Transplantation of nerve cells for treatment of neurodegenerative states

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed group of inventions relates to medicine, namely to neurosurgery and biotechnology, and can be used for cultivation of nerve stem cells of mammal, excluding embryonic human cells, and for treatment of spasticity, rigidity, spinal cord or amniotrophic state in subject, requiring such treatment. For cultivation of said cells preliminarily incubated in culture vessel is solution, which contains poly-D-lysine in concentration from 0.1 mkg/ml to 1 mg/ml during from 5 minutes to 3 hours. After that, culture vessel is rinsed and dried. Nerve stem cells are inoculated into said culture vessel without serum. After that, solution of fibronectin and, at least, one mitogen is added into culture vessel and nerve stem cells are cultivated without serum. Then, cultivated nerve stem cells are passed until fusion. For treatment of said states nerve stem cells, cultivated be claimed method, are concentrated. Therapeutically efficient amount of said concentrated nerve stem cells is introduced into region of patient's central nervous system tissue with decreased level of GABA-producing or glycin-producing neurons.

EFFECT: group of inventions ensures efficient method of cultivating mammalian nerve stem cells, excluding embryonic human cells, which makes it possible to achieve in vitro higher degree of neuronal differentiation to the level sufficient for treatment of said states and improvement of survival in vivo of such cells, as well as efficient treatment of spasticity, rigidity or amniotrophic state in subject due to introduction into their central nervous system tissues with lower level of GABA-producing or glycin-producing neurons of cells, able to differentiate into sufficient number of GABA-producing or glycin-producing neurons in said tissue.

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The methods described are methods of treatment of disease through transplantation of cells, which are the only favorable for the indicated treatments. In particular, the methods described are methods of treating neurodegenerative conditions using neural stem cells (NSC).

Neurodegenerative disorders are characterized by States with neuronal damage in the result of disease, hereditary conditions or injuries, such as traumatic or ischemic injury to the spinal cord or brain.

Spinal circuit that controls the contraction of skeletal muscles of the limbs, includes excitatory motor neurons and inhibiting GABA-ergicheskie (i.e. producing GABA) and glycinergic (i.e. producing glycine) inserted neurons. Motor neuron is a nerve that originates from the anterior horn of the gray matter of the spinal cord. The axon of the motor neuron comes from a segment of the spinal cord as efferent motor fibers, which innervates muscle fibers. The impulses that holds the motor neuron stimulates contraction of the muscle fibers. GABA, gamma-aminobutyric acid, is a natural metabolite of the nervous system of mammals, of which the first acts as a neurotransmitter, inhibiting or decreasing the conduction of nerve electrical potential. Loss of GABA-eliteskin inserted neurons leads to dysregulation of inhibitory tone caused by motor neuron muscle contractions. In the absence of control by any abscopal inserted neurons against excitatory neurons, there is the excitement of excitatory neurons, leading to uncontrolled spastic reduction or uncontrolled rigidity of the muscles of the limbs. The loss of motor neurons leads to flaccid paraplegia, in which the subjects are not able to contract the muscles and as a result is not able to move.

One of the cases in which the GABA-ergicheskie inserted neurons of the spinal cord damage is a complication associated with transient cross-clamping of the descending thoracic or thoracoabdominal aorta. The specified clamping is a necessary step in surgical interventions on vessels to correct an aneurysm of the thoracic or thoracoabdominal aorta. During the compression part of the spinal cord deprived of its blood supply and may become ischemic. Depending on the duration of ischemia and subsequent neurodegenerative dysfunction can be expressed neurodegenerative, as prepares or as fully developed spastic or flaccid couples who plegia.

While the mechanism leading to induced ischemia, degeneration of neurons, known only partially and may include excessive release/activity of excitatory amino acids, prostaglandins and/or oxygen free radicals, the population of neurons of the spinal cord, which affects transient ischemic stroke, is well known. For example, histopathological analysis of the spinal cord, taken from animals with fully developed spastic paraplegia, shows a selective loss of small inhibitory neurons; however, alpha-motoneurons persist in previously ischemic spinal segments. Described is similar to the pathology of spinal cord neurons in people with ischemic damage to the spinal cord.

In contrast, in animals with flaccid paraplegia observed pankraticheskoi neurodegenerative changes, affecting both small and inhibiting excitatory inserted neurons and ventral motor neurons. During the period of neuronal degeneration after spinal ischemia may also occur caused by damage to local activation of microglia and inflammatory changes, such as infiltration by macrophages, as with focal or global cerebral ischemia. Depending on the prevalence of corruption inflammatory changes usually peak between the at two and seven days after ischemic stroke and then there is a gradual disappearance of the elements of inflammation within two to four weeks postischemic period.

In the last two or three decades, considerable effort has been made to experimental animals to evaluate therapeutic potential of transplanted into the spinal cord of a number of materials. Thus, cell lines or badly selected tissue of the spinal cord of the fetus was delivered in damaged areas, and also used a direct spinal gene therapy to facilitate neurodegenerative dysfunction in multiple models of spinal cord injury, including traumatic mechanical damage, chemical damage to the spinal cord, or genetically modified animals with progressive α-motoneuronal degeneration (ALS-transgenic mice or rats).

In General, studies demonstrate long-term survival and preservation of neuronal phenotypes in grafts derived from fetal tissue, but not from precursor cells of neurons, which were multiplied in vitro. Actually been demonstrated only limited neuronal differentiation and maturation of progenitor cells, neurons razmnozhavshikhsya in vitro and transplanted in mechanically or chemically damaged spinal cord. Cells were differentiated mainly in cell types not related to neurons. While the mechanism specified, mainly non-neuronal, differentiation is not well understood, there is a hypothesis that is probably the local release of proinflammatory cytokines (such as TNFα and TGFβ) at the site of previously existing damage.

The neurodegeneration is a particularly problematic biological environment for cell therapy and signals cell death that occurs during the steady-state neurodegenerative disease (Rothstein et al., 1992; Howland et al., 2002; Turner et al., 2005), may not be compatible with survival of the graft. In addition, the spinal cord of an adult organism is considered as lost cells and/or signals, providing regeneration (Park et al., 2002), and most studies of NSC transplantation showed weak or limited differentiation (Cao et al., 2002; Yan et al., 2004).

One of the main problems of cell therapy is the low survival rate of transplanted cells (less than 5%). All the transplanted cells now dying in large quantities soon after injection in vivo. So, with the purpose of delivering effective doses of the end cells, the dose should be injected at least 20 times. This, in turn, requires a much larger scale cells, which creates additional regulatory and economic the ski obstacles. In addition, the survival of these cells in vivo, it was impossible to save. Failure to demonstrate reproducible introduction of effective doses of cell therapy prevents obtaining permission to apply that treatment from the government and other regulatory bodies, such as the Management under the control over products and medicines of the USA.

Additional problems arise in the treatment of neurodegenerative diseases and conditions, which capture a large area of the body, tissue or organ, such as the nervous system in General, and not a single localized area. For example, in the case of the ALS neurodegeneration includes a slow death of motor neurons throughout the spinal cord, and neurons of the motor cortex. Similarly, when the majority of lysosomal diseases neuronal destruction captures most of the areas of the brain and spinal cord. Alzheimer's captures a large part of the brain. Even if more localized neurodegenerative diseases such as Parkinson's disease and Huntington's disease, the affected area striped body is quite large, much larger than the area of transplantation, which can be achieved surgically. Thus, the stem cell therapy for neurodegenerative diseases, the AK is expected, would require the implementation of more extensive procedures transplantation.

Thus, there is a need for improved methods of treatment of neurodegenerative conditions. There is a need also to improved methods of cultivation and transplantation of neural stem cells, excluding embryonic human cells, and undifferentiated precursor cells, neurons after transplantation will overcome all previously observed limitations and to bring a functional use. Thus, this method of treatment of neurodegenerative conditions, in vivo, provides a reliable differentiation of nerve cells, prolonged survival of neurons at various degenerative conditions and maturation in therapeutically important subpopulations of neurons in the tissues of an adult organism, which do not have incentives for development, and creates a wide therapeutic interval compared with the localization of the cells.

The INVENTION

The described methods include methods of treating neurodegenerative conditions. In particular, the described methods include transplantation to a subject who needs it, NSC, undifferentiated precursor cells neurons or progenitor cells neurons, which were multiplied in vitro in such a way that cells can improve neurodegeneration the condition. In one embodiment, the present invention describes methods include the identification, selection, reproduction and preparation of donor cells for use as a treatment for neurodegenerative condition. Donor cells intended for transplantation, can be subjected to selection for compliance with the elements or no elements that contribute to the condition, its symptoms and/or effects.

Cells of the described methods include cells, after transplantation generate neurons in sufficient quantity in order to integrate into the neural infrastructure for improving the painful status or condition. In one embodiment, the present invention describes methods include the treatment of neurodegenerative diseases or conditions through transplantation multipotent undifferentiated precursor cells, neurons or neural stem cells that were isolated from the Central nervous system of a mammal and were multiplied in vitro. For example, transplantation multiplied neural stem cells can be used to improve the function of distance from the subject suffering from various forms of myelopathy with symptoms of spasticity, rigidity, seizures, paralysis or any other hyperactive the STI muscles.

The method of treatment may include the delivery of a damaged nerve region, through transplantation, the appropriate number of NSC, which can differentiate into a sufficient number of GABA-producing neurons and/or glycine-producing neurons, to reduce defective neural circuits, including hyperactive neuronal circuits.

In one embodiment, the present invention describes methods involve the restoration of motor function in the disease of motor neurons. Appropriate amount or therapeutically effective amount NSC or undifferentiated precursor cells of neurons that are able to differentiate into motor neurons, can be delivered in at least one area of neurodegeneration, such as degenerative spinal cord for motor function recovery. NSC exert their therapeutic effect by replacing the degenerated nerve-muscle synapses.

In combination or the alternative, NSC exert their therapeutic effect through the expression and release of trophic molecules that protect neurons degenerating tissue so that a greater number of them survive for a longer period of time. Received from NSC neurons can be sent in ventral roots, so in order to envirovet muscles, and in this case, NSC participate in the extensive reciprocal connections with motor neurons of the owner in subjects with degenerative disease of motor neurons. Thus, in one embodiment, the present invention NSC from fetal spinal cord can be transplanted into the lumbar spinal cord, where these cells can undergo differentiation into neurons, which form synaptic contacts with neurons of the owner and expressyour and release growth factors motor neurons.

In one embodiment, the present invention describes methods include obtaining neural stem cells or undifferentiated precursor cells neurons that integrate into the host tissue and deliver one or more growth factors for neurons of the host, protecting them, therefore, from the degenerative influences present in the tissue. The methods include the introduction of a sufficient number NSC or undifferentiated precursor cells neurons in the spinal-cord region so that NSC secretively effective amount of at least one growth factor.

In one embodiment, the present invention describes methods include a method of applying experimental animals as m is Delhi for pre-clinical evaluation of stem cells to replace cells in neurodegenerative conditions.

In one embodiment, the present invention describes methods include increasing the efficiency of differentiation of transplanted NSC in neurons. The method includes the reproduction of highly enriched NSC or undifferentiated precursor cells of neurons in their undifferentiated state, so that after transplantation of a sufficient amount, such as 20%, of the cells in the transplant later turned into neurons.

In one embodiment, the present invention describes methods include increasing the number of differentiated cells without increasing the number NSC or undifferentiated precursor cells of neurons destined for transplantation. In one embodiment of the present invention the method includes obtaining a reproduced donor population so that after transplantation of NSC or undifferentiated precursor cells neurons continued to share in vivo up to ten times without forming tumors and effectively increasing the total number of delivered cells.

Cells of the described methods can be selected or obtained from fetal, neonatal, juvenile, adult, or post-mortem tissue of a mammal, non-human. Cells described methods can plot the t or get of the Central nervous system, blood or any other suitable source of stem cells that differentiate into neurons. Cells can also be obtained from embryonic stem cells. For example, in one embodiment of the present invention cells include neuroepithelial cells isolated from the developing fetal spinal cord. In some cases, the precursor cells of neurons may represent an undifferentiated precursor cells, neurons isolated from specific cobblestay the Central nervous system.

According to the described methods, neural stem cells propagated in culture. In one embodiment of the present invention, the precursor cells of neurons can represent multipotent NSC, are able to multiply in culture and after differentiation to generate both neurons and stroma.

At the time of transplantation of the cells may be undifferentiated, prodifferentiating or fully differentiated. In one embodiment of the present invention, the cells are induced to differentiate into neuronal lineage. Cells described methods can undergo neuronal differentiation in situ in the presence of proinflammatory cytokines and other environmental factors existing in damages the Noah fabric.

Using these methods, it is possible to treat nerve chain by transplantation or injection of cells into the appropriate areas to facilitate disease, disorder or condition. Usually occurs transplantation in the nervous tissue or in tissue, non-nervous tissues, which contribute to the survival of the transplanted cells. The NSC grafts used in the described methods, survive well in neurodegenerative environment, where NSC can have a powerful clinical effects in the form of a delay in the onset and progression of neurodegenerative conditions or diseases.

In some cases it may occur transplantation in remote areas of the body, and cells can migrate in the place designed for them. Accordingly, the described methods can also include partial transplantation of NSC. Used herein, the term "partial transplantation" may refer to implantation multiplied NSC only in part, or in less than the entire area of neurodegeneration. For example, partial transplantation of NSC in the lumbar segments of the spinal cord. At least part of the effect of NSC on degenerating motor neurons involves the delivery of neurotrophic agents and trophic cytokines to degenerating motor neurons owner is via the classical cellular mechanisms. With this purpose, the NSC, partially transplanted in the lumbar segments of the spinal cord using the above methods, as was shown in transgenic animals, which served as a model disease of motor neurons survive, undergo extensive neuronal differentiation, promote the survival and functioning of motor neurons in the immediate field of transplantation, as well as in areas distant from the field of transplantation.

