the neurotransplant replaces the functions of dead brain cells, which is the substrate for improving both the motor and mental functions of patients.

The effect of embryonic nerve tissue transplant on the reorganization of interneuronal interconnections was studied experimentally. The authors studied the patterns of restoration of intermodular axonal connections in the area of mechanical damage to the cerebral cortex in white rats with and without transplantation of embryonic nerve tissue using the fluorescent lipophilic label DIL (1,1-dioctadecyl-3,3,33'-tetramethylindocarbocyanine perchlorate) and confocal laser scanning. It was found that the introduction of embryonic nerve tissue into the damage area ensures the growth of axons, which after passing through the transplant connect with the adjacent brain tissue, whereas without transplantation of embryonic nerve tissue the damage area is an insurmountable obstacle for growing axons. In this work, transplantation of embryonic (15-17th day of gestation) neocortex was performed. The results obtained by the authors are further evidence in favor of the active influence of the embryonic nerve tissue transplant on the post-traumatic reorganization of interneuronal relationships of neighboring structural and functional modules of the cerebral cortex. Transplantation of embryonic nerve tissue provides partial restoration of connections between damaged areas of the cerebral cortex by creating favorable conditions for axon growth in the zone of action of the transplant's neurotrophic factors. The existence of such an effect has been proven experimentally and is discussed in the literature as evidence of high plastic capabilities of the damaged brain of sexually mature animals. In this regard, cell transplantation is currently considered as an optimal therapeutic strategy for restoring the function of the damaged human CNS.

The data obtained by the authors on the efficiency of using embryonic nervous tissue of the brain as an exogenous transplant medium for axon growth confirm the prospects of targeted creation of communication links between intact adjacent areas of the brain. The work on studying the effect of transplantation of nervous tissue on the dynamics of functional parameters of the central nervous system seems relevant. The task of the work was to investigate the effect of transplantation of the embryonic locus coeruleus (LC) on the morphofunctional indices of LC neurons and locomotor activity of recipients. The recipients were female Wistar rats, and the donors were 18-day-old embryos of rats of the same line. Transplantation of embryonic LC was performed into the cavity of the third ventricle of the brain. Histologically, engraftment of the graft was detected in 75% of recipient animals. In cases of engraftment, the graft was adjacent to the ventricular wall, filling 1/5-2/5 of its lumen, and was viable. At 1 and 6 months after the operation, the transplanted nervous tissue, according to its morphological characteristics, represented structures that would have arisen during their normal ontogenetic development, i.e., LC structures. The data obtained by the authors indicate that in animals to which the embryonic LC anlage was transplanted, the dynamic activity changes and the matrix activity of the chromatin of the LC cell nuclei increases. Consequently, the activity of neurons of their own LC intensifies, but the engrafted transplant is also functionally active. It is known that the so-called locomotor region of the midbrain practically coincides with the localization of LC. The authors believe that the basis for the change in the motor activity of recipient rats is the activation of LC cells, both their own and the transplant, with the release of a large amount of norepinephrine, including in the spinal cord segments. Thus, it is assumed that the increase in motor activity under conditions of LC transplantation into the intact brain of animals is due to the presence of a functionally active transplant integrated with the recipient's brain and contributing to the activation of locomotor activity in rats.

In addition, it was shown that transplanted neuroepithelial cells of the embryonic rudiments of the neocortex and spinal cord survive and differentiate into neuroblasts, young and mature neurons within 1-2 months after their transplantation into the damaged sciatic nerve of mature rats. When studying the dynamics of development of NADPH-positive neurons of the embryonic rudiments of the neocortex and spinal cord of rats in heterotopic allografts (15-day rat embryo), engraftment of 70 to 80% of neurografts was revealed on longitudinal sections through the sciatic nerves of recipient rats, which depended on the observation period. Uni- and bipolar neuroblasts with rounded light nuclei and one or two nucleoli began to form in the grafts one week after surgery, which was accompanied by the formation of clusters. The authors failed to detect cells containing NADPH diaphorase (NADPH-d) among neuroblasts. After 7 days, only cellular elements of blood vessels were NADPH-positive - capillary endothelial cells in the thickness of the transplant, as well as endothelial and smooth muscle cells of the recipient's sciatic nerve vessels. Since in vascular smooth muscle cells the induction of NO synthase (NOS) occurs under the influence of IL-1, the authors associate the appearance of NADPH-positive smooth muscle cells in the blood vessels of the sciatic nerve with the presence of IL-1 synthesized in damaged nerve trunks. It is known that neurogenesis under conditions of transplantation of embryonic brain rudiments occurs synchronously with the development of neurons in situ. The results of morphological studies indicate that the differentiation of some neural elements of the transplants seven days after transplantation corresponds to the differentiation of cells in similar parts of the brain of newborn rats. Thus, under conditions of heterotopic transplantation into the peripheral nerve, the transplanted embryonic nerve cells exhibit the ability to synthesize NADPH-d. In this case, more neurons containing NADPH-d are found in spinal cord transplants than in neocortex transplants, but nitric oxide synthesis begins in the transplanted neurons later than during in situ development. In the CNS of vertebrates, NOS-positive cells appear already in the prenatal period. It is believed that NO promotes the formation of synaptic connections in the developing brain, and the presence of NOS-positive nerve afferent fibers that provide NO synthesis in cerebellar neuroblasts stimulates the migration and differentiation of neurons, due to which normal brain cytoarchitecture is formed. An important role of NO in synapsogenesis has been established in the tectum - only those neurons that had synaptic connections with retinal cells turned out to be NOS-positive.

It is known that nitric oxide is one of the regulators of brain activity, where it is formed from arginine under the influence of NO synthase, which has diaphorase activity. In the central nervous system, NO is synthesized in endothelial cells of blood vessels, microglia, astrocytes and neurons of various parts of the brain. After traumatic brain injury, as well as during hypoxia and ischemia, an increase in the number of neurons containing NO, which is one of the regulators of cerebral blood flow, is observed. Given the ability of NO to induce synapsogenesis, the study of the formation of NO-containing cells in conditions of neurotransplantation against the background of traumatic damage to the recipient's nervous tissue is of particular interest.