Accordingly, the described methods relate to a method of treating spasticity, rigidity, or States of hyperactivity of the muscles. The method includes allocating at least one neural stem cell in a mammal, and reproduction in vitro neural stem cell to the state of replicated populations. The method also includes the concentration multiplied populations and the introduction of a therapeutically effective amount multiplied the population in at least one region of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in the spinal cord of the recipient.

In one embodiment of the present invention the condition is caused by traumatic spinal cord injury, ischemic spinal cord injury, traumatic brain injury, stroke, multiple, skle the oz, cerebral palsy, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, chronic ischemia, hereditary conditions, or any combination thereof.

In one embodiment, the present invention neural stem cells isolated from a source selected from the group consisting of Central nervous system, peripheral nervous system, bone marrow, peripheral blood, umbilical cord blood and at least one embryo.

In one embodiment, the present invention gestational age of the developing mammal is from about 6.5 to about 20 weeks.

In one embodiment, the present invention neural stem cells isolated from fetal spinal cord of a mammal, not a person.

In one embodiment, the present invention propagation neural stem cells comprises culturing neural stem cells in the absence of serum.

In one embodiment, the present invention propagation neural stem cells include neural stem cells, at least one growth factor.

In one embodiment of the present invention, the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combined the Nations.

In one embodiment, the present invention is a therapeutically effective amount multiplied the population is able to generate at least 1000 GABA-producing neurons in vivo.

In one embodiment, the present invention is a therapeutically effective amount multiplied the population is able to generate at least 1000 glycine-producing neurons in vivo.

In one embodiment of the present invention at least 40% of the population multiplied capable of generating neurons in the spinal cord.

In one embodiment of the present invention the introduction of a therapeutically effective amount multiplied population includes injecting at least part of a therapeutically effective quantity in many areas of the spinal marrow of the recipient.

In one embodiment, the present invention is at least 30% multiplied populations are able to differentiate into neurons in vitro.

In another embodiment, the present invention derived neural stem cell. Neural stem cell is able to treat spasticity, rigidity, or state of hyperactivity of the muscles. Neural stem cells secrete from the mammal and propagated in vitro to the state of replicated populations. Multiplied the population, enabling the Yu stem cells, concentrate and implement a therapeutically effective amount multiplied the population in at least one region of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in the spinal cord of the recipient.

In another embodiment, the above methods is described a method for the treatment of chronic pain. The method includes allocating at least one neural stem cell from the mammal, and reproduction in vitro neural stem cell to the state of replicated populations. The method also includes the concentration multiplied populations and the introduction of a therapeutically effective amount multiplied the population in at least one region of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in the spinal cord of the recipient.

In one embodiment, the present invention chronic pain caused by traumatic spinal cord injury, ischemic spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, chronic ischemia, hereditary conditions, or any combination thereof.

In one embodiment, the present izaberete the Oia therapeutically effective amount multiplied the population is able to generate at least 1000 GABA-producing neurons in vivo.

In one embodiment, the present invention is a therapeutically effective amount multiplied the population is able to generate at least 1000 glycine-producing neurons in vivo.

In one embodiment of the present invention at least 40% of the population multiplied capable of generating neurons in the spinal cord.

In one embodiment of the present invention the introduction of a therapeutically effective amount multiplied population includes injecting at least part of a therapeutically effective quantity in many areas of the spinal marrow of the recipient.

In one embodiment of the present invention the area include the dorsal horn.

In one embodiment of the present invention the area include the intrathecal space.

In yet another embodiment of the present invention derived neural stem cell. Neural stem cell is able to treat chronic pain. Neural stem cells secrete from the mammal and propagated in vitro to the state of replicated populations. Multiplied the population, including stem cells, concentrate, and introducing a therapeutically effective amount multiplied the population in at least one region of the spinal marrow of the recipient. For men is our least 20% multiplied population capable of generating neurons in the spinal cord of the recipient.

In another embodiment, the described methods are described method of treating the degeneration of motor neurons. The method includes allocating at least one neural stem cell from the mammal, and reproduction in vitro neural stem cell to the state of replicated populations. The method also includes the concentration multiplied populations and the introduction of a therapeutically effective amount multiplied the population in at least one region of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in the spinal cord of the recipient.

In one embodiment, the present invention degeneration of motor neurons caused by traumatic spinal cord injury, ischemic spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, chronic ischemia, hereditary conditions, or any combination thereof.

In one embodiment of the present invention the method includes the allocation of neural stem cells from the area that contains many cells of at least one neuronal subtype, in which neuronal subtype produces a growth factor, effektivnye improve motor deficits.

In one embodiment, the present invention multiplied population includes the number of neural stem cells that can differentiate into neurons, is sufficient for secretion therapeutically effective amount of at least one growth factor.

In one embodiment of the present invention the method includes the allocation of neural stem cells from the area that contains many motor neurons.

In yet another embodiment of the present invention derived neural stem cell, capable of treating syringomyelia. Neural stem cells secrete from the mammal and propagated in vitro to the state of replicated populations. Multiplied the population, including stem cells, concentrate, and introducing a therapeutically effective amount multiplied the population in at least one region of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in the spinal cord of the recipient.

In another embodiment, the described methods are described method of treating those of syringomyelia. The method includes allocating at least one neural stem cell from the mammal, and reproduction in vitro neural stem cell to the state of replicated populations. The method also includes the concentration of p is snounou population and the introduction of a therapeutically effective amount multiplied populations in pathological cavity of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in pathological cavity of the spinal marrow of the recipient.

In one embodiment of the present invention, syringomyelia caused by traumatic spinal cord injury, ischemic spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, chronic ischemia, hereditary conditions, or any combination thereof.

In one embodiment of the present invention the method includes the allocation of neural stem cells from the area that contains many cells of at least one neuronal subtype, in which neuronal subtype produces growth factor effective to facilitate those of syringomyelia.

In one embodiment of the present invention the method includes the allocation of neural stem cells from the area that contains many motor neurons.

In one embodiment, the present invention multiplied population includes the number of neural stem cells that can differentiate into neurons, is sufficient for secretion therapeutically effective amount of at least one growth factor.

One in which the version of the implementation of the present invention is a therapeutically effective amount multiplied the population is able to generate at least 1000 neurons.

In one embodiment of the present invention at least 100,000 neural stem cells propagated populations introduced in pathological cavity of the spinal marrow of the recipient.

In yet another embodiment of the present invention derived neural stem cell, capable of treating syringomyelia. Neural stem cells secrete from the mammal and propagated in vitro to the state of replicated populations. Multiplied the population, including stem cells, concentrate, and introducing a therapeutically effective amount multiplied populations in pathological cavity of the spinal marrow of the recipient. At least 20% multiplied population capable of generating neurons in pathological cavity of the spinal marrow of the recipient.

In an additional embodiment, the above methods is described a method of propagation in vitro of at least one neural stem cell to the state of replicated populations of neural stem cells. Each propagation neural stem cells exceeds thirty cell doublings without differentiation. The method includes the separation of neural stem cells from the tissue of the Central nervous system and the introduction of at least one extracellular protein in the culture vessel. Extracellular protein comprises at least about 10 MK is/ml poly-D-lysine and approximately 1 mg/ml fibronectin. The method also includes the cultivation of dissociated neural stem cells in the culture vessel in the absence of serum and added to the culture vessel at least one growth factor. The growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof. The method also includes the passage of cultured cells to merge.

In one embodiment, the present invention multiplied neural stem cells are able to differentiate into neurons.

In one embodiment, the present invention propagation neural stem cells involves the addition of fibronectin in the culture medium as a soluble factor.

In one embodiment of the present invention, the dissociation of the cells, transfer cells include enzymatic dissociation.

In one embodiment of the present invention the enzymatic dissociation involves the impact on the cells with trypsin.

In one embodiment, the present invention is a therapeutically effective amount multiplied the population of introducing at least one region of the nervous system of the recipient for treatment of neurodegenerative conditions.

Thus, the advantage of these methods over the existing farmacologiche the Kimi strategies is the creation of a method to facilitate the ability of transplanted NSC to secrete trophic molecules, which can be delivered to the degenerating motor neurons in conditions of optimal bioavailability.

Another advantage of the present invention is a method of cultivation and breeding NSC from fetal spinal cord of a mammal to facilitate the successful engraftment of transplanted NSC in the lumbar spinal cord.

Another advantage of the described methods includes a method of achieving a higher proportion of neuronal differentiation of NSC population.

Another advantage of the above methods includes the achievement of clinical effects from partial transplantation of NSC.

Other characteristics and advantages of the described methods are described and will be apparent from the following detailed description and drawings.

BRIEF DESCRIPTION of DRAWINGS

FIGURE 1. Reproduction spinal stem cells. The spinal line undifferentiated progenitor cells (also known as NSC) was isolated from the tissue of the spinal cord 7-8 days post mortem (dead) embryos and produced serial passage for approximately 130 days total culture period. At each passage, the number of collected cells was divided by the initial number of cells at seeding to obtain fold increase in the number of cells. Cumulative fold increase (left axis ) was obtained by multiplying fold increase at each passage. The doubling time (right Y-axis) the number of cells at each passage was calculated by dividing fold increase in the number of cells in each culture period (X axis). This process was repeated three times (serial multiplication 1, 2 and 3).

FIGURE 2. Morphology multiplied spinal stem cells. (A) Phase-contrast photograph fixed, unpainted, breeding culture, the 20x lens. (C) Staining interestincome antibodies.

FIGURE 3. Characterization of differentiated cultures obtained from the reproduced spinal stem cells. Growing cells of passage 15-16 differentiated approximately 14 days in culture were fixed and stained using various specific for neuronal antibodies. (A) Tau and MAR; (C) tubulin beta type 3; (C) GABA; (D) acetylcholinesterase.

FIGURE 4. The proliferation of stem cells of the mid-brain. Line undifferentiated precursor cells of the mid-brain (also known as NSC) was isolated from the tissue of the mid-brain 7-8-day of the dead (post mortem) embryos and produced serial passage for about 170 days total culture period. At each passage, the number of collected cells was divided by the initial number of cells at seeding to obtain fold increase in the number of cells. The cumulative crtn the increase (Y-axis) was obtained by multiplying fold increase at each passage.

FIGURE 5. The capture of dopamine propagated by stem cells of the mid-brain. Active transport of dopamine (DAT) in living cells was determined in line stem cells of the mid-brain and one of its clonal compared, which were differentiated for 22 or 44 days during the study. Cells were incubated with radioactively labeled dopamine in the presence (+) or absence (-) of the DAT inhibitor of nomifensine (10 μm). Cells were washed to remove unincorporated dopamine and literally obtaining scintillation cocktail. Then determined the total radioactivity of cells pulse/min (dpm) using a scintillation counter.

6. The influence of exogenous factors on the induction of neuronal differentiation and dopaminergic differentiation of the stem cell line of the mid-brain. Stored in a frozen state neural stem cells from two lines of stem cells of the mid-brain (527RMB and 796RMB) were thawed and seeded with a density of 40 000 cells per well in 4-chamber slides in the presence of bFGF and allowed to proliferate for 6 days. Then bFGF was removed and the cells were allowed to differentiate for an additional 8 days. The cells were divided into four groups depending on the timing and duration of exposure in air-conditioned environment for Sertoli cells (SCCM, at a dilution of 1:1, N2). Od is in the group was subjected to SCCM during proliferation and differentiation (condition 1); the second was subjected to only during proliferation (condition 2); the third was subjected to only during differentiation (condition 3); and the fourth was not subjected to SCCM (control). Medium was changed every other day, and mitogen was added daily during the proliferative phase. For each condition had four holes for painting on many markers. After differentiation, the cells were fixed using 4% paraformaldehyde and immunologically stained using antibodies to 2b (FIGA) and tyrosine hydroxylase (6), and GFAP and GalC. Immunologically stained cells was counted by using a 40x lens and in each well was counted at least three fields. Revealed little or no identified cells GFAP+ or GalC+ in the analysis of cells in each condition, so the antigens were excluded from the analysis.

7. Reducing spasticity/rigidity and motor deficits in rats after transplantation of spinal stem cells. Rats with spastic manifestations were obtained using ischemic injury of lumbar spinal cord. In one group (black circle) rats (n=9) transplanted spinal stem cells propagated in culture (passage 16), while the other control group (white circle, n=7) received only cf the water without cells. Immunosuppressant, FK506, was introduced in quantities of 1 mg/kg daily for both groups during the entire duration of the study (8 weeks). Motor coordination of individual animals was assessed using the BBB scale once a week.

FIG. Reducing spasticity/rigidity and motor deficits in rats after transplantation of spinal stem cells. Rats with spastic manifestations were obtained using ischemic injury of lumbar spinal cord. In one group (black circle and a black square) rats (n=13) transplanted spinal stem cells propagated in culture (passage 16), while the other control group (filled triangle, n=6) received only medium without cells. Immunosuppressant, FK506, was injected in an amount of 3 mg/kg daily for both groups during the entire duration of the study (12 weeks). Motor coordination of individual animals was assessed using the BBB scale once a week.

FIG.9. The influence of treatment NSC on the severity of the disease of motor neurons in rats G93A SOD-1, shown with progressional analysis (a-b), as well as analysis of the final results (C-E) clinical and pathological parameters in the case of transplants living cells (L, red) and grafts (blue) dead cells (control, C).

A-C. Part And p is ecstasy a graph showing Kaplan-Meier, showing significant separation between experimental and control animals during the observation period (P=0,0003). Part b shows the separation of the two main parameters of muscle weakness (number of points for BBB and inclined plane) between the two groups (P=0,00168 and 0,00125 respectively).