No less important is the study of the effect of neurotransplantation on the conditioned reflex stereotype of behavior. In experiments on the study of the effect of intracerebral and distant (between CII and CIII) transplantation of embryonic locus coeruleus tissue (17-19 days of gestation) on memory processes and catecholamine content in rats with the destruction of the frontotemporal neocortex, it was shown that electrolytic damage to the frontotemporal cortex of the brain disrupts the stereotype of the conditioned reflex emotional reaction of avoidance (memory), weakens physiological activity, reduces the content of norepinephrine in the zone of coagulated neocortex, but increases its level in the hypothalamus, where a decrease in the concentration of adrenaline is observed, although its amount in the blood and adrenal glands increases.

As a result of intracerebral transplantation of embryonic locus coeruleus tissue, the stereotype of the conditioned reflex emotional avoidance reaction, disrupted by electrolytic damage to the frontotemporal regions of the cerebral cortex, is restored in 81.4% of animals, the content of adrenaline in the reticular formation of the midbrain, hypothalamus and neocortex is normalized, and its level in the hippocampus even increases, which is combined with a decrease in the concentration of adrenaline in the blood.

Remote transplantation of embryonic locus coeruleus tissue not only restores the disrupted stereotype of the conditioned reflex emotional avoidance reaction in rats with electrolytic damage to the frontotemporal cortex, but also increases the content of norepinephrine and adrenaline, mainly in the hypothalamus, blood, adrenal glands and heart. It is assumed that this is due to vascularization of the transplant, penetration of neurotransmitters into the bloodstream, their passage through the blood-brain barrier and activation of the mechanisms of reuptake of adrenaline and norepinephrine by types uptake 1, 2, 3. The authors believe that long-term stabilization of the norepinephrine level under conditions of engraftment and functioning of the transplant can be considered as a phenomenon of its progressive release in minimal doses by neurons of the locus coeruleus.

Positive clinical effects of embryonic nerve tissue transplantation may also be due to the ability of the latter to influence the processes of vascular neoplasm, in the regulation of which growth factors and cytokines directly participate. Vasculogenesis is activated by angiogenic growth factors - vascular endothelial growth factor (VEGF), FGF, PDGF and TGF, which are synthesized during ischemia, which acts as the initiating moment of angiogenesis. It has been proven that the depletion of the vascular growth potential occurs during the aging process of the body, which plays a significant role in the pathogenesis of diseases such as coronary heart disease and obliterating atherosclerosis of the lower extremities. Tissue ischemia also develops in many other diseases. The introduction of angiogenic factors into ischemic zones (therapeutic angiogenesis) stimulates the growth of blood vessels in ischemic tissues and improves microcirculation due to the development of collateral circulation, which, in turn, increases the functional activity of the affected organ.

VEGF and FGF are considered the most promising for clinical use. The results of the first randomized studies were encouraging, especially if the optimal dosages and methods of administration of angiogenic factors were chosen correctly. In this regard, an experimental assessment of the angiogenic activity of an extract isolated from human embryonic brain tissue was carried out. The work used aborted material obtained at the twentieth week of pregnancy and processed according to the method of I. Maciog et al. (1979) as modified by the IC ANRF. This drug is an analogue of “Endothelial cell growth supplement” (“Sigma”) and is a natural mixture of human angiogenic factors, which includes VEGF and FGF. The experiments were performed on rats with models of hindlimb and myocardial tissue ischemia. Based on the study of alkaline phosphatase activity in experimental animals given the extract of embryonic nerve tissue, an increase in the number of capillaries per unit area of the myocardium was found - both in longitudinal and transverse sections of the heart. The angiogenic activity of the preparation was manifested by direct administration to the ischemic zone, as well as in the case of systemic (intramuscular) administration, which led to a decrease in the average area of the post-infarction scar.

In any variant of embryonic nervous tissue transplantation, it is extremely important to correctly select the gestational age of the transplanted embryonic material. Comparative analysis of the efficiency of cell preparations from the embryonic ventral mesencephalon of 8-, 14- and 16-17-day rat embryos three months after intrastriatal neurotransplantation to mature rats with parkinsonism in the automated test of apomorphine-induced motor asymmetry revealed a significantly higher efficiency of CNS cell preparations from 8-day embryos and the lowest efficiency from 16-17-day embryonic nervous tissue. The obtained data correlated with the results of histomorphological analysis, in particular, with the size of the transplants, the severity of the glial reaction and the number of dopaminergic neurons in them.

Differences in the therapeutic effect of embryonic nervous tissue cells may be associated with both the degree of immaturity and commitment of the cells themselves and their different responses to growth factors released in the area of induced damage to dopaminergic neurons. In particular, the effect of EGF and FGF2 on the development of telencephalic neural stem cells in vivo occurs at different stages of embryogenesis. Neuroepithelial cells of 8.5-day-old mouse embryos, when cultured in vitro in a serum-free medium, proliferate in the presence of FGF2, but not EGF, to which only populations of stem cells isolated from the brain of embryos at later stages of development respond. At the same time, neural stem cells proliferate in response to each of these mitogens and additively enhance growth in the case of adding EGF and FGF2 to a culture with a low cell seeding density. EGF-reactive neural stem cells from the germinal zones of 14.5-day-old mouse embryos are considered to be linear descendants of FGF-reactive neural stem cells that first appear after 8.5 days of gestation. The potential phenotype of neural stem and progenitor cells depends on the complex effect of their microenvironment. Immunophenotyping of neural cells from the periventricular and hippocampal zones of 8-12- and 17-20-week-old human embryos by flow cytofluorometry revealed significant variability associated with both the gestational age and individual constitutional features of the donor biomaterial. When these neural progenitor cells are cultivated in a selective serum-free medium with EGF, FGF2, and NGF, neurospheres are formed at a rate that significantly depends on the gestational age. Cells from different parts of the brain of 5-13-week-old human embryos, when briefly cultured with FGF2 in monolayer culture on a laminin substrate in the presence of trace amounts of growth factors, maintain proliferation for 6 weeks with a high percentage of nestin-positive cells against the background of spontaneous formation of cells with markers of all three lines of neural differentiation. Cells isolated from the mesencephalon of a human embryo at a gestation period exceeding 13 weeks, proliferate under the influence of EGF and also form neurospheres. A synergistic effect was achieved by using a combination of EGF and FGF2. The most intense proliferation of neural stem cells with the formation of neurospheres is observed when culturing the cerebral cortex tissue of 6-8-week-old human embryos in the presence of EGF2, IGF1 and 5% horse serum on a substrate with fibronectin.

It should be noted that the questions concerning the gestational age and the section of the embryonic CNS, the tissue of which is preferable to use for the purpose of neurotransplantation, remain open. The answers to them should be sought in the neurogenesis of the developing brain, which continues throughout the prenatal period - at a time when the epithelium of the neural tube forms a multilayer structure. It is believed that the source of stem cells and new neurons is radial glia, consisting of elongated cells with long processes radially directed relative to the wall of the brain vesicles and contacting the inner surface of the ventricles and the outer pial surface of the brain wall. Previously, radial glia was endowed only with the function of a neuronal tract along which neuroblasts migrate from the ventral region to the superficial sections, and it was also assigned a skeletal role in the process of forming the correct laminar organization of the cortex. Today it has been established that as development proceeds, radial glia transdifferentiate into astrocytes. A significant part of it in mammals is reduced immediately after birth, however, in those animal species in which radial glia is preserved until adulthood, neurogenesis actively occurs in the postnatal period.

In culture, radial glial cells from the embryonic neocortex of rodents formed neurons and glial cells, with neurons being predominantly formed at the 14- to 16-day gestational age of embryo development (the period of maximum intensity of neurogenesis in the cerebral cortex of mice and rats). On the 18th day of embryogenesis, differentiation shifted toward the formation of astrocytes with a significant decrease in the number of newly formed neurons. In situ labeling of radial glial cells with GFP made it possible to detect asymmetric division of labeled cells in the cavity of the cerebral vesicles of 15- to 16-day-old rat embryos with the appearance of daughter cells with immunological and electrophysiological characteristics of neuroblasts. It is noteworthy that, according to the results of dynamic observations, the emerging neuroblasts use the mother cell of radial glial cells for migration to the pial surface.

The endogenous marker of radial glia is the intermediate filament protein nestin. Using the method of fluorescent flow sorting of cells labeled with a retrovirus associated with GFP and expressed under the control of nestin, it was shown that stem cells of the dentate gyrus and hilus of the human hippocampus (the material was obtained during operations for epilepsy) express nestin. Therefore, they belong to the radial glia, which in humans, as in other mammals, is preserved only in the dentate gyrus.

At the same time, the efficiency of cell transplantation is determined not only by the high viability of donor cells, their differentiation potential and ability to replace defective cells, but, first of all, by their directed migration. Full functional integration of transplanted cells depends on their migration ability - without disrupting the cytoarchitecture of the recipient's brain. Since radial glia undergoes almost complete reduction in the postnatal period, it was necessary to find out how donor cells can move from the transplantation zone to the site of brain damage in adult recipients. There are two variants of cell migration to the CNS that do not depend on radial glia: the phenomenon of tangential migration or the movement of neuroblasts during the development of the cerebral cortex perpendicular to the radial glia network, as well as migration "in a row" or "along a chain". In particular, the migration of neural progenitor cells from the rostral subventricular zone to the olfactory bulb occurs as a sequence of tightly adjacent cells surrounded by glial cells. It is believed that these cells use partner cells as a migration substrate, and the main regulator of such intercellular interactions is PSA-NCAM (polysialylated neural cell adhesion molecule). Therefore, neuronal migration does not necessarily require the participation of radial glia or pre-existing axonal connections. The extraradial form of cell movement in a “string” along the rostral migration tract is maintained throughout life, which indicates a real possibility of targeted delivery of transplanted neural progenitor cells to the mature nervous system.

There is a hypothesis about the presence of a stem cell line in the ontogenesis of the brain, according to which, in the early stages of brain development, the stem cell is a neuroepithelial cell, which, as it matures, transdifferentiates into radial glia. In adulthood, the role of stem cells is performed by cells that have the characteristics of astrocytes. Despite a number of controversial points (contradictions regarding the stem cells of the hippocampus, as well as deep parts of the brain that do not have a layered cortex and develop from the thalamic tubercles, where radial glia is absent), a clear and simple concept of a consistent change in the phenotype of stem cells throughout ontogenesis looks very attractive.

The influence of microenvironmental factors on the determination and subsequent differentiation of neural differentiate cells has been clearly demonstrated by transplantation of mature rat spinal cord stem cells into different regions of the mature nervous system. When stem cells were transplanted into the dentate gyrus or into the region of neuronal migration in the olfactory bulbs, active migration of transplanted cells was observed, with the formation of numerous neurons. Transplantation of stem cells into the spinal cord and the Ammon's horn region resulted in the formation of astrocytes and oligodendrocytes, whereas transplantation into the dentate gyrus resulted in the formation of not only glial cells, but also neurons.

In a mature rat, the number of dividing cells in the dentate gyrus can reach several thousand per day - less than 1% of the total number of granule cells. Neurons account for 50-90% of cells, astocytes and other glial elements - about 15%. The remaining cells do not have antigenic features of neurons and glia, but contain endothelial cell antigens, which indicates a close relationship between neurogenesis and angiogenesis in the dentate gyrus. Supporters of the possibility of differentiation of endothelial cells into neuronal precursor cells refer to the ability of endothelial cells in vitro to synthesize BDNF.

The speed of self-assembly of neural circuits is impressive: during differentiation, the precursor cells of granule cells migrate to the dentate gyrus and form processes growing towards the SAZ zone of the Ammon's horn and forming synapses with pyramidal glutamatergic and intercalary inhibitory neurons. Newly created granule cells are integrated into existing neural circuits within 2 weeks, and the first synapses appear as early as 4-6 days after the emergence of new cells. By frequent administration of BrdU or 3H-thymidine (one of the methods for identifying adult stem cells) to mature animals, a large number of labeled neurons and astrocytes were found in the Ammon's horn, which indicates the possibility of forming new neurons not only in the dentate gyrus, but also in other parts of the hippocampus. Interest in the processes of division, differentiation and cell death in the dentate gyrus of the hippocampus of the mature brain is also due to the fact that the neurons formed here are localized in one of the key areas of the hippocampus, responsible for learning and memory processes.