C-E. the Analysis of the final results of survival (S), time to onset of the disease (D) and the number of motor neurons (E) in experimental and control rats. Part C shows a significant 11-day difference in life expectancy between the two groups (P=0.0005). Part D shows a significant 7-day difference in time to onset of the disease between the two groups (P=0.0001). Part E shows the difference in 3212 cells in the lumbar protrusion between the living and the dead NSC (P=0.01). Bottom inset (E) shows the difference in survival of motor neurons between representative experimental (top) and control (bottom) rat at the age of 128 days; the arrows indicate the lateral group of motor neurons. The size of the slices: 150 microns.

DETAILED DESCRIPTION

The described methods relate to the treatment of neurodegenerative conditions. In particular, the described methods include methods of obtaining neural stem cells for transplantation to a subject who needs it. Obtaining cells for transplantation may include reproduction is quantitative in vitro specific cell population to a level sufficient for commercial use as a tool for treatment of neurodegenerative conditions. In one embodiment of the present invention a method for the treatment of degenerated or damaged neural region includes shipping in the area of effective number of neural stem cells, sufficient to improve the neurodegenerative condition.

Used herein, the term "neurodegenerative condition" can include any disease or disorder, or symptoms, or causes or their effects, including damage to or destruction of neurons. Neurodegenerative conditions may include, without limitation, the disease Alexander disease Alpers, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia-telangiectasia, Canavan disease, syndrome Cockayne, corticobasal degeneration, a disease of Creutzfeldt-Jakob disease, Huntington's disease, Kennedy disease, Krabbe disease, dementia Lewy Body disease Machado-Joseph, multiple sclerosis, Parkinson's disease, Pelizaeus-Merzbacher disease Neumann-Peak, primary lateral sclerosis, a disease of Resume, disease Sandhoff, disease Shilder, disease, Steele-Richardson-Olzewski, the " dryness " of the spinal cord or any other state associated with neuronal damage. Other neurodegenerative what condition may include or be caused by traumatic spinal cord injury, ischemic spinal cord injury, stroke, traumatic brain damage and hereditary conditions.

The described methods include the use of NSC to improve neurodegenerative condition. Used herein, the term "NSC" can also refer to a nerve or neuronal undifferentiated cells-the precursors or neuroepithelial cells predecessors. NSC can be functionally defined by their ability to differentiate into each of the three major cell types of the CNS: neurons, astrocytes and oligodendrocytes.

In one embodiment, the present invention NSC are multipotential, so that each cell has the ability to differentiate into neuron, astrocytomas or oligodendrocyte. In one embodiment, the present invention NSC is bipotential, so that each cell has the ability to differentiate into two of the three cell types of the CNS. In one embodiment, the present invention NSC include at least bipotential cells generating both neurons and astrocytes in vitro, and include at least commitirovannah cells that generate neurons in vitro.

Growing conditions can affect the direction of cell differentiation towards one or the other tile is knogo type, that indicates that the cells are not commitirovannah in the direction of a single cell line. Under culture conditions that are favorable for neuronal differentiation, cells, particularly of the Central nervous system of a mammal, are almost completely bepotastine in respect of neurons and astrocytes, and differentiation into oligodendrocytes is minimal. Thus, the differentiated cell cultures described methods can turn into neurons and astrocytes. In one embodiment of the present invention, the ratio of neurons and astrocytes may be closer to 50:50.

The described methods include obtaining the NSC in such areas of the Central nervous system of mammals, as the neuroepithelium. Other parts of the Central nervous system, from which you can select the NSC include ventricular and subventricular areas of the CNS and other parts of the CNS that contain mitotic precursors and postmitotic neurons. In one embodiment of the present invention in the described methods can be used NSC, in parts of the developing CNS of mammals.

In one embodiment, the present invention NSC is derived from the area, which is naturally neurogenic desirable for the population of neurons. The desired population of cells may include ina cell-specific neuronal phenotype, which can replace or Supplement the specified phenotype, lost or inactive neurological condition.

A number of different neuronal subtypes, including subtype, suitable for the specific treatment of neurodegenerative diseases or conditions, can be obtained by separating the NSC from different regions or areas of the CNS and at different gestational ages during fetal development. NSC isolated from different regions or areas of the CNS and in different gestational age, use for optimum reproduction and the ability to neuronal differentiation. One of the hallmarks of the Central nervous system of mammals is the diversity of neuronal subtypes. A single population of NSC, for example, can spontaneously generate only a few neuronal subtypes in the culture. In addition, cells in a fetal gestational age can establish the physiological relevance of cultured cells.

In one embodiment, the described methods cells for transplantation subjects derived from fetal equivalent of damaged neural region. In one embodiment, the present invention NSC isolated from the plots of fetal CNS in gestational age from approximately 6.5 to approximately 20 weeks. In one embodiment of the present invention the cells of Malinovo spinal cord isolated in gestational age from approximately 7 to approximately 9 weeks. You should take into account that the proportion of the population susceptible to allocation of neural stem cells may vary depending on the age of the donor. The ability of cell populations to reproduction may also vary depending on the age of the donor. Such regional and temporal specificity NSC indicates that NSC behave as constrained in their further fate of undifferentiated precursor cells, but not as "cell blank or a single population of cells.

The share of the population in vitro, including GABA-producing neurons, is usually constant and is approximately 5-10%.

NSC ventral mid-brain, for example, differ from NSC derived from the spinal cord at the same gestational stage. In particular, the NSC from the ventral mid-brain give rise exclusively dopaminergic neurons expressing tyrosine hydroxylase, while the NSC from the spinal cord to generate exclusively cholinergic neurons that produce acetylcholine. Both cell types, however, at the same time generate more widespread glutamate - and GABA-producing neurons. Thus, in one embodiment, the present invention describes methods include receiving NSC from the ventral mid-brain to treat conditions, obligee is s or weaken, at least in part, by the implantation of dopaminergic neurons expressing tyrosine hydroxylase. Describes the methods additionally include receiving NSC of the spinal cord for treatment of neurodegenerative conditions that facilitate or weaken, at least in part, by the implantation of cholinergic neurons that produce acetylcholine.

Thus, for the treatment of movement disorders such as Parkinson's disease, which is characterized by loss of dopaminergic neurons, a variant implementation of these methods involves the use of NSC derived from areas such as the ventral midbrain, which is important neurogenesis of dopaminergic neurons. In addition, NSC can be obtained at gestational age of fetal development, during which important is the neurogenesis of dopaminergic neurons. Accordingly, in one embodiment, the present invention describes methods include receiving NSC from the ventral mid-brain, obtained in gestational age from approximately 7 to approximately 9 weeks, for the treatment of movement disorders.

For the treatment of motor neuron diseases such as amyotrophic lateral sclerosis or flaccid paraplegia caused by the loss of ventral motor neurons, the wasp is estline these methods involves the use of NSC, derived from areas such as the spinal cord, in which neurogenesis ventral motor neurons is essential, and received in this gestational age, fetal development, during which neurogenesis ventral motor neurons is essential. Accordingly, in one embodiment, the present invention NSC isolated from the spinal cord in gestational age from approximately 7 to approximately 9 weeks for the treatment of motor neuron diseases.

You should note, however, that in some cases the limits specified regional specificity for practical purposes are quite broad. Thus, NSC from various areas of the spinal cord, such as cervical, thoracic, lumbar and sacral segments can be used interchangeably for implantation and treatment in other locations than the corresponding place of origin NSC. For example, NSC derived from the cervical spinal cord, can be used for the treatment of spasticity and/or rigidity by transplantation of cells in the lumbar segments of the patient.

NSC also can be isolated from body tissues after birth and tissues of an adult organism. NSC derived from the tissues of the body after birth and tissues of an adult organism, are quantitatively equivalent to t is his view of their capacity for differentiation into neurons and stroma, as well as the characteristics of their growth and differentiation. However, the effectiveness of in vitro selection NSC from various tissues of the Central nervous system of the body after birth and adult organism can be significantly lower than the efficiency of the allocation of NSC from fetal tissues, which are home to the most abundant population of NSC. However, as in the case of NSC from fetal tissues, the described methods provide at least about 30% NSC is derived from neonatal and adult organisms, for differentiation into neurons in vitro. Thus, the tissues of the body after birth and tissues of an adult organism can be used as described above for NSC derived from fetal tissues, but the use of fetal tissue is preferred.

Different neuronal subtypes can be obtained using the manipulation of embryonic stem cells proliferating in vitro. Thus, specific neuronal subtypes, based on the above methods, you can select and clear of other, irrelevant or unwanted cells to improve the results, if necessary, and can be used for treatment of the same neurodegenerative conditions.

NSC in the described methods can be obtained from one area and transplanted to another area of the same subject as autograft. In addition, NSC the described methods can be obtained from a genetically identical donor and transplant as istranslated. Also NSC in the described methods can be obtained from a genetically non-identical member of the same species and to transplant as an allograft. Alternatively, the NSC can be obtained from various species of animals and transplant as a xenograft. With the development of powerful immunosuppressants allograft and xenograft precursors of nerve cells, such as precursors of nerve cells pigs, can be transplanted to other mammals.

The tissue sample can be dissociate any standard method. In one embodiment of the present invention the fabric dissociate careful mechanical grinding using a pipette and saline phosphate buffer containing no divalent cations, to obtain a suspension of dissociated cells. Desirable dissociation enough for mostly single cells, in order to avoid excessive local density of cells.

For successful commercial use NSC preferably maintaining a healthy and sustainable crops that exhibit stable characteristics of reproduction and differentiation during many successive passages. As described above, methods of cultivation can be optimized to achieve long-term, stable time is norene individual cell line NSC from different areas and at different ages of development, at the same time preserving their distinct properties of undifferentiated precursors.

When implementing these goals unexpectedly, it was found that stimulation of NSC adhesion to the substrate contributes to the accelerated rate of mitosis NSC or undifferentiated precursor cells, resulting in getting a stronger culture of NSC or undifferentiated precursor cells. In particular, in addition to avoiding excessive local density of cells and the maintenance of mitogen concentrations, it was found that the concentration of proteins of the extracellular matrix affect the long-term ability of the NSC to mitosis and differentiation. Extracellular matrix proteins may include poly-D-lysine, poly-L-lysine, poly-D-ornithine, poly-L-ornithine, fibronectin, and combinations thereof. Other proteins of the extracellular matrix may include various isotypes, fragments, recombinant forms, or synthetic mimetics of fibronectin, lamina, collagen, and combinations thereof. Alternatively, or in addition, one should take into account that the described methods may include any other suitable substance, which is capable of stimulating an effective adhesion of cells so that each cell attached to the culture substrate throughout the duration of cultivation, not demonstrate what irua cytotoxicity or delay cell division.

Although extracellular matrix proteins can be effectively used to stimulate cell adhesion, various amino acid polymers, such as poly-L/D-ornithine or poly-L/D-lysine, can be toxic to cells at certain concentrations for each cell line.

The duration of incubation can also affect the final amount of the polymer deposited on the surface of the Cup, which affects cell viability. For NSC, used in the described methods, the concentration of the polymer can be between about 0.1 μg/ml to about 1 mg/ml In one embodiment, the present invention 100 µg/ml poly-D-lysine are dissolved in 0,01M HEPES buffer or in water at a neutral pH value and applied to the culture vessel. The culture vessel is incubated at room temperature for 1 hour. The culture vessel is then thoroughly rinsed with water and dried before use.

The described methods can also include a double coating culture vessels with a protein of the extracellular matrix. In one embodiment of the present invention the culture vessel is treated with fibronectin or derived fibronectin followed by the application of poly-L/D-ornithine or poly-L/D-lysine as described above. In one embodiment, is sushestvennee of the present invention use fibronectin protein, derived from human plasma. You should note, however, that you can use any other suitable form or source fibronectin protein, such as porcine or bovine fibronectin, recombinant fibronectin fragments fibronectin proteins, synthetic peptides and other chemical mimetics of fibronectin. In one embodiment, the present invention can be applied from approximately 0.1 μg/ml to about 1 mg/ml fibronectin.

In one embodiment, the present invention includes a reproduction of the NSC of the tissue of the spinal cord, the culture vessel is treated with 100 μg/ml poly-D-lysine over a period of time sufficient to extracellular protein associated and formed a coating on the culture vessel. The specified time period may range from five minutes to three hours. The culture vessel can then be rinsed with water. After air drying the culture vessel can be treated approximately 25 mg/ml of fibronectin during the period of time from about five minutes to several hours at room temperature, or about 1 mg/ml of fibronectin during the period of time from about 1 hour to several days at 37°C. Subsequently, fibronectin can be removed, and the culture vessel can be washed is at least once or stored in PBS until use.

Alternatively, fibronectin can be added in the growth medium as a soluble factor that is directly supplied to the cells. In this embodiment, the present invention NSC can be reproduced by adding 1 μg/ml of fibronectin in the growth environment, in addition to or instead of processing the culture vessels by fibronectin. Adding protein, facilitating the attachment in the growth medium as a soluble factor at the time of seeding cells on tablets is particularly advantageous for the cultivation of NSC on an industrial scale due to the relatively short period of storage vessels with fibronectin coating. This method is also suitable for the production line of neural stem cells, requiring compliance with considerably the exact conditions and ensure reproducibility as required for cGMP protocols and for the production line of neural stem cells for therapeutic applications.

In one embodiment, the present invention allocated NSC added to the culture vessel with a density of from about 1000 to about 20,000 cells per square see the Specified density promotes uniform dispersion and adhesion of individual cells in the culture vessel, allowed to avoid localized concentrations of cells and to enrich the culture for NC.

In one embodiment, the present invention NSC multiply in the absence of serum. In one embodiment, the present invention NSC cultured in particular, does not contain serum, environment, to avoid impact on NSC concentrations of serum, sufficient to destabilize the ability of NSC to mitosis and differentiation. In addition, the impact on NSC certain growth factors such as leukemia inhibitory factor (LIF) or ciliary neurotrophic factor (CNTF), can also destabilize the NSC and should be avoided.

Mitogens can be added to the culture at any stage of cultivation to enhance growth NSC. Mitogens may include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), growth factor epidermal (EGF), transforming growth factor-alpha (TGFα), and combinations thereof.