Thus, it has been established today that neural progenitor cells originate from the cells of the subependymal zone of the lateral ventricle of mature rodents. They migrate along the rostral migration tract formed by longitudinally oriented astroglial cells to the olfactory bulb, where they are embedded in the granule cell layer and differentiate into neurons of this structure. Migration of progenitor neural cells has been detected in the rostral migration tract of adult monkeys, which indicates the possibility of forming new neurons in the olfactory bulb of primates. Neural stem cells have been isolated from the olfactory bulb of an adult human and transferred into lines, the cloned cells of which differentiate into neurons, astrocytes, and oligodendrocytes. Stem cells have been found in the hippocampus of the mature brain of rats, mice, monkeys, and humans. Neural stem cells of the subgranular zone of the dentate fascia are a source of progenitor cells migrating to the medial and lateral limbs of the hippocampus, where they differentiate into mature granule cells and glial elements. Axons of de novo formed neurons of the dentate fascia are traced to the CA3 field, which indicates the participation of newly formed neurons in the implementation of hippocampal functions. In the association areas of the neocortex of the adult monkeys, neuronal progenitor cells migrating from the subventricular zone were found. In layer VI of the neocortex of the mouse brain, new pyramidal neurons are detected 2-28 weeks after induced damage and death of native neurons of this layer due to the migration of previously dormant progenitor cells of the subventricular zone. Finally, the reality of postnatal neurogenesis in the human brain is evidenced by a twofold increase in the number of cortical neurons, which continues during the first 6 years after birth.

Of no small importance for practical cell transplantology is the issue of regulation of the processes of reproduction and differentiation of neural stem and progenitor cells. The most important factors suppressing the proliferation of neural progenitor cells are glucocorticoids, which sharply reduce the number of divisions, while removal of the adrenal glands, on the contrary, significantly increases the number of mitoses (Gould, 1996). It is noteworthy that the morphogenesis of the dentate gyrus in rodents is most intense during the first two weeks of postnatal development during the period of absence of reaction to stress against the background of a sharp decrease in the production and secretion of steroid hormones of the adrenal cortex. Corticosteroids inhibit the migration of granule cells - new neurons are not embedded in the granular layer of the dentate gyrus, but remain in the hilus. It is assumed that the processes of formation of synaptic connections are simultaneously disrupted. The protection of cells from such “steroid aggression” is carried out by minimal expression of mineralocorticoid and glucocorticoid receptors on proliferating granule cells not only during the development of the dentate gyrus, but also in mature animals. However, of all the neurons of the brain, it is the neurons of the hippocampus that are characterized by the highest content of glucocorticoid receptors, which causes the stress effect on the hippocampus. Psychoemotional stress and stressful situations inhibit neurogenesis, and chronic stress sharply reduces the ability of animals to acquire new skills and learn. A more pronounced negative effect of chronic stress on neurogenesis is quite understandable if we take into account the predominantly dormant state of neural stem cells. When immobilizing pregnant rats (for rodents - an extremely strong stress factor), it was found that prenatal stress also causes a decrease in the number of cells in the dentate gyrus and significantly inhibits neurogenesis. It is known that glucocorticoids participate in the pathogenesis of depressive states, the morphological equivalent of which is inhibition of neurogenesis, pathological reorganization of neurons and interneuronal connections, and death of nerve cells. On the other hand, antidepressant chemotherapy agents activate the formation of neurons de novo, which confirms the connection between the processes of formation of new neurons in the hippocampus and the development of depression. Estrogens have a significant effect on neurogenesis, the effects of which are opposite to the action of glucocorticosteroids and consist in supporting the proliferation and viability of neural progenitor cells. It should be noted that estrogens significantly increase the learning ability of animals. Some authors associate cyclic changes in the number of granule cells and their excess number in females with the influence of estrogens.

It is known that neurogenesis is controlled by EGF, FGF and BDNF, however, the mechanisms of the effect of external signals on stem cells from mitogens and growth factors have not been sufficiently studied. It has been established that PDGF in vitro maintains the neuronal direction of differentiation of progenitor cells, and ciliary neurotrophic factor (CNTF), like triiodothyronine, stimulates the formation of predominantly glial elements - astrocytes and oligodendrocytes. Pituitary adenylate cyclase-activating protein (PACAP) and vasoactive intestinal peptide (VIP) activate the proliferation of neural progenitor cells, but at the same time inhibit the processes of differentiation of daughter cells. Opioids, especially in the case of their long-term exposure, significantly inhibit neurogenesis. However, opioid receptors have not been identified in stem cells and neural progenitor cells of the dentate gyrus (they are present in differentiating neurons of the embryonic period), which does not allow us to assess the direct effects of opioids.

The needs of practical regenerative-plastic medicine have forced researchers to pay special attention to the study of pluri- and multipotency of stem cells. The implementation of these properties at the level of regional stem cells of an adult organism could in the future ensure the production of the necessary transplant material. It was shown above that epigenetic stimulation of neural stem cells allows obtaining proliferating cells already preformed according to neural phenotypes, which limits their number. In the case of using the totipotent properties of an embryonic stem cell, proliferation until a sufficient number of cells is obtained occurs earlier than neural differentiation, and the multiplied cells are easily converted into a neural phenotype. To obtain neural stem cells, ESCs are isolated from the inner cell mass of the blastocyst and cultured in the obligatory presence of LIF, which preserves their totipotency and the ability to unlimited division. After this, neural differentiation of ESCs is induced using retinoic acid. Transplantation of the resulting neural stem cells into the striatum damaged by quinolin and 6-hydroxydopamine is accompanied by their differentiation into dopaminergic and serotonergic neurons. After injection into the ventricles of the rat embryonic brain, the ESC-derived neural progenitor cells migrate to various regions of the recipient brain, including the cortex, striatum, septum, thalamus, hypothalamus, and cerebellum. The cells remaining in the ventricular cavity form epithelial structures resembling a neural tube, as well as individual islands of non-neural tissue. In the brain parenchyma of the recipient embryo, the transplanted cells produce the three main cell types of the nervous system. Some of them have elongated apical dendrites, pyramidal cell bodies, and basal axons projecting into the corpus callosum. Astrocytes of donor origin extend processes to nearby capillaries, and oligodendrocytes closely contact myelin muffs, participating in the formation of myelin. Thus, neural progenitor cells obtained from ESCs in vitro are capable of directed migration and regional differentiation adequate to microenvironmental signals, providing many areas of the developing brain with neurons and glia.

Some authors consider the possibility of de- and transdifferentiation of regional stem cells of an adult organism. Indirect confirmation of cell dedifferentiation in culture with expansion of their potentials is provided by data on engraftment of mouse neural stem cells in the red bone marrow with subsequent development of cell lines from them, yielding functionally active cells of the peripheral blood. In addition, transplantation of genetically labeled (LacZ) neurosphere cells obtained from the mature or embryonic brain into the brain of irradiated mice with suppressed hematopoiesis led to the formation of not only neural derivatives from stem cells, but also caused the generation of blood cells, which indicates the pluripotency of neural stem cells, realized outside the brain. Thus, a neural stem cell is capable of differentiating into blood cells under the influence of signals from the bone marrow microenvironment with preliminary transformation into a hematopoietic stem cell. On the other hand, when transplanting bone marrow hematopoietic stem cells into the brain, their differentiation under the influence of the brain tissue microenvironment into glial and neural cells was established. Consequently, the differentiation potential of neural and hematopoietic stem cells is not limited by tissue specificity. In other words, factors of the local microenvironment, different from those characteristic of the brain and bone marrow tissues, are able to change the direction of differentiation of these cells. It was shown that neural stem cells introduced into the venous system of irradiated mice create populations of myeloid, lymphoid and immature hematopoietic cells in the spleen and bone marrow. In vitro, the effect of bone marrow morphogenetic proteins (BMPs) on the survival and differentiation of neural stem cells was established, determining, as in the early stages of embryogenesis, their development in the neural or glial directions. In neural stem cell cultures from 16-day-old rat embryos, BMPs induce the formation of neurons and astroglia, whereas in stem cell cultures derived from perinatal brain, only astrocytes are formed. In addition, BMPs suppress the generation of oligodendrocytes, which appear in vitro only with the addition of the BMP antagonist noggin.

Transdifferentiation processes are species-nonspecific: human bone marrow hematopoietic stem cells transplanted into the striatum of mature rats migrate to the white matter of the external capsule, ipsi- and contralateral neocortex, where they form astrocyte-like cellular elements (Azizi et al., 1998). When bone marrow stem cells are allotransplanted into the lateral ventricle of newborn mice, the migration of hematopoietic stem cells can be traced to the structures of the forebrain and cerebellum. In the striatum and molecular layer of the hippocampus, the migrated cells are transformed into astrocytes, and in the olfactory bulb, inner granule cell layer of the cerebellum, and reticular formation of the brainstem, they form neuron-like cells with a positive reaction to neurofilaments. Following intravenous administration of hematopoietic cells to adult mice, GFP-labeled micro- and astrocytes were detected in the neocortex, thalamus, brainstem, and cerebellum.

In addition, bone marrow mesenchymal stem cells, which give rise to all types of connective tissue cells, can also undergo neural transdifferentiation under certain conditions (recall that the embryonic source of mesenchyme is neural crest cells). It has been shown that human and mouse bone marrow stromal cells cultured in vitro in the presence of EGF or BDNF express the marker of neural progenitor cells nestin, and the addition of various combinations of growth factors leads to the formation of cells with markers of glia (GFAP) and neurons (nuclear protein, NeuN). Labeled syngeneic mesenchymal stem cells transplanted into the lateral ventricle of the brain of newborn mice migrate and localize in the forebrain and cerebellum without disrupting the cytoarchitecture of the recipient brain. Bone marrow mesenchymal stem cells differentiate into mature astrocytes in the striatum and molecular layer of the hippocampus, and populate the olfactory bulb, granular layers of the cerebellum, and reticular formation, where they transform into neurons. Human bone marrow mesenchymal stem cells are capable of differentiating into macroglia in vitro and integrating into rat brain structures after transplantation. Direct transplantation of bone marrow mesenchymal stem cells into the hippocampus of adult rats is also accompanied by their migration into the brain parenchyma and neuroglial differentiation.

It is assumed that transplantation of bone marrow stem cells may expand the possibilities of cell therapy of CNS diseases characterized by excessive pathological death of neurons. It should be noted, however, that not all researchers recognize the fact of mutual transformation of neural and hematopoietic stem cells, especially in vivo, which is again due to the lack of a reliable marker for assessing their transdifferentiation and further development.

Stem cell transplantation opens new horizons for cellular gene therapy of hereditary neurological pathology. Genetic modification of neural stem cells involves the insertion of genetic regulatory constructs, the products of which interact with cell cycle proteins in the automatic regulation mode. Transduction of such genes into embryonic progenitor cells is used to multiply neural stem cells. Most genetically modified cell clones behave like stable cell lines, showing no signs of transformation in vivo or in vitro, but have a pronounced ability for contact inhibition of proliferation. When transplanted, the multiplied transfected cells are integrated into the recipient tissue without disrupting the cytoarchitecture and without undergoing tumor transformation. Donor neural stem cells do not deform the integration zone and compete equally for space with the host progenitor cells. However, on the 2nd-3rd day, the intensity of division of transfectant cells decreases sharply, which corresponds to contact inhibition of their proliferation in vitro. Embryos-recipients of neural stem transfectants do not have abnormalities in the development of the central nervous system, all areas of the brain in contact with the transplant develop normally. After transplantation, clones of neural stem cells quickly migrate from the injection zone and often go beyond the corresponding embryonic zones along the rostral tract, adequately integrating with other areas of the brain. The integration of genetically modified clones and transfected cell lines of neural stem cells into the brain of the host organism is characteristic not only of the embryonic period: these cells are implanted into numerous areas of the central nervous system of the fetus, newborn, adult, and even aging recipient organism and demonstrate the ability for adequate integration and differentiation. In particular, after transplantation into the ventricular cavity of the brain, transfected cells migrate without damaging the blood-brain barrier and become integral functional cellular components of the brain tissue. Donor neurons form appropriate synapses and express specific ion channels. With the integrity of the blood-brain barrier preserved, astroglia, a derivative of transfectant neural stem cells, extend processes to cerebral vessels, and donor-derived oligodendrocytes express myelin basic protein and myelinate neuronal processes.