NSC describes ways you can grow and multiply at least two different cultural forms. One form of culture includes aggregated form, usually called a cluster, aggregated form, called the suspension culture. Another form of culture includes dispergirovannoyj, non-aggregated form, called attached culture.

In dispersed attached culture NSC described methods cells clicks the form a monolayer, in which individual cells are first contacted with the culture substrate directly. Eventually, after a period of incubation, the cells may sporadically to form clusters in which at least one additional layer of cells is formed on a lower layer, at the same time, when the cells of the lower layer individually attached to the substrate. The specified clusters is observed especially when the culture inoculant with a high density of cells or allow the culture to achieve a high density of cells, which in one embodiment of the present invention is minimized for optimum reproduction NSC or undifferentiated precursor cells or to maintain optimal multipotential ability NSC. In dispersed attached to the culture of one possible implementation of the described methods NSC provide an opportunity to share with a frequency of less than about one time in four days.

Another hallmark attached dispersed culture is that NSC described ways to share with the formation of daughter cells, each of which retains its multipotential ability. In one embodiment of the present invention attached dispersed culture NSC described methods assortment of the company includes the ability to reproduce, component of at least 20 doublings of cells in the absence of significant differentiation. Most NSC can be propagated with a speed greater than at least 50 cell doublings before they will lose their neurogenic potential. In one embodiment, the present invention NSC, multiplied dispersed in attached culture described methods, demonstrate enhanced neuronal differentiation, providing in one embodiment of the present invention at least about 30% neuronal differentiation. In many cases, at least 50% of NSC differentiated into neurons. Despite the fact that the dispersed attached form of culture is the preferred form of culture, the different methods of cultivation can afford to allocate different in vivo cell populations with different potentials of differentiation both in vitro and in vivo.

This method also allows clonal selection NSC from a number of sources without genetic modification or inclusion of feeder cells. So, a very small amount, preferably less than 1000 cells per square centimeter cells may be seeded onto the tablet for culturing cells, prepared as described above.

For several days after sowing NC cells can form a well-isolated colonies. Colonies can be grown to the desired size, such as at least about 250 to about 2000 cells. In one embodiment of the present invention at least one colony of cells are selected manually and inoculant individually on a new tablet for culturing cells, for example in advance of the tablet.

Selected clonal population can be propagated serial passirovannym and be used to create multiple lines of neural stem cells. Many of these clonal cell lines were isolated from various regions of the CNS of a mammal, including the spinal cord, midbrain and hind brain. Clonal cell lines are suitable for the enrichment of specific cell phenotype, for example, to ensure a higher proportion of neuronal subtypes. For example, clonal cell lines enriched in respect of dopaminergic neurons expressing tyrosine hydroxylase, GABA-eliteskin neurons, cholinergic neurons and other neurons of specific phenotypes can be selected by using the described methods.

In one embodiment, the present invention polyclonal or monoclonal line neural stem cells can be induced in the direction of further enrichment in regard to specific is adtype neurons. A number of growth factors, chemical agents and natural substances have been screened to identify effective inducers of specific neurons, such as dopaminergic neurons expressing tyrosine hydroxylase, and cholinergic neurons that produce acetylcholine, NSC middle of the brain or spinal cord. Factor, or chemical agent, or a combination of them can be entered during the mitotic phase and/or phase of differentiation of NSC. In one embodiment of the present invention the line of neural stem cells to dopaminergic phenotype additionally enriched as a donor population for the treatment of Parkinson's disease.

Different neuronal subtypes can be obtained by selection of stem cells having the desired pattern of differentiation in vitro. The results of in vitro can largely be reproduced in vivo. This means that the potential efficacy of stem cells in vivo can be predicted on the pattern of differentiation of stem cells in vitro. After injection alive postnatal subjects, NSC, both in undifferentiated and predifferentiation condition, in many ways the in vivo pattern of differentiation observed in vitro. Thus, NSC, giving rise to neurons, producing tyrosine hydroxylase in vitro, also generate neurons, producing tyrosine-hydroxylase is, in vivo. On the contrary, NSC, not giving rise to neurons, producing tyrosine hydroxylase in vitro, does not produce neurons, producing tyrosine hydroxylase in vivo.

However, the signals of differentiation present in vitro is limited compared to in vivo conditions. Thus, a significant fraction of differentiated neurons may not Express the main neurotransmitter phenotype. Additional signals, such as signals from afferent or efferent neurons, or agents that mimic these natural signals can be used for reconfiguration of differentiated phenotypes during the mitotic stage of the NSC, and during their differentiation. NSC have the ability to respond to signals that are present in vivo and in vitro. So, after transplantation into the damaged ischemia of the spinal cord spinal NSC generate a significantly higher proportion of GABA-producing neurons than in vitro. Thus, the NSC have plasticity. The specified plastic nature NSC is a characteristic of their multipotentiality, and as such is specified plasticity can be used to identify phenotype-inducing agents and conditions, which you can then combine with a population of NSC to redirect its properties.

In one embodiment, the present invention is similar to the reprogramming on the host of the impact on the NSC from the tissue of the spinal cord for obtaining enhanced expression of phenotypes motor neurons. The exposure conditions include joint cultivation of NSC or differentiated cells with a variety of muscle cells or cells derived from the peripheral nervous system, such as neural crest cells or ganglion neurons. On NSC can also be affected by mixtures of molecules known that they are expressed and produced in motor neurons or in the spinal cord to enhance the expression of NSC phenotypes motor neurons.

In order to induce increased expression of NSC dopaminergic phenotype mid-brain, NSC affected by such molecules as lithium, GDNF, BDNF, pleiotrophin, erythropoietin, conditioned medium from cells such as Sertoli cells, or any other suitable chemical agents or cells obtained by screening, or combinations thereof. The specified incentive may encourage the transplanted NSC to Express and maintain dopaminergic phenotype in vivo.

In one embodiment, the present invention NSC described methods may include predifferentiated cells for transplantation. For maximum collection of cells and simplicity for transplantation collect fused culture, which includes mainly the population of undifferentiated cells. Should be concerned with the ü in mind, however, that minimum population of cells which has just started to spontaneously differentiate, can also exist due to the high density of cells.

In one embodiment of the present invention the passage NSC includes the collection or release of cells from the substrate. In one embodiment, the present invention describes methods include the collection or release of cells from the substrate using at least one enzyme. The enzymatic treatment can be avoided if the time cell cycle NSC is short enough to inactivate mitogen receptors on the cell surface.

In one embodiment of the present invention after collecting the cells from the cell culture concentrate by brief centrifugation. The cells are then optionally washed and resuspendable end, suitable for clinical use, the solution, such as saline solution, saline solution, phosphate buffer, or, alternatively, to resuspending in solution for storage or hibernation mode. Alternatively, cells can be resuspendable environment for freezing, such as environment with dimethylsulfoxide or any other suitable cryoprotectant and frozen for storage.

The solution for hibernation manufacture of avleat thus, to support the viability of living cells over an extended period of time. In one embodiment of the present invention, the storage solution can be adapted to use for the transport of living cells in the form of ready to use compositions in the surgical transplantation for immediate use. Suitable conditions for the transport of living cells in a distant institution also include insulating device which can maintain a stable temperature in the range of from approximately 0°to approximately 20°C for at least 24 hours. Live cells were kept at a temperature ranging from approximately 0°to approximately 8°C for from about 24 hours to about 48 hours, are suitable for transplantation for the treatment of diseases or conditions.

In one embodiment of the present invention, the cells are concentrated in the solution, such as is suitable for clinical use, hibernation, or storage of the solutions described above. In one embodiment of the present invention, the cells are concentrated to a suitable density of cells, which may be the same as the density of cells for introduction, or different from it. In one embodiment, the implementation of whom tvline of the present invention, the density of cells for injection can be varied from approximately 1000 cells per microliter to about 1,000,000 cells per microliter, depending on such factors as the place of injection, neurodegenerative status of the site of injection, the minimum dose required for optimal effect and considerations in toxicity and side effects. In one embodiment, the present invention describes methods include injecting cells with cellular density of approximately 5000 cells per microliter to about 50,000 cells per microliter.

Volume environments, in which the reproduced cells are suspended for delivery to the treatment, can herein be called the volume of injection. The volume of injection depends on the injection site and degenerative status of the tissue. More specifically, the lower limit of the amount of injection can be determined by practical manipulation with viscous suspensions with a high density of cells using fluid and given the tendency to the formation of cell clusters. The upper limit of the amount of injection may be determined by the limits of the compression pressure caused by the volume of injection, which is necessary to avoid damage to the host tissue, as well as practical time of surgical intervention.

Low survival of donor cells using known methods have made it necessary to deliver a large number of cells in a relatively small area in the try is effective treatment. The volume of injection, however, is a hydrostatic pressure that occurs in the tissue of the host, and for a long time injections associated with large volume injection, increases surgical risk. In addition, excessive injection of donor cells leads to compression and subsequent damage to parenchymal host tissue. In an attempt to compensate for the limitations imposed by the volume of new ways demanded the manufacture of suspensions for injection with a high density of cells. However, the high density of cells contributes to the formation of dense clusters of transplanted cells and inhibits the migration or proliferation of cells, preventing effective treatment outside of the restricted area and smooth integration into the host tissue.

On the contrary, due to improved survival in vivo cells, obtained by the described methods, for injections, fewer cells. In fact, three to four times the number of injected cells, as has been shown, there are six months after injection, which shows a significant quantitative survival when using these methods. Also thanks quantitative survival can be achieved reproducible introduction of desirable doses of cells. Accordingly, in one embodiment, the present invention glue the key concentrate to a density of from about 1000 to about 200,000 cells per microliter. In one embodiment of the present invention from about 5000 to about 50,000 cells per microliter used for efficient engraftment. In another embodiment, the present invention uses approximately 10,000 to 30,000 cells per microliter. In one embodiment of the present invention, the cells can be delivered to the treatment, suspended in the volume of injection is less than approximately 100 microliters one injection site. For example, in the treatment of neurodegenerative conditions in humans, when it is possible to perform multiple injections bilaterally along the spinal path, you can use the amount of injection from 0.1 to approximately 100 microlitres at the injection site.

Any suitable device for injection of cells into the desired area can be used in the described methods. In one embodiment of the present invention use a syringe capable of delivering submicroliter volumes within a certain period of time when almost constant flow rate. Cells can be loaded into the device through a needle or a flexible catheter, or any other suitable transmission device.

In one embodiment of the present invention the desired injection for the treatment of neurodegenerative from the situation includes at least one region of the spinal cord. In one embodiment of the present invention, the cells are implanted in at least one particular segment or region of the spinal cord, such as cervical, thoracic, lumbar or sacral part of the spinal cord. In the lumbar spine, for example, only five pairs of nerve pathways cross bone canal of the vertebrae; each pair of the nerve pathways exist in the spine at each lumbar level, distributed over a vast area. Because of the lower density of nerve pathways in the lumbar spinal cord lumbar spine is particularly well suited to select the safe zone for injection of cells. In one embodiment of the present invention, the cells are implanted into the staging area of the parenchyma of the spinal cord.

In one embodiment, the present invention cells injected approximately 5-50 areas. In one embodiment, the present invention cells injected approximately 10-30 areas on each side of the cord. At least two sections may be separated by a distance of from about 100 microns to about 5000 microns. In one embodiment of the present invention the distance between the sites of injection is from about 400 to about 600 microns. The distance between the sites of injection can define the e l e C reasons almost continuous and related to the presence of donor cells for spinal segments and the average volume of injection, which was shown to be approximately 2-3-month survival in experimental animals such as rats or pigs. In one embodiment, the present invention cells are injected along both sides of the middle line of the spinal cord within the length of at least several of the lumbar segments, suitable for the treatment of this symptom as spasticity/rigidity, or for the survival of motor neurons. The actual number of injections for men can be extrapolated on the basis of results obtained in experimental animals.

In one embodiment of the present invention the injection site, which is the target, is located in the gray matter of the spinal cord. Inside the gray matter of the needle tip can be positioned in such a way as to place the NSC on the specific levels of the arc plates of the spine. For example, for delivery GABA/glycine-producing neurons for the treatment of spasticity/rigidity NSC deliver in the region covering plate of the arc of the spine V-VII. Alternatively, the NSC can be delivered in the dorsal horn of gray matter of different spinal segments, from cervical to lumbar, or its vicinity, with a view the completion of the treatment of neuropathic pain or chronic pain. Alternatively, the NSC can be delivered in the ventral horn of gray matter of different spinal segments, from cervical to lumbar, or close to it for the treatment of motor neuron diseases such as ALS.

Cells described methods can generate large numbers of neurons in vivo. When the NSC are not openly predifferentiated before transplantation, NSC can proliferate up to two to four divisions in vivo before differentiation, which further increases the number of effective donor cells. After differentiation of neurons secrete specific neurotransmitters. In addition, neurons secrete into the environment surrounding the graft in vivo, growth factors, enzymes and other proteins or substances that are beneficial for various conditions. Accordingly, the number of States can be treated by the described methods due to the ability of cardiac cells to generate large numbers of neurons in vivo and due to the fact that neurodegenerative condition may be caused or result in the loss of items, including derived from neuronal elements. Thus, subjects suffering from degeneration of the tissues of the Central nervous system because of the loss of these derived from neuronal elements, such as growth factors, enzymes and other proteins, can be treated effectively described with the persons.

Condition that responds to growth factors, enzymes and other proteins or substances that are excreted implanted neurons include hereditary lysosomal diseases, such as disease, and Tay-Sachs disease Neumann Peak disease batten, Krabbe disease, ataxia and other diseases.

In addition, the described methods of treatment include the implantation of cells propagated in vitro, which can replace a damaged or degenerated neurons exert an inhibitory or stimulatory effect on other neurons and/or to release trophic factors that contribute to the regeneration of neurons.

Variant implementation of the present invention includes the provision of additional motor neurons as replacement of damaged or degenerated neurons. For example, the described methods include the provision of sufficient neural infrastructure within pathological cavity of the spinal cord to fill the formed cavities. Neuronal infrastructure is sufficient if it can slow the increase of pathological cavity associated with syringomyelia caused by traumatic spinal cord injury, hereditary conditions or any other cause. You should take into account that adequate nevr the school infrastructure also helps to facilitate further complications, resulting from degeneration of the spinal cord.

Not all NSC are therapeutic for the disease. Types of neuronal populations affected in various diseases may be different. Thus, therapeutically effective donor population NSC contributes to the replacement of lost neuronal element. For example, treatment of spasticity, seizures, movement disorders and other disorders associated with hyperactivity of the muscles, may include providing a therapeutically effective amount of cells capable of differentiating in inhibiting neurons that produce GABA or glycine. Different populations can be assessed in vitro by examining the differentiated neuronal phenotype. Pattern differentiation in vitro is then used to predict the efficiency of the cells in relation to the generation of the corresponding phenotype in vivo, not only from the point of view of the appropriate neurotransmitter phenotype, but also from the point of view of a suitable morphology, migration, and other phenotypic characteristics of neurons.

In one embodiment of the present invention implanted NSC, which is capable of generating neuronal subtype corresponding to the damaged or destroyed neuronal subtypes associated with the etiology of the symptoms. For example, Ki is reaktivnosti excitatory circuits in subjects may be caused by a genetic condition or neuronal damage due to spinal cord injury, surgical interventions on the thoracic/thoracoabdominal aorta, stroke, epilepsy, brain injury, Huntington's disease, urinary incontinence, hyperactive peristalsis of the intestines and other uncontrolled contractions of the muscles caused by damage or a genetic condition. Spasticity, seizures or other hyperactivity observed in the brain, in contrast to the spinal cord, have different etiological nature. Focal epilepsy, for example, it is believed, causes deregulirovania hyperactivity due to lack of controlled GABA tone control circuit. For this purpose the described methods include providing the affected areas any abscopal neurotransmitters, such as GABA or glycine by transplantation of in vitro propagated NSC. In the case of spasticity, seizures and hyperactivity, for example, the number of NSC, which are able to differentiate in inhibiting neurons, such as the GABA-producing or glycine-producing neurons generated in vitro for transplantation in order to alleviate at least one hyperactive neural circuits associated with spasticity, seizures and other neuronal hyperactivity. The described methods can thus be used for the treatment of epilepsy and similar neurodegenerative sostenibilidad.

The described methods can also be used for the treatment of paresis, paralysis, spasticity, rigidity, or any other motor, speech, or cognitive symptoms caused by cerebral ischemia. Cerebral ischemia may occur as a result of stroke, brain or heart attack, in which blood circulation to the brain is interrupted for a significant period of time. This ischemia, thus, is similar to spinal cord ischemia, as described above. Some of the subjects of stroke develop seizures of Central origin, as well as other deficits, such as memory loss, paralysis or paresis. These deficits after cerebral ischemia is also likely to be associated with a selective loss of inhibitory inter neurons in the hippocampus and/or other areas of the brain. Thus, the described methods can be used to treat subjects with stroke, suffering from paresis, paralysis, spasticity, or other motor, speech and cognitive symptoms.

In the case of paralysis, flaccid paraplegia and other conditions associated with loss of control of muscle contractions, such as condition caused by ALS, traumatic spinal cord injury, ischemic injury or hereditary conditions described what these ways include providing neuronal implantation for obtaining significant trophic effects, slowing the loss of motor neurons. In particular, the described methods facilitate the ability of transplanted NSC to secrete trophic molecules that can be delivered to the degenerating motor neurons in conditions of optimal bioavailability. These trophic molecules include victims of exocytosis superoxide dismutase, such as superoxide dismutase (SOD-1), lysosomal enzymes and non-protein molecules, such as produced by cells of antioxidants. Other trophic factors secreted transplanted cells may include generally obtained from cells derived neurotrophic factor (GDNF), obtained from brain-derived neurotrophic factor (BDNF), vascular growth factor, epidermal (VEGF), pleiotrophin, vascular endothelial growth factor (VEGF), erythropoietin, Malkin, insulin, insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2), or any other favorable trophic element.

Another factor that contributes to the ability of these methods to treat a wide variety of neurodegenerative conditions, includes the ability of NSC-differentiated cells to migrate extensively along the existing neuronal fibers. Migration of transplanted cells leads to global distribution and integration of donor the neurons and/or glia and the emergence of dispersed provider of therapeutic element, secreted by the specified cell.

Wide migration of cells makes possible global and stable delivery of key therapeutic proteins and substances throughout the nervous system and the body of the subject who needs it. Thus, the cells of the described methods are effective delivery vehicles for therapeutic proteins and substances. For the purposes specified delivery described methods include the transplantation of cells in different parts of the nervous system, including the CNS parenchyma, ventricles, subdural, intrathecal and epidural space, in areas of the peripheral nervous system, as well as in areas outside the nervous system, including the intestines, muscles, endovascular system and subcutaneous sites.

Example 1. The propagation neural stem cell/undifferentiated precursor cells of the spinal cord

Receive the spinal cord from at least one donor at gestational age approximately 7-8,5 weeks. Only adjacent tissue of the spinal cord dissociate in physiological solution with phosphate buffer containing no CA++and mg++using mechanical grinding. The obtained cell suspension is then seeded on the plates for tissue culture, pre-coated with poly-L-ornithine or poly-D-lysine and human is m fibronectin or other proteins of the extracellular matrix. Processed tablets for tissue cultures were incubated with 100 μg/ml poly-D-lysine at room temperature for 1 hour. Then they were washed three times with water and dried. Then they were incubated with 25 mg/ml at room temperature for 5 minutes. Sometimes used 10 mg/ml fibronectin at room temperature for 1 hour. Sometimes used 1 mg/ml fibronectin at 37°C for 18 hours. Culture medium consisted of N2 (DMEM/F12 plus insulin, transferrin, selenium, putrescine and progesterone), was supplemented with human recombinant basic fibroblast growth factor (bFGF). In one embodiment, the present invention can be used within 0.1 ng/ml-100 ng/ml In one embodiment, the present invention optimally use 10 ng/ml bFGF.

The initial culture consists of postmitotic neurons and proliferating NSC in the monolayer. Subsequently, after approximately five to twenty days of cultivation, dividing nestin positive NSC dominate culture over non-dividing neurons or slowly dividing glia. These culturing conditions selectively contribute to the reproduction of the NSC. The breeding population of NSC passedout by mild enzymatic action, for example, using trypsin. Cells cultured in media not containing the x serum or containing no serum. Despite the fact that low-concentration serum, the cells can carry, it is best to avoid exposure of serum to cells, because serum contains many cytokines, such as LIF and CNTF, which promote glial differentiation of NSC. Thus, during the passage of the used enzyme is stopped by the addition of a specific inhibitor of the enzyme, such as trypsin inhibitor, and not serum. At each passage counts the number of collected cells and the fraction re-seeded for further reproduction. As shown in figure 1, when using the method according to the present invention NSC can be propagated in a population in excess of 1018again, at the same time keeping their properties, growth and differentiation. Cells can be propagated in a reproducible manner. As shown in figure 1, the serial passage of the cells was repeated three times with reproducible growth curve and doubling time of the cells. During the breeding almost all cells Express nestin in vivo marker of mitotic neuroepithelial cells and do not contain antigens of differentiated neurons and glia, such as tubulin type 3-beta and GFAP. Cells immunological not turn on PSA-NCAM, a possible marker commiteeman neuronal precursors, 04 and GalC, markers of oligodendrocytes, and RC2, a marker of radial glia. So the m way according to the results of immunological staining NSC stably maintain their profile of expression of antigens during prolonged breeding period. Example morphology and expression nectin shown in figa and respectively.

Example 2. The differentiation of neural stem cell/undifferentiated precursor cells of the spinal cord

At any time during the breeding NSC culture can be differentiated by withdrawal from the culture mitogen, such as bFGF. The NSC differentiation occurs within approximately 1-3 days after mitogen withdrawal, and differ from each other heterogeneous cell morphology are apparent. Approximately 4-7 days differentiation of neuron-specific antigens, such as Mars, Tau and beta-tubulin type III, can be visualized by immunological staining. Approximately 12-14 days long, rising in the form of a bundle of axonal processes are clearly visible in the culture with a clear polarization of the movement of subcellular protein. Approximately 28 day of synaptic proteins, such as synapsin and synaptophysin, localized in the axonal endings, which is manifested in the form of spotty dyeing. Additional supply sublayer astrocytes can be provided for further stimulate long-term maturation of neurons. As shown is figure 3, the differentiation of spinal NSC generates a mixed culture of neurons and glia, in which neurons reliably Express a neuron-specific antigens, such as Tau, 2b (A) and beta-tubulin type 3 (C), and includes approximately 50% culture. As shown in figs, culture spontaneously generates long, fasciculate the spermatic cord axons, which are within a few centimeters. As shown in figs, a significant proportion of neurons is GABA-ergicheskoi. Cholinergic motor neurons are also present in the culture (fig.3D). The presence in the culture of a significant number of GABA-eliteskin neurons predicts the suitability of spinal NSC for the treatment of various neurological conditions caused by decreased production of GABA in a particular circuit. Similarly, the presence of cholinergic neurons shows that spinal NSC capable of differentiation into motor neurons, and predicts their suitability for the treatment of various diseases of motor neurons caused by progressive degeneration of motor neurons. To ensure NSC propagated in the presence or in the absence of other conditions that increase the phenotype, harvested and injected in neuronal area of deficit.

Example 3. The propagation neural stem cell/undifferentiated precursor cells of the mid-brain

Get fabric mid-brain from a single donor in gestational age 7-8,5 weeks. NSC fabric mid-brain gain, as described in example 1. Cells serially passedout within 160 days of the culture period, and the resulting multiplication is shown in figure 4. During the breeding period NSC stably retain their multipotentiality and neurogenic potential, and the potential for differentiation into dopaminergic neurons. Dopaminergic neurons assessed by neuronal expression of tyrosine hydroxylase (TH) and dopamine Transporter (DAT).

The DAT expression is a marker of dopamine-producing neurons. The expression of DAT neurons can be assessed by determining their ability to transport radioactive labelled dopamine via synaptic membrane of differentiated neurons in culture. The function of DAT differentiated NSC mid-brain and monoclonal received NSC mid-brain estimate in the analysis of capture radioactively labeled dopamine (figure 5). The results of the analysis show high functional dopaminergic activity NSC mid-brain. Moreover, they show that dopaminergic phenotype can be enhanced by selection of a monoclonal population of NSC, which are especially prone to the formation of a higher proportion of dopaminergic neurons after differentiation (figure 5).

NSC, GE is Araruama enriched dopaminergic neurons, are particularly suitable for the treatment of Parkinson's disease. Similarly, NSC, reprogrammed at the time of selection of fabric for enhanced differentiation into specific phenotype, can be used to highlight other specific desirable neurons, such as cholinergic neurons in the forebrain that are suitable for the treatment of Alzheimer's disease, spinal cholinergic neurons that are suitable for the treatment of motor neuron diseases such as ALS, serotonergic neurons that are suitable for the treatment of depression, and GABA-ergicheskie neurons that are suitable for the treatment of epilepsy and Huntington's disease.

Example 4. The differentiation of neural stem cell/undifferentiated precursor cells of the mid-brain

NSC/undifferentiated precursor cells of the mid-brain can be differentiated as described in example 2. During the mitotic period NSC or during their differentiation proportion of the desired phenotype can enrich impact on the culture of exogenous factors. An example of these factors is able to enrich the dopaminergic phenotype of NSC medium of the brain, demonstrated by the conditioned media from Sertoli cells, as shown in figa and 6B.

In the studies shown in figa and 6B, stored frozen stem cells are thawed and C is sevali with a density of 40,000 cells per well in 4-cell sectional slides in the presence of mitogen and allowed the cells to proliferate within 6 days then mitogen was removed, and the cells were allowed to differentiate for 8 days. The cells were divided into four groups based on the time and duration of exposure to conditioned medium from Sertoli cells (SCCM, a dilution of 1:1 in N2a). One group was subjected to SCCM during proliferation and differentiation (state 1); the second was subjected to only during proliferation (state 2); the third was subjected to only during differenziali (condition 3); and the fourth was not subjected to SCCM (control or pin). Medium was changed every other day, and mitogen was added daily during the proliferative phase. In each state there were four holes for coloring on many markers, and studied three cell lines: 796MB, 527MB and 566SC. Cells 566SC that were obtained from the spinal cord, which had no detectable TH-positive neurons in the figure ignored.

After differentiation, the cells were fixed using 4% paraformaldehyde and immunologically stained using antibodies to MAR [clone AR (Sigma), which recognizes subtypes 2b], neuron-specific β-tubulin [TuJI (Covance)], and tyrosine hydroxylase (Pel-Freez), and GFAP (Dako) and GalC (Chemicon). Immunologically stained cells was calculated using the 40x lens, and in each well was counted by men is her least three fields. Little or no cells GFAP+ or GalC+ was analyzed in cells cultured under all conditions, therefore, these antigens were excluded from the analysis. In addition, cells 566SC after cultivation had too much density, preventing the counting described conditions and were not included in the final analysis.

The analysis shows that NSC middle of the brain can be influenced by exogenous factors to find the protein factor or chemical agents suitable for further enrichment in the direction of dopaminergic neurons. Thus, the new synthetic/natural chemical and protein factors can be effectively subjected to screening using the NSC, to obtain the population, especially suitable for the treatment of specific indications, such as Parkinson's disease.