In addition, neural stem cells are transfected for use as cellular vectors. Such vector-genetic constructs provide in vivo stable expression of foreign genes involved in the development of the nervous system, or are used to correct existing genetic defects, since the products of these genes are capable of compensating for various biochemical abnormalities of the central nervous system. High migration activity of transfected stem cells and adequate implantation into the germinal zones of various areas of the developing brain allow us to hope for complete restoration of hereditary deficiency of cellular enzymes. In modeling ataxia-telangiectasia syndrome (mutant mouse lines pg and pcd), Purkinje cells disappear from the cerebellum of experimental animals during the first weeks of postnatal development. It has been shown that the introduction of neural stem cells into the brain of such animals is accompanied by their differentiation into Purkinje cells and granular neurons. In pcd mutants, movement coordination disorders are partially corrected and tremor intensity is reduced. Similar results were obtained by transplanting cloned human neural stem cells into primates in which Purkinje cell degeneration was induced using onconase. After transplantation, donor neural stem cells were found in the granular, molecular, and Purkinje cell layers of the cerebellar parenchyma. Therefore, genetic modification of neural progenitor cells can provide a stable committed modification of the phenotype that is resistant to external influences. This is especially important in pathological processes associated with the development of factors in the recipient that prevent the survival and differentiation of donor cells (e.g., during immune aggression).

Mucopolysaccharidosis type VII in humans is characterized by neurodegeneration and progressive intellectual disability, which is modeled in mice by a deletion mutation in the beta-glucuronidase gene. After transplantation of transfected neural stem cells secreting beta-glucuronidase into the cerebral ventricles of newborn defective recipient mice, the donor cells are found first in the terminal zone and then spread throughout the brain parenchyma, stably correcting the integrity of lysosomes in the brain of mutant mice. In a model of Tay-Sachs disease, retrovirus-transduced neural stem cells, when in utero administered to mouse fetuses and transplanted into newborn mice, provide efficient expression of the beta-subunit of beta-hexosaminidase in recipients with a mutation leading to pathological accumulation of beta2-ganglioside.

Another direction of regenerative medicine is stimulation of the proliferative and differentiating potential of the patient's own neural stem cells. In particular, neural stem cells secrete NT-3 during spinal cord hemisection and brain asphyxia in rats, express NGF and BDNF in the septum and basal ganglia, tyrosine hydroxylases in the striatum, as well as reelin in the cerebellum and myelin basic protein in the brain.

However, the issues of stimulation of neurogenesis are clearly not given enough attention. A few studies suggest that the functional load on the nerve centers responsible for distinguishing odors is reflected in the formation of new neurons. In transgenic mice with a deficit of neuronal adhesion molecules, a decrease in the intensity of neurogenesis and a decrease in the number of neurons migrating to the olfactory bulbs was combined with an impairment of the ability to distinguish odors, although the threshold of odor perception and short-term olfactory memory were not impaired. The functional state of the cells of the dentate gyrus plays an important role in the regulation of neurogenesis: a weakening of the effect of glutamate on granule cells after the destruction of the entorhinal cortex promotes proliferation and differentiation of neurons, and stimulation of the fibers of the perforant pathway (the main afferent input to the hippocampus) causes inhibition of neurogenesis. NMDA receptor antagonists activate the processes of new neuron formation, while agonists, on the contrary, reduce the intensity of neurogenesis, which in effect resembles the action of glucocorticosteroids. Contradictory research results are found in the literature: information on the experimentally proven inhibitory effect of the excitatory neurotransmitter glutamate on neurogenesis is inconsistent with data on the stimulation of the proliferation of progenitor cells and the appearance of new neurons with an increase in seizure activity in the hippocampus of animals with experimental caine and pilocarpine models of epilepsy. At the same time, in the traditional model of epilepsy caused by multiple subthreshold stimulation of a certain area of the brain (kindling) and characterized by less pronounced neuron death, the intensity of neurogenesis increases only in the late phase of kindling, when damage and death of neurons are observed in the hippocampus. It has been shown that in epilepsy, seizure activity stimulates neurogenesis with abnormal localization of new granule neurons, many of which appear not only in the dentate gyrus but also in the hilus. Such neurons are of great importance in the development of mossy fiber sprouting, since their axons form normally absent reverse collaterals that form numerous synapses with neighboring granule cells.

The use of regional neural stem cells opens up new prospects for the application of cell transplantation in the treatment of metabolic and genetic neurodegenerative diseases, demyelinating diseases and post-traumatic disorders of the central nervous system. Before performing replacement cell transplantation according to one of the methods, selection and expansion of the required type of neural progenitor cells ex vivo is carried out with the purpose of their subsequent introduction directly into the damaged area of the brain. The therapeutic effect in this case is due to the replacement of damaged cells or the local release of growth factors and cytokines. This method of regenerative-plastic therapy requires the transplantation of a sufficiently large number of cells with predetermined functional characteristics.

Further studies of the molecular characteristics and regenerative-plastic potential of mature brain stem cells, as well as the ability of regional stem cells of different tissue origin to transdifferentiate, should also be considered appropriate. Today, screening of antigens of bone marrow hematopoietic stem cells has already been carried out, with the determination of a marker combination of cells capable of transdifferentiating into neural stem progenitor cells (CD 133+, 5E12+, CD34-, CD45-, CD24). Cells have been obtained that form neurospheres in vitro and form neurons when transplanted into the brain of newborn immunodeficient mice. Of interest to cellular xenotransplantology are the results of studies on the possibility of cross-transplantation of stem cells in individuals of evolutionarily distant taxa. The results of neural stem cell implantation into the brain tumor area remain without proper interpretation: transplanted cells actively migrate throughout the tumor volume without going beyond its limits, and when cells are introduced into the intact part of the brain, their active migration toward the tumor is observed. The question of the biological significance of such migration remains open.

It should be noted that successful transplantation of neural stem cells, as well as other neural progenitor cells obtained from ESCs, is possible only when using highly purified neural progenitor cells, since undifferentiated embryonic stem cells are inevitably transformed into teratomas and teratocarcinomas when transplanted to an adult immunocompetent recipient. Even a minimal amount of poorly differentiated cells in the donor cell suspension sharply increases the tumorigenicity of the transplant and unacceptably increases the risk of tumor development or the formation of non-neural tissue. Obtaining homogeneous populations of neural progenitor cells is possible when using cells that arise at certain stages of normal embryogenesis as an alternative source of donor tissue. Another approach involves careful elimination of unwanted cell populations by lineage-specific selection. The use of ESCs for neurotransplantation after their insufficient exposure in vitro to growth factors is also dangerous. In this case, a failure of the neural differentiation program with the formation of structures inherent in the neural tube cannot be ruled out.