Example 5. Treatment of spasticity and rigidity in rats by transplantation of neural stem cell/undifferentiated precursor cells of the spinal cord

In order to cause a transient ischemia of the spinal cord, used a method previously described by Taira (1996). Rats Sprague Dawley (SD) gave anesthetized with halothane gas (1.5 percent). The catheter 2 Fr Fogarty® was performed through the left femoral artery and through the descending thoracic aorta to the level of the left subclavian artery. For measurement of distal blood pressure (DBP) below the level of occlusion of the aorta caudal artery was Coulibaly polyethylene catheter (PE-50).

Ischemia of the spinal cord induced by inflating intraorale balloon catheter 0.05 ml of physiological solution. Systemic hypotension during the period of occlusion of the aorta reproduced by the withdrawal of a part of the arterial blood (10,5-11 cubic cm) of the carotid artery, kanilirovannoy catheter RE-50. Using this method it is possible to induce systemic hypotension approximately 40 mm Hg On the effectiveness of occlusion testified immediate and continuing fall in DBP measured in tail artery. After about 10 minutes induced ischemia of the spinal cord balloon was blown away, and the blood withdrawn from the carotid artery, was repusively. After stabilization of blood pressure (within 20-30 minutes after reinfused) arterial catheters were removed, and the wound sutured.

After the induction of ischemia of the spinal cord was assessed by the recovery of motor function with approximately 2-day intervals, using locomotor scale open field from 21 points (BBB). In the experimental group for the study of transplantation were selected only those animals for which the sum of the scores on the BBB was 0-4.

Approximately 7-21 days after ischemic injury in rats with symptoms of spasticity, in which the sum of the scores of BBB was 0-4, gave anesthesia to 1.5-2% halothane gas in the air and placed the do in the spinal unit. Then did a partial laminectomy of the vertebrae Thll-L2. Glass capillary with a tip diameter of 80-100 μm was connected to microinjector with adjustable pressure. Rats were injected with a 0.5 μl of cell suspension containing 5000, 10000, 15000 or 20000 neural stem cell/undifferentiated precursor cells per injection. Each rat received in the amount of 6-8 injections on each side of the spinal cord (left or right), evenly distributed between segments L2-L6. Centre injections were sent to the Central gray matter (laminae V-VII) (the distance from the dorsal surface of the spinal cord at the level of L3: 1 mm). After implantation, the incision was saniroval 3% H2O2and a mixture of penicillin/streptomycin and sutured in 2 layers. Then the rats were left to recover.

Immunodepressant treatment with FK-506 (Prograf; Fujisawa; 1 mg/kg; in b/W) started all animals approximately 3 days before spinal transplant. After transplantation, the animals received immunodepressant treatment daily for the entire period of survival. Immune rejection of these grafts can be effectively prevented with the use of FK-506. Rats survive for approximately 2 or 7 weeks (n=5 for each time interval).

At the end of the survival period the rats were given anaesthesia pentobarbital (40 mg/kg; in b/W) and transcardial the but was perfesional them within 1-2 minutes of heparinized saline solution, and then 4% paraformaldehyde in 0.1m phosphate buffer (PB). The spinal cord was dissected and re-fixed in the same fixative overnight at 4°C. After secondary fixation of the tissue of the spinal cord subjected cryoprotective in the gradient of sucrose (10, 20 and 30%) during the three days. Then carried out the front, parasagittal or horizontal slices of the spinal cord (10-30 μm). For immunohistochemical analysis of free-floating sections (30 μm) were placed in PBS, 0.1 m (pH 7.4)containing 5% normal goat serum (NGS), and 0.2% Triton X100 (TX), for two hours at room temperature to block nonspecific protein activity. Followed by incubation overnight at 4°C With different primary specific antibodies.

After incubation with primary antibodies, the sections were washed 3 times in PBS and incubated with secondary goat anti-rabbit or artemisinine antibodies conjugated with a fluorescent marker (Alexa 488 or 594; 4 μl/ml; Molecular Probes). All blocking or antibody-based test and preparations were made in 0,1M PBS/0.2%of MV/5% NGS. For experiments with doubly labelled primary antibodies from different species were used simultaneously, followed by the application of secondary antibodies conjugated with different fluorescent markers. In control experiments, primary antibodies were applied. For General poison the aqueous coloring to the final solution of the secondary antibody was added DAPI (3 μl/ml). After staining, the sections were dried at room temperature and covered with a set of Prolong anti-fade kit (Molecular Probes).

Slides were examined using a fluorescent microscope Leica. Pictures (512×512 pixels) were done with a Olympus digital camera and were processed using the computer program Adobe Photoshop 5.5 (Adobe Systems, Mountain View, CA). To confirm the joint localization of different antibodies on sections with double staining was obtained images using deconvolution microscope DeltaVision, including Photometries CCD camera mounted on a Nikon microscope (Applied Precision, Inc.). In General, received sixty-optical slices at intervals of 0.1 or 0.2 μm. Used lens 20x, 40x and 60x (NA 1.3). Performed the deconvolution of data sets and analyzed using computer software SoftWorx software (Applied Precision, Inc.) on the workstation, Silicon Graphics Octane.

The total number of transplanted neurons immunoreactive for nuclear antibodies NUMA, was determined using stereological, random and systematic sampling. Every tenth pre-painted slice taken from spinal segments L2-L6, used for stereological quantitative assessment after application of fractional sampling design. Optical image (thickness 1 μm) were obtained using a microscope Leca DMLB with an oil immersion 100x lens with a numerical aperture of 1.3. Optical images were obtained using a digital camera (Olympus) and a computer program ImagePro (Media Cybernetics)with controlled StagePro motorized platform Z (Media Cybernetics). The total number of transplanted cells was then calculated using the fractionation formula N=Q × 1/hsf × 1/asg × 1/ssf, where N represents the total number of positive cores, Q represents the sum of the counted cells, hfs is the height sampling fraction, asf is the area sampling fraction, a ssf represents the fraction of particles in the sample.

In order to obtain the spatial reconstruction of the transplanted NSC in ischemic spinal cord, used a pre-stored images of serial sections of the spinal cord. On average, about 60-100 serial sections were used for spatial reconstruction. In the first stage, a set of serial images were opened using a computer program Ellipse and has ranked using using a specially designed module Allign (VidiTo, SK). The continuity of the series consisted of identifying the two basic morphological points in all serial images of the spinal cord (the first point: the center of the Central channel; the second point: the medial border of the dorsal horn) and subsequent computer build number is all images. To identify the boundaries of the dorsal and ventral horns lowered line kits are pre-ranked images using module Laminar Maps (Ellipse). Ultimately, the set of pre-ranked and processed using Laminar Maps images used for the spatial reconstruction using three-dimensional design (Media Cybernetics).

Culture of spinal NSC on rat astrocytes within two to three weeks showed time-dependent maturation and development of the neuronal phenotype in most cultures. This is confirmed by staining with species-specific antibodies against NSE or MOS. Identified numerous neurons with well developed axodendritic tree. The majority (85-90%) NSE-positive neurons are GABA-positive. In some MOS-positive neurons observed expression of synaptophysin in axona and dendrites.

There is a reliable survival of cells transplanted through twenty-one days after ischemic injury. This is expressed in the form of clearly identifiable bilateral grafts, immunologically reactive against NUMA, WA, or NSE, all species-specific antibodies. Analysis of horizontal slices taken from segments of the spinal cord, received the transplant, showed distinct migration NUMA is positive cells between the individual parts of the injection. Most NUMA-positive cells showed joint localization with MOS-immunoreactivity.

Double label sections of the spinal cord using antibodies against NUMA and GABA, were analyzed using confocal microscopy revealed on average 25-35% of GABA-positive cells. Stable expression of GABA-ergicheskoe phenotype was observed in all animals that received a transplant within seven weeks of survival.

At the same time (i.e. seven weeks after transplantation) double staining using antibodies against synaptophysin and NUMA showed dense synaptophysin-positive network within the grafts. Only occasionally NUMA-positive cells will show a joint localize with GFAP antibody. These cells are usually located at the periphery of the grafts.

Stereological evaluation of NUMA-positive cells shows the average 75460±5697 persistent transplanted cells within individual grafts. This is an average of 3-3 .6 times more cells than the original injected cells. The antigen of the cell cycle, Ki67 is a marker of cells with active mitosis. Staining of sections of the spinal cord in two to seven weeks after transplantation shows immunoreactivity against hKi67 only two weeks after transplantation. Only occasionally (1-2 cells/10 slices)cells are C-positive after seven weeks of survival. These results show that the transplanted spinal NSC and their precursors proliferate in an amount equivalent to average approximately three doublings of cells during the initial two-week period, then they are in a postmitotic state and stably integrated.

Three-dimensional reconstruction of segments L3-L5, which were transplanted, was performed using image 40 μm serial sections of the spinal cord, dyed MOS antibodies and DAPI (the total number of 150-200 slices). Three-dimensional reconstruction of the graft shows a well-recognized rostrocaudally oriented MOS-positive implant, distributed within the gray matter. As shown in Fig.7, Fig, the functional effect of spinal NSC assess the engraftment of the transplanted cells and the determination of favorable actions on motor function at the BBB. With regard to the degree of behavioral recovery observed in the present study, applicants after transplantation was observed by three principal groups. First: the animals that showed the strongest recovery and the ability to walk (BBB<16); second: animals that showed improvement in the active mobility of all three joints of the lower extremities, but were not str is obny stand (BBB about 8), and a third group in which the animals did not show any improvement (i.e. did not respond to treatment). While the reason for the differences in response to transplantation is not clear, the applicants believe that the subtle differences in the location of grafts with regard to disinhibitory primary afferents and/or α-motoneurons, may play a role. In addition, it should be noted that animals could survive only within 3 months of this study. Applicants believe that long-term survival after transplantation and continued physical rehabilitation, it may be associated with a higher degree of functional recovery. However, in contrast to the experimental group was not observed significant recovery none of the animals that were injected with only Wednesday.

Example 6. Treatment of diseases of the motor neurons in rats by transplantation of spinal NSC

NSC described methods provide clinical and biological benefits that are powerful and reliable. With this purpose, the described methods allow for the treatment of conditions as disseminated throughout the Central nervous system, such as ALS, and localized in a specific area, as in ischemia of the spinal cord, as described above. In ALS, although transpla the procedures in the lumbar spinal cord can miss other vital part of the segmental motor system, i.e. cervical nerve bundle motor neurons responsible for respiratory movements, describes how the NSC implantation into the spinal cord to facilitate the release of BDNF and GDNF other factors of transplanted cells in the CSF (CSF - approx. books, where you may experience a wider effect on the motor neurons of the host around the spinal cord.

Unexpectedly, it was found that partial NSC grafts in the lumbar segments of neurodegenerative environment of the spinal cord survive, undergo extensive neuronal differentiation and promote the survival and function of motor neurons in the implantation site and in other areas. NSC significantly delay the onset of symptoms and extend the life of rats G93A sod-1, model of human ALS (amyotrophic lateral sclerosis).

Rat G93A sod-1 is a comprehensive model of neurological pathology and clinical symptoms particularly aggressive form of ALS [(Nagai et al., 2001; Rowland et al., 2002)]. NSC from fetal spinal cord of mammals can be transplanted into the lumbar spinal cord of rats and mice G93A sod-1, where there is extensive differentiation of neurons and where differentiated neurons subsequently form synaptic contacts with neurons of the host and Express and release GDNF and BDNF. Rat G93A sod-1 model, for example, harakteryzuyutsya fulminant disease of motor neurons, can be used to study and demonstrate favorable effects NSC in this disease. To this end, the NSC grafts used in the described methods, survive well in neurodegenerative environment and provide a powerful clinical effects. At least part of these effects is related to the ability of these grafts to Express and release the growth factors of motor neurons. Accordingly, the transplanted NSC described methods delay the start and progression of fulminant disease of motor neurons and prolong the life of the specified animals more than ten days, in spite of the limited scheme transplantation, which was limited to the lumbar ledge.

NSC of the tissue of the spinal cord of the fetus at gestational age of eight weeks, obtained post mortem, propagation in free serum medium containing fibroblast growth factor (FGF-2), in the amount of approximately 10-12 passages before transplantation (Jone et al., 1996). The fate of these cells reliably traced using antibodies against nuclear antigens (Yan et al., 2003). All surgical procedures that use these cells made in accordance with the protocols included in this document as a reference, which were approved by the Committee on the content and use of animals medicine the institutions Johns Hopkins, using gas anesthesia (anfluran:oxygen:nitrogen=1:33:66) and methods of asepsis.

Alive or dead NSC transplanted into the lumbar protrusion (L4 and L5) rats G93A sod-1 both sexes at the age of nine weeks (220-300 g), fixed on the spinal stereotactic device Kopf under the control of the microscope. Dead cells were obtained by repeated freezing and thawing prior to transplantation. Cell suspension was taken under aseptic conditions by approximately eight injections aimed at the ventral horn on both sides of the ventral horn (5×104NSC at one injection site, four injection site on one side), with elongated-beveled glass micropipette connected via silicone tubes with 10 μl of microspace Hamilton. All rats received FK-506 (1 mg/kg/p) to prevent immune rejection, Osmose pilot data indicates that untreated animals or animals receiving cyclosporine, the survival of the graft does not exceed one month.

Rats were tested for motor strength and body weight twice a week. Tests on the motor force included locomotor rating scale Basso, Beattie and Bresnahan (BBB) (Basso et al., 1995) and the scale inclined plane (Rivlin and Tator, 1977). For testing on a scale BBB animals studied for about 4Or 5 minutes in the open field. All locomotor activity was recorded and evaluated in accordance with the scale. For testing on a scale inclined plane rats were placed on an inclined flat Mat and the angle is changed to the maximum value, in which the animals were able to stabilize its position for approximately 5 seconds. This angle is then recorded as the score of the animal on the scale of an inclined plane. Scores in the BBB and the scale of the inclined plane were analyzed by MANOVA with subsequent test Fisher LSD post hoc. Onset was defined as the point at which the body weight began to decline sharply. The disease as an effect of the type of transplant (transplant living or dead cells) was analyzed by comparison of age of onset and age of death in the two groups (using t-student test), and also by means of survival analysis Kaplan-Meier estimates, with subsequent long-rank criterion.

Rats were killed by perfusion-fixation, when their scores in the BBB (see below) was less than 3, at a stage at which only one joint was observed or not observed movements in General, and the animal was regarded as dying.

Tissue was obtained from animals that were perfesional 4% paraformaldehyde with a neutral buffer. Thoracic-lumbar segments of the spinal cord with attached roots and easycinema nerves additionally fixed by immersion in the same fixative for another four hours. Blocks containing transplantirovannam the entire area, plus 1 mm on the edges above and below the block, subjected cryoprotective and frozen for further processing. Roots L3-S1 were treated separately as solid preparations or after the separation of the roots using glass pipettes with melted ends. Of the blocks were made of slices in the transverse or sagittal plane (35 microns). Survival and differentiation of NSC studied using immunofluorescence double label, which unites in most cases, HNu, species-specific marker, with another cell marker, and carried out the study, as described by Yan et al., 2004.

To study the differentiation of NSC used metereological way of counting the total number of HNu(+) cells and cells labeled with doubly labelled HNu and phenotypic marker, on a randomly selected fields immunofluorescence preparations applicants with strong magnification (100x). From each animal used one box in each of the six sections of the field of transplantation, separated from each other by intervals of ~1 mm, Number of HNu(+) and profiles with double mark from all six calculated for each case fields were combined and grouped for the Protocol of the experiment. Dismissing the average number of cells with single and double label for each experimental group (n=6 in g is the SCP).

To assess the survival of motor neurons in rats, which transplanted living or dead cells (n=4 for each case), studied tissue from animals, dead at the age of 128 days. Selected every sixth cut in the field L3-S1 from each animal for stereological analysis (Yan et al., 2004) and calculated α-motor neurons, identified as multipolar cells with a distinct nucleus and a body diameter of >35 μm, using an optical fractionator, as described by Yan et al., 2004. Differences between animals that transplanted living or dead cells, were analyzed using t-student test.

To determine the neurotrophic factors of motor neurons using ELISA animals gas anesthesia selected CSF from the 4th ventricle 25G syringe. Tissue samples containing areas of transplantation and adjacent areas, across excised from 1 mm fresh slices of the spinal cord. CSF or tissue samples are processed and the first measure total protein, as described by Sheng et al., 2003. The levels of GDNF and BDNF were determined in CSF and in samples of the spinal cord using the system for immunological analysis of E-Mach ImmunoAssay system (Promega, Madison, WI). Absorption TMB-Chromogen was read at 450 nm. Analysis of variance for concentrations between samples from live transplants, areas adjacent to the graft is m, and grafts of dead cells was performed using one-way ANOVA, followed by the test according to the criterion of multiple comparisons Tukey. Differences in CSF concentrations between animals, which transplanted living or dead cells, were analyzed using t-student test.

For Western blotting of neurotrophic factors in motor neuron protein samples of CSF and spinal cord, prepared for ELISA were subjected to electrophoresis with molecular weight markers and transferred to membranes made of nitrocellulose. Spots were blocked in TBS, pH 7.4, containing 5% donkey serum, and then incubated with antibodies against GDNF and BDNF (1:500; over night, 4°C)and then with HRP-linked donkey anticosti IgG (GDNF) and anti-rabbit IgG (BDNF) (1:2000; Jackson ImmunoResearch) (1 hour, RT). All antibodies were diluted in TBS containing 5% donkey serum. Spots showed using a chemiluminescent substrate SuperSignal (Pierce) and overexposed film Kodak-XAR (Eastman Kodak, Rochester, NY). The stain was then removed and re-blotting was performed using antibodies against β-actin (1:500, Sigma) and HRP-linked donkey artemisinin IgG (1:10000, Jackson ImmunoResearch). Immunoreactive bands were analyzed using the computer program Bio-Rad Quantity One (Bio-Rad Laboratories, Hercules, CA). The ratio of the density of the spots (GDNF or BDNF: β-actin) was calculated for the animal and the average values for the groups were subjected to statistical analysis, as in the case of experiments using ELISA.

NSC in the spinal cord of rats G93A sod-1 through 22 weeks after transplantation identified by immunological staining of species-specific antibodies. Positive by staining the cells, as shown, has survived in the ventral horn (A) and was painted in the majority of markers of neuronal line, such as is associated with microtubules epitope TUJ-1. NSC identified by the signature of a nuclear protein, and their phenotypic fate was monitored using a double immunocytochemical analysis of nuclear protein and epitopes that are specific for neuronal precursor of neuronal and glial cells. By the end of the experiments on rats G93A sod-1 cells NSC showed robust engraftment of the graft and excellent long-term survival. Most of HNu(+) cells (70,4±6,4%) differentiated into neuronal line on the basis of their joint localization with TUJ-1. Approximately one fifth (19,2±5,6%) HNu(+) cells were localized together with mestinon and very few (1,3±0,9%) HNu(+) cells were positive for GFAP.

The ability of NSC integrated into the circuit of the owner studied using perikaryal markers for cells of the graft/host and markers, selective regarding endings as the host and graft. The sections were stained for nuclear protein set for the effect of the origin of the graft, on TUJ-1 for the establishment of neuronal differentiation and monoclonal antibody on the presynaptic protein Bassoon (BSN), which recognizes rat and mouse epitopes but does not recognize other epitopes. A large number of HNu(+) cells, TUJ-1(+) cells, located in the parenchyma was found to communicate through synaptic buds of rat origin.

According to the results of confocal microscopy obtained from NSC neuronal cells with TUJ-1(+) cytoplasm communicate through the rat endings. On the contrary, preparations stained with TUJ-1 and speciesspecific synaptophysin, find a dense terminal field of small buds intertwined with the neurons of the host, especially with large and small motor neurons. Motor neuron master is in contact with a large amount of graft buds. Horizontal sections stained for nuclear protein and NF-70, demonstrate a large number of graft axons emerging from the graft to the left and passing mainly along white matter ventral cord. Cells/processes with ChAT-immunoreactivity was used to differentiate between gray matter from white matter of the ventral horn. A large number of axons labeled species-specific antibody against neurofilament epitope NF70, as had the tableno, was in connection with places transplantation, which suggests that many NSC differentiated into planned neurons; these axons show a preference for white matter of the ventral horn. The NSC grafts in the lumbar spinal cord of rats G93A sod-1 increase lifespan and delay the death of motor neurons, as well as the beginning and progression of the disease. Progressional analysis of clinical and pathological parameters in cases of transplantation of living cells (L) and dead cells (control) are shown in Fig.9. Animals who transplanted living NSC, showed significantly higher survival as the test results of Kaplan-Meier and analysis of the final results. The graph of the Kaplan-Meier (figa) shows significant separation between experimental and control animals during the observation period (P=0,0003). Graphics score of BBB open field and test with an inclined plane in a time-dependent (figure 9) show a significantly slower progression of muscle weakness in animals, which transplanted living NSC, compared with animals that received the dead NSC.

The effect of NSC on the survival of motor neurons in the lumbar protrusion (L3-S1) rats TD studied on a small group of animals that receive the whether living or dead NSC and were euthanized at the age of 128 days. The average life expectancy of animals that are transplanted dead NSC was 138 days, while rats that transplanted living NSC, lived 149 days. Thus, a significant 11-day difference in life expectancy was observed between the experimental and control rats (P=0.0005). The average time to onset of the disease was 115 days for animals that have been dead cells, and 122 days for the animals, which transplanted living NSC. Significant 7-day difference in time to onset of the disease was observed between the two groups (P=0.0001).

Stereological set amount of α-motor neurons was 6418 for animals that received live NSC, and 3206 for the animals, which transplanted the dead NSC, i.e. in the lumbar protrusion experimental animals had twice as many neurons than in control animals of the same age. The difference in 3212 cells in the lumbar protrusion between the living and the dead NSC was observed (P=0.01) representative experimental and control rats at the age of 128 days.

Potential mechanisms of neuroprotection provided by NSC degenerating motor neurons, include the expression and release of two peptides with classical trophic effect on motor neurons in mammals [BDNF and GDNF] (Henderson et al., 1994; Koliatss et al., 1993). Determined the expression and release of GDNF and BDNF in the spinal cord of rats G93A sod-1-treated grafts. In the preparations of the spinal cord and CSF samples were determined BDNF and GDNF using Western blotting and ELISA. The concentration of GDNF in the parenchyma and CSF of rats, which transplanted living cells (L1 and L2), and animals that are transplanted dead cells (C)were determined using ELISA. L1 represents the concentration at the site of transplantation, while L2 reflects the concentration in the tissues of one segment above or below. The variance among groups is significant and is called the big difference between the groups L1 or L2 and C.

The difference of concentrations in CSF between experimental (living cells, L) and control (dead cells) groups is also significant according to the results of t-test. ELISA showed a concentration 0,912±0,050 PG/μg in the area of transplantation and 0,819±0,115 PG/μg in one segment later in the spinal cord of animals who transplanted living NSC. In animals, which transplanted the dead NSC specified concentration was 0,368±0,026 PG/μg in the segments of the spinal cord containing the graft. In CSF concentration of GDNF was 0,027±0,012 PG/ál in experimental animals and 0.006±0,002 PG/μl (control animals). These data show a three-fold increase in the expression and release of GDNF in the spinal cord and fivefold increased the E. secretion of GDNF in the CSF of animals living NSC.

Western blotting also shows a higher normalized concentration of GDNF in animals, which transplanted living NSC. GDNF Western blotting confirms the pattern of increase in ELISA by identifying protein 16 kDa. Western blotting also shows the normalized density GDNF 0,860±0,007 in the grafts of living cells and 0,708±0.052 in the grafts of dead cells.

Was determined by ELISA BDNF staining in the parenchyma and CSF of experimental rats and controls. The ELISA analysis showed a concentration of 0,086±0,014 PC/μg in the area of transplantation (L1) and 0,054±0,009 PG/μg in one segment further from the graft in experimental animals (L2). In control rats, the concentration of BDNF was 0,010+0,003 PC/μg in the segments containing the graft. The difference between experimental and control concentrations in CSF is significant. In CSF concentration of BDNF was 0,041±0,013 PC/µl in experimental animals and 0.010±0,008 PC/ml in controls. These data show an eight-fold increase in the concentration of BDNF in the spinal cord and a fourfold increase in CSF of experimental animals. Together ELISA data suggest a more widespread secretion of GDNF compared to BDNF in animals, which transplanted living NSC, especially in CSF.

Immunocytochemical analysis also found that the vast majority of transplantion the HNu(+) cells expresses GDNF. Source of GDNF in animals with live grafts are transplanted cells. In preparations with double staining of HNu (red) and GDNF (green) shows the excess of GDNF immunoreactivity in the cytoplasm of transplanted NSC. In animals that received live transplants, intensive GDNF immunoreactivity in all cytoplasmic structures resembling secretory vesicles in motor neurons of the host.

Confocal microscopy motor neuron owner painted GDNF and synaptophysin (the latter for marking obtained from transplant endings)shows the localization of GDNF in bubble structures, but not derived from the graft ends, located on the surface shown motor neuron owner. The sections stained with antibodies against synaptophysin (for tagging all endings transplant, Innervate motor neurons of the owner) and GDNF, show the absence of any joint localization of the two proteins in the buds that come in contact with motor neurons of the host.

Rats live transplants demonstrate the development paths originating from the graft and Innervate structures in the Central channel and around it. In contrast to the absence of GDNF protein in the endings in contact with the engine in Linyi neurons of the owner, the vast majority derived from NSC axons that Innervate the Central channel is localized together with GDNF-immunoreactivity. Widespread joint localization synaptophysin and GDNF was observed in obtained from transplant endings that Innervate cells ependyma in the Central channel. Specified anatomical patterns show that GDNF may, is captured by the endings of motor neurons of the owner, which innerviews graft through retrograde transport and are not delivered to the specified neurons through transsynaptic transfer (Rind et al., 2005).

The apparent resistance of the transplanted NSC to the ongoing degenerative process in the ventral horn of rat G93A sod-1 is a particularly promising. Survival and extensive differentiation of NSC reported in this document, is a powerful indication that the inflammatory/excitotoxicity alarm, which involves motor neurons containing G93A sod-1 (Rothstein et al., 1992; Howland et al., 2002; Turner et al., 2005), have no apparent toxic effect on the cells. This is only one factor encouraging in regard to future cellular strategies using grafts to restore motor function in degenerative disease of the motor is output neurons.

Example 7. Treatment of traumatic spinal cord injury by transplantation of neural stem cell/undifferentiated precursor cells of the spinal cord.

The treatment of those of syringomyelia

Reproduced spinal stem cells were injected with adult female rats of Sprague-Dawley with depressed immunity or Nude naked rats with immunodeficiency. Contusion injury With4-5was applied in both groups one month prior to transplantation. Recipients of grafts (n=24) had survived for 60-150 days after transplantation. Received NSC was formed large cell aggregates consisting of neurons, astrocytes and oligodendrocytes. These grafts consistently and completely filled every injury. Immature form of NeuN+/ core+neurons often included 50% of the population of donor cells. These neurons were sent neurofilament+shoots through the gray and white matter at a distance of at least 2 cm from the site of transplantation. Intense immunoreactivity for synaptophysin was noted in the vicinity of neurons as the host and graft, and, apparently, non-reactive GFAP+cells were placed next to the donor neurons. Further, these grafts have supported the growth of T+and T+fibers, which apparently arose from the sources of the owner. This line NSG, therefore, as pre is supplied, primary fetal CNS-like qualities conducive to interspinales recovery of the gray matter.

It should be understood that various changes and modifications of the preferred embodiments of the present invention, described herein, will be understood by specialists. These changes and modifications can be effected without departure from the idea and scope of the described methods and without reducing their plan benefits. Thus, it is expected that these changes and modifications will be covered by the attached claims.