Today it is quite obvious that neural stem cells exhibit tropism for pathologically altered areas of the central nervous system and have a pronounced regenerative-plastic effect. The microenvironment in the site of nervous tissue cell death models the direction of differentiation of transplanted cells, thus replenishing the deficiency of specific neural elements within the zone of CNS damage. In some neurodegenerative processes, neurogenic signals arise for the recapitulation of neurogenesis, and neural stem cells of the mature brain are able to respond to this instructive information. Numerous experimental data serve as a clear illustration of the therapeutic potential of neural stem cells. Intracisternal administration of a clone of neural stem cells to animals with ligation of the middle cerebral artery (a model of ischemic stroke) helps to reduce the area and volume of the destructively altered area of the brain, especially in the case of transplantation of neural stem cells together with FGF2. Immunocytochemically, migration of donor cells to the ischemic zone with their subsequent integration with intact recipient brain cells is observed. Transplantation of immature cells of the mouse neuroepithelial line MHP36 into the brain of rats with experimental stroke improves sensorimotor function, and the introduction of these cells into the cerebral ventricles enhances cognitive function. Transplantation of neurally preformed hematopoietic cells of human bone marrow to rats eliminates dysfunction of the cerebral cortex caused by ischemic damage. In this case, xenogeneic neural progenitor cells migrate from the injection site to the zone of destructive changes in brain tissue. Intracranial transplantation of homologous bone marrow cells in traumatic damage to the cerebral cortex in rats leads to partial restoration of motor function. Donor cells engraft, proliferate, undergo neural differentiation into neurons and astrocytes and migrate towards the lesion. When injected into the striatum of adult rats with experimental stroke, cloned human neural stem cells replace damaged CNS cells and partially restore impaired brain function.

Human neural stem cells are mainly isolated from the embryonic telencephalon, which develops much later than more caudally located parts of the nerve trunk. The possibility of isolating neural stem cells from the spinal cord of a 43-137-day-old human fetus has been shown, since in the presence of EGF and FGF2 these cells form neurospheres and exhibit multipotency at early passages, differentiating into neurons and astrocytes. However, long-term cultivation of neural progenitor cells (over 1 year) deprives them of multipotency - such cells are capable of differentiating only into astrocytes, i.e., they become unipotent. Regional neural stem cells can be obtained as a result of partial bulbectomy and, after reproduction in culture in the presence of LIF, transplanted to the same patient with neurodegenerative changes in other parts of the central nervous system. In the clinic, replacement cell therapy using neural stem cells was first performed to treat patients with stroke accompanied by damage to the basal ganglia of the brain. As a result of transplantation of donor cells, an improvement in the clinical condition of most patients was noted.

Some authors believe that the ability of neural stem cells to engraft, migrate and integrate into various areas of nervous tissue in case of CNS damage opens up unlimited possibilities for cell therapy of not only local, but also extensive (stroke or asphyxia), multifocal (multiple sclerosis) and even global (most inherited metabolic disorders or neurodegenerative dementias) pathological processes. Indeed, when cloned mouse and human neural stem cells are transplanted into recipient animals (mice and primates, respectively) with degeneration of dopaminergic neurons in the mesostriatal system induced by the introduction of methyl-phenyl-tetrapyridine (model of Parkinson's disease) 8 months before transplantation, donor neural stem cells integrate into the recipient's CNS. A month later, the transplanted cells are localized bilaterally along the midbrain. Some of the resulting neurons of donor origin express tyrosine hydrolase in the absence of signs of an immune reaction to the transplant. In rats administered 6-hydroxydopamine (another experimental model of Parkinson's disease), the adaptation of transplanted cells to the microenvironment in the host brain was determined by the conditions of culturing neural stem cells before their transplantation. Neural stem cells, rapidly proliferating in vitro under the influence of EGF, compensated for the deficiency of dopaminergic neurons in the damaged striatum more effectively than cells from 28-day cultures. The authors believe that this is due to the loss of the ability to perceive the corresponding differentiation signals during the process of cell division of neural progenitor cells in vitro.

In some studies, attempts were made to increase the effectiveness of the impact on the processes of reinnervation of the damaged striatum by transplanting embryonic striatum cells into this area as a source of neurotrophic factors with simultaneous transplantation of dopaminergic neurons of the ventral mesencephalon. As it turned out, the effectiveness of neurotransplantation largely depends on the method of introducing embryonic nervous tissue. As a result of studies on the transplantation of embryonic nervous tissue preparations into the ventricular system of the brain (in order to avoid injury to the striatum parenchyma), information was obtained on their positive effect on the motor defect in Parkinsonism.

However, in other studies, experimental observations have shown that transplantation of embryonic nervous tissue preparations of the ventral mesencephalon containing dopaminergic neurons into the cerebral ventricle, as well as transplantation of GABA-ergic embryonic neural elements into the striatum of rats with hemiparkinsonism, does not promote restoration of the impaired functions of the dopaminergic system. On the contrary, immunocytochemical analysis confirmed the data on the low survival rate of dopaminergic neurons of the ventral mesencephalon transplanted into the striatum of rats. The therapeutic effect of intraventricular transplantation of embryonic nervous tissue of the ventral mesencephalon was realized only under the condition of simultaneous implantation of a preparation of embryonic striatal cells into the denervated striatum. The authors believe that the mechanism of this effect is associated with the positive trophic effect of GABA-ergic elements of the embryonic striatum on the specific dopaminergic activity of intraventricular ventral mesencephalon transplants. A pronounced glial reaction in the transplants was accompanied by a slight regression of the apomorphine test parameters. The latter, in turn, correlated with the GFAP content in the blood serum, which directly indicated a violation of the permeability of the blood-brain barrier. Based on these data, the authors concluded that the GFAP level in the blood serum can be used as an adequate criterion for assessing the functional state of the transplant, and increased permeability of the blood-brain barrier for neurospecific antigens such as GFAP is a pathogenetic link in the development of transplant failure due to autoimmune damage to the recipient's nervous tissue.

From the point of view of other researchers, the engraftment and integration of neural stem cells after transplantation are stable and lifelong, since donor cells are found in recipients for at least two years after transplantation and without a significant decrease in their number. Attempts to explain this by the fact that in an undifferentiated state neural stem cells do not express MHC class I and II molecules at a level sufficient to induce an immune rejection reaction can be considered true only in relation to low-differentiated neural precursors. However, not all neural stem cells in the recipient's brain are preserved in an immature dormant state. Most of them undergo differentiation, during which MHC molecules are expressed in full.