1. Method of culturing neural stem cells of a mammal, excluding embryonic human cells, comprising: providing a neural stem cell of the mammal; pre-incubation in the culture vessel of a solution containing poly-D-lysine at a concentration of from 0.1 μg/ml to 1 mg/ml for 5 min to 3 h; rinsing and drying the culture vessel;
the seeding neural stem cells in the specified culture vessel in the absence of serum; adding a solution of fibronectin and at least one mitogen in the culture vessel and culturing neural stem cells in the absence of serum; and the passage of cultured neural stem cells to merge.

2. the procedure according to claim 1, in which mitogen includes basic fibroblast growth factor (bFGF).

3. The method according to claim 1, in which a solution of poly-D-lysine has a concentration of poly-D-lysine approximately 100 µg/ml.

4. The method according to claim 1, in which stage of incubation of the culture vessel with a solution of poly-D-lysine runs approximately 1 hour

5. The method according to claim 1, in which the solution of fibronectin has a concentration of fibronectin approximately 1 µg/ml.

6. The method according to claim 1, in which the solution of fibronectin and at least one mitogen added to the culture vessel with a preliminary coating simultaneously with seeding neural stem cells.

7. The method according to claim 1, in which neural stem cells are tissue cells of the Central nervous system.

8. The method according to claim 7, in which the tissue of the Central nervous system includes the spinal-cord tissue.

9. The method according to claim 8, in which the tissue of the Central nervous system obtained from post-mortem of the embryos of mammals, non-human, in gestational age from approximately 6.5 to 20 weeks.

10. The method according to claim 1, in which neural stem cells of a mammal is provided from the embryo and fetus, non-human.

11. The method according to claim 1, in which neural stem cells isolated from a source selected from the group consisting of Central nervous system, periferiche the coy nervous system, bone marrow, peripheral blood and umbilical cord blood.

12. The method according to claim 1, wherein the mammal is a developing mammal.

13. The method according to item 12, in which the gestational age of the developing mammals is from about 6.5 to 20 weeks.

14. The method according to claim 1, wherein the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and their combinations.

15. A method for the treatment of spasticity, rigidity, spinal cord or amyotrophies state of the subject in need of such treatment, comprising: a concentration of neural stem cells cultured according to claim 1, getting concentrated neural stem cells; introducing a therapeutically effective amount of the above concentrated neural stem cells into the region of tissue of the Central nervous system of the patient with a reduced level of GABA-producing or glycine-producing neurons; and generating a GABA-producing or glycine-producing neurons in the specified fabric.

16. The method according to item 15, in which amyotrophies condition includes amyotrophic lateral sclerosis or sluggish paraplegia.

17. The method according to item 15, in which stage the introduction of a therapeutically effective amount of the above concentrated neural stem cells comprises injecting parts of concentrated allnightlong cells.

18. The method according to 17, in which concentrated neural stem cells in a therapeutically effective amount is injected through multiple injection.

19. The method according to p, in which at least two injections of a specified set of injections performed in the areas of injection at a distance of from about 100 microns to about 5000 microns from each other.

20. The method according to item 15, in which condition caused by traumatic spinal cord injury, ischemic spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, cerebral palsy, epilepsy, Huntington's disease, amyotrophic lateral sclerosis, chronic ischemia, hereditary conditions, or any combination thereof.

21. The method according to item 15, in which a therapeutically effective amount multiplied the population is able to generate at least 1000 GABA-producing neurons in vivo.

22. The method according to item 15, in which a therapeutically effective amount multiplied the population is able to generate at least 1000 glycine-producing neurons in vivo.

23. The method according to item 15, in which at least 40% of the population multiplied capable of generating neurons in the spinal cord.

24. The method according to item 15, in which the introduction of a therapeutically effective amount multiplied ass is acii includes injecting at least part of a therapeutically effective quantity in many areas of the spinal cord of the patient.

25. The method according to item 15, in which at least 30% multiplied populations are able to differentiate into neurons in vitro.



 

Same patents:

FIELD: medicine.

SUBSTANCE: method includes filling work chamber with suspension of tumour cells and their aggregates. After that carried out is complex processing of suspension of tumour cells and their aggregates by high-amplitude low-frequency ultrasound and ozone/NO - containing gas mixture, introduced immediately into volume of insonifies and bubbled suspension, as well as ozone/NO-containing solution, which is formed at barbotage of suspension with ozone/NO-containing gas mixture.

EFFECT: invention makes it possible to increase efficiency of process of disintegration of suspension of tumour cells and their aggregates for maximally possible separation from them of biologically active substances.

6 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method of cell expansion includes culturing adhesive mesenchymal stromal cells from placenta or adipose tissue in three-dimensional conditions of culturing, which contribute to expansion of cells.

EFFECT: invention allows obtaining adhesive mesenchymal stromal cells of placenta or adipose tissues, which can by applied for supporting hematopoetic stem cells, for immunosuppression, for obtaining conditioned media in transplantation.

22 cl, 22 dwg, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method includes opening of abdominal cavity, cannulating of v.portae, through which two-step perfusing is performed, mechanical processing of liver, purification of hepatocytes, filtration and differential centrifuging, preliminary incubation and inoculation of hepatocytes in Goryaev chamber, primary perfusing being performed with calcium-free Hanks solution, the secondary one being performed three-four times with Hanks solution, which contains calcium ions and 1 type collagenase in concentration 0.02-0.05% for 20 minutes at perfusate temperature 15-16°C; purification of hepatocytes in ficoll gradient is performed three-four times for 15 minutes on centrifuge with 1000 rev/min at temperature 20°C with addition of 0.5-1.15 M of saccharose, differential centrifuging is carried out with 700 rev/min with 2 minute exposition; preliminary incubation of hepatocytes is carried out at 37°C for 20 minutes.

EFFECT: invention makes it possible to increase percentage of output of viable hepatocytes of birds of genus Columba livia, and can be used for studying mechanisms of cell functioning, in diagnostics of viral infections, in production of vaccines, correction of functions of injured liver tissues.

3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: described is method of cell analysis by means of biochip, containing immobilised molecules of substances, able of binding with molecules, which are on the surface of cells, includes incubation of biochip with suspension of cells. Then, washing of biochip from nonspecifically bound cells is carried out. After that, fixation and staining of cells, reading of results and estimation of quantity of cells, which bound in one or several parts of biochip of the given area, are performed. In conclusion, analysis of the image of bound cells is carried out. Before carrying out fixation from biochip surface excess of liquid is removed, without permitting it to dry.

EFFECT: improvement of technology.

4 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: method includes cultivation of pluripotent stem cells in culture medium, which does not contain substance, stimulating activation of canonical signal way Wnt, during period of time between beginning of differentiation induction and 24 hours before period of increased expression of genes of canonical Wnt. After that carried out is cultivation of pluripotent stem cells in culture medium, which contains substance, stimulating activation of canonical signal way Wnt, during period of time from 24 to 96 hours, starting from 24 to 0 hours before period of increased expression of genes of canonical Wnt.

EFFECT: method makes it possible to induce cardiomyocyte differentiation efficiently and selectively.

15 cl, 15 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: method includes cultivation of pluripotent stem cells in culture medium, which does not contain substance, stimulating activation of canonical signal way Wnt, during period of time between beginning of differentiation induction and 24 hours before period of increased expression of genes of canonical Wnt. After that carried out is cultivation of pluripotent stem cells in culture medium, which contains substance, stimulating activation of canonical signal way Wnt, during period of time from 24 to 96 hours, starting from 24 to 0 hours before period of increased expression of genes of canonical Wnt.

EFFECT: method makes it possible to induce cardiomyocyte differentiation efficiently and selectively.

15 cl, 15 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: method of cryoconservation of multipotent mesenchymal stromal cells (MMSC) includes preparation of culture mixture in culture medium, mixing cell culture in culture medium with cryoprotector, step-by-step controlled cooling and freezing mixture of culture medium with cryoprotector to storage temperature and further storage of frozen mixture at low temperatures. As cryoprotector for mixture saturation used is xenon gas, introduced till saturation is achieved into prepared mixtute of cell culture MMSC in DMEM medium, which is in open cryo test tubes by blowing culture medium with xenon at 0°C with further cooling and formation of primary monolith of culture medium with cryoprotector, directed later at cooling to storage temperature.

EFFECT: invention ensures preservation of cells in cryopreservation.

4 cl, 2 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: described is method of obtaining population of stem cells, which come from dental follicle of human, named FENC (coming from follicles stem cells of embryonic nerve crest), which include: a) collection of follicle sac in sterile conditions, splitting, growing and grafting of initial culture; b) optional amplification; c) sorting out on FAC - sorter by the following stem characteristics: SSEA-4, TRA 1-60, TRA 1-81, OCT-4+; d) analysis of RNA with respect to positivity by transcriptional factors Nanog and Rex-1.

EFFECT: invention makes it possible to obtain homogenous population of stem cells.

10 cl, 6 ex

FIELD: medicine.

SUBSTANCE: method provides the following stages: (a) preparing a matrix, (b) preparing isolated hepatocytes, (c) inoculating a matrix of density 2 to 4x103 mm-2 with hepatocytes, (d) leaving the cell-coated matrix for 10-180 minutes so that to enable the cells to adhere to the matrix, (e) washing the nonadherent cells from the cell-coated matrix, (f) leaving the cell-coated matrix for max. 180 minutes, and (g) freezing the cell-coated matrix in a freezing medium, (h) storing the frozen cell-coated matrix, (i) defrosting the frozen cell-coated matrix, (j) washing the nonadherent cells from the cell-coated matrix, (k) coating the defrozen cell-coated matrix with a layer of a second matrix, and (1) reculturing the cells built-in between the matrixes.

EFFECT: invention allows providing an increased percentage of viable hepatocytes in the sandwich-type cultures.

12 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves increasing biomass of human embryo stem cells of the feeder-less hESKM-05 with the use of a base mTeSR medium in the bottles coated with 0.1 % gelatin. An increasing procedure is enabled with the use and daily change of a base coDMEM medium containing 10% serum substitute SR, 100 mcg/ml of kanamycin sulphate, 1 mM L-glutamine, 4 ng/ml of a base fibroblast growth factor (bFGF), 1 mM essential amino acids. A conditioned medium is produced from a culture of mice embryo neuronal cells. The increased biomass is transferred by means of 0.5 % collagenase in bottles containing a prepared collagen-chitosan matrix in the conditioned medium or in a complete nutrient medium with neuronal factor N2 added. The medium is changed every three days.

EFFECT: invention allows producing the neuronal matrix suitable for direct transplantation.

16 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of formula (I), in which X denotes N or CR3, M denotes (CH2)m; m equals 0 or 1, R1 denotes H or lower alkyl which can be substituted with a group selected from a group consisting of mono- or di-lower alkylamino and -O-lower alkyl, R2 denotes H or lower alkyl, R3 denotes H or lower alkyl substituted with a group selected from a group consisting of halogen, mono- or di-lower alkylamino and cyclic amino, R41 denotes H or pyridine which can be substituted with a cyano group, R42 denotes a bridged polycyclic hydrocarbon or a bridged azacyclic hydrocarbon, each of which can be substituted, R5 denotes a group selected from a group consisting of halogen, cyano, lower alkyl-carbonyl, lower alkyl-oxycarbonyl, hydroxycarbonyl, formyl, amidinooxycarbonyl, guanidinooxycarbonyl, guanidino, carbamoyl, -C(=O)-5- or -6-member heterocycloalkyl, -C(=O)-5- or -6-member heteroaryl, lower alkyl, lower alkenyl, -O-lower alkyl, 5- or 6-member heterocycloalkyl and 5-member heteroaryl, each of which can be substituted, provided that when R5 denotes a 5-member heteroaryl, X denotes -CR3; or R41 and R15 can be bonded through a defined functional group to form divalent groups shown below: (I-A) (I-B) or (I-C), in which RA denotes H or acyl, which can be substituted, provided that the term "substituted" with respect to R4 and/or R5 denotes substitution with one or more substitutes selected from a group comprising the following substitutes: (a). halogen; (b) -OH, -O-R2, -O-phenyl, -OCO-RZ-OCONH-RZ oxo (=O); (c) -SH, -S-R2, -S-phenyl, -S-heteroaryl, -SO-R2, -SO-phenyl, -SO-heteroaryl, -SO3H, -SO2-RZ, -SO2-phenyl, - SO2-heteroaryl, sulphamoyl, which can be substituted with one or two RZ groups; (d) amino, which can be substituted with one or two RZ groups, -NHCO-RZ, -NHCO-phenyl, -NHCO2-RZ, -NHCONH2, -NHCONH-RZ, -NHSO2-R0, -NHSO2-phenyl, -NHSO2NH2, -NO2, =N-O-RZ; (e) -CHO, -CO-RZ, -CO2H, -CO2-RZ, carbamoyl, which can be substituted with one or two RZ groups, -CO-cyclic amino, -COCO-RZ, cyano; (f) RZ; (g) phenyl, which can be substituted with one or more groups selected from substitutes described above in paragraphs from (a) to (f), a 5- or 6-member heterocycloalkyl, a 5- or 6-member heteroaryl, a 5- or 6-member heterocycloaryl; or pharmaceutically acceptable salts thereof. The invention also relates to a method of producing compounds of formula II, a pharmaceutical composition based on said compounds which is a Janus kinase 3 inhibitor, a method of treating and/or preventing different immunopathological diseases, including autoimmune diseases, inflammatory diseases and allergic diseases.

EFFECT: novel compounds are obtained and described, which can be used as an active ingredient of an agent for treating or preventing diseases caused by undesirable cytokine signal transmission or diseases caused by pathological cytokine signal transmission.

14 cl, 579 ex, 72 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to novel pyrimidine derivatives or their pharmaceutically acceptable salts, possessing inhibiting activity with respect to glycogensintase kinase-3 (GSK3). In compound of formula