In particular, the insufficient efficiency of using intrastriatal transplantation of embryonic ventral mesencephalon preparations containing dopaminergic neurons for the treatment of experimental parkinsonism is associated with the low survival rate of transplanted dopaminergic neurons (only 5-20%), which is caused by reactive gliosis accompanying local trauma of the brain parenchyma during transplantation. It is known that local trauma of the brain parenchyma and concomitant gliosis lead to disruption of the integrity of the blood-brain barrier with the release of antigens of nervous tissue, in particular, OCAR and neuron-specific antigen, into the peripheral blood. The presence of these antigens in the blood can cause the production of specific cytotoxic antibodies to them and the development of autoimmune aggression.

V. Tsymbalyuk and co-authors (2001) report that the traditional point of view still holds, according to which the central nervous system is an immunologically privileged zone isolated from the immune system by the blood-brain barrier. In their review of the literature, the authors cite a number of works indicating that this point of view does not fully correspond to the essence of immune processes in the mammalian brain. It has been established that labeled substances introduced into the brain parenchyma can reach deep cervical lymph nodes, and after intracerebral injection of antigens, specific antibodies are formed in the body. Cells of the cervical lymph nodes respond to such antigens by proliferation, beginning on the 5th day after the injection. The formation of specific antibodies has also been revealed during skin transplantation into the brain parenchyma. The authors of the review provide several hypothetical pathways for antigen transport from the brain to the lymphatic system. One of them is the transition of antigens from the perivascular spaces to the subarachnoid space. It is assumed that the perivascular spaces localized along the large vessels of the brain are the equivalent of the lymphatic system in the brain. The second path lies along the white fibers - through the ethmoid bone into the lymphatic vessels of the nasal mucosa. In addition, there is an extensive network of lymphatic vessels in the dura mater. The impermeability of the blood-brain barrier for lymphocytes is also quite relative. It has been proven that activated lymphocytes are capable of producing enzymes that affect the permeability of the structures of the brain "immune filter". At the level of postcapillary venules, activated T-helpers penetrate the intact blood-brain barrier. The thesis about the absence of cells in the brain that represent antigens does not stand up to criticism. At present, the possibility of representing antigens in the CNS by at least three types of cells has been convincingly proven. Firstly, these are bone marrow-derived dendritic cells that are localized in the brain along large blood vessels and in the white matter. Secondly, antigens are capable of presenting endothelial cells of brain blood vessels, and in association with MHC antigens, which supports the clonal growth of T cells specific to these antigens. Thirdly, micro- and astroglia cells act as antigen-presenting agents. Participating in the formation of the immune response in the central nervous system, astrocytes acquire the properties of an immune effector cell and express a number of antigens, cytokines and immunomodulators. When incubated with y-interferon (y-INF), astroglial cells in vitro express MHC class I and II antigens, and stimulated astrocytes are capable of antigen presentation and maintenance of clonal proliferation of lymphocytes.

Brain tissue trauma, postoperative inflammation, edema and fibrin deposits accompanying embryonic nerve tissue transplantation create conditions for increased permeability of the blood-brain barrier with impaired autotolerance, sensitization and activation of CD3+CD4+ lymphocytes. Presentation of auto- and alloantigens is performed by astrocytes and microglial cells that respond to y-INF by expressing MHC molecules, ICAM-1, LFA-I, LFA-3, costimulatory molecules B7-1 (CD80) and B7-2 (CD86), as well as secretion of IL-la, IL-ip and y-INF.

Consequently, the fact of longer survival of embryonic nervous tissue after intracerebral transplantation than after its peripheral administration can hardly be associated with the absence of initiation of transplant immunity. Moreover, monocytes, activated lymphocytes (cytotoxic CD3+CD8+ and T-helper cells) and the cytokines they produce, as well as antibodies to antigens of the peripheral transplant of embryonic nervous tissue play a major role in the process of its rejection. A low level of expression of MHC molecules in embryonic nervous tissue is of certain importance in creating conditions for longer resistance of neurotransplants to T-cell immune processes. This is why in the experiment, immune inflammation after transplantation of embryonic nervous tissue into the brain develops more slowly than after skin grafting. Nevertheless, complete destruction of individual transplants of nervous tissue is observed after 6 months. In this case, T-lymphocytes restricted by MHC class II antigens are predominantly localized in the transplantation zone (Nicholas et al., 1988). It has been experimentally established that during xenological neurotransplantation, depletion of T-helpers (L3T4+), but not cytotoxic T-lymphocytes (Lyt-2), prolongs the survival of rat nervous tissue in the brain of recipient mice. Rejection of the neurotransplant is accompanied by its infiltration by host macrophages and T-lymphocytes. Consequently, host macrophages and activated microglial cells act in situ as antigen-presenting immunostimulating cells, and increased expression of donor MHC class I antigens enhances the killer activity of recipient cytotoxic T-lymphocytes.

There is no point in analyzing the numerous speculative attempts to explain the fact of neurotransplant rejection by the reaction of the recipient's immune system to the donor's endothelial cells or glial elements, since even pure lines of neural progenitor cells are subject to immune attack. It is noteworthy that the expression of Fas ligands by brain cells that bind apoptosis receptors (Fas molecules) on T lymphocytes infiltrating the brain and induce their apoptosis plays an important role in the mechanisms of longer transplant survival within the CNS, which is a typical protective mechanism of trans-barrier autoimmunogenic tissues.

As V. Tsymbalyuk and co-authors (2001) rightly note, transplantation of embryonic nervous tissue is characterized by the development of inflammation with the participation of cells sensitized to brain antigens and activated cells, antibodies, and also as a result of local production of cytokines. An important role in this is played by the pre-existing sensitization of the body to brain antigens, which occurs during the development of CNS diseases and can be directed at transplant antigens. This is why the really long-term survival of histoincompatible neurotransplants is achieved only by suppressing the immune system with cyclosporine A or by introducing monoclonal antibodies to the recipient's CD4+ lymphocytes.

Thus, many problems of neurotransplantation remain unresolved, including those related to the immunological compatibility of tissues, which can only be resolved after targeted fundamental and clinical research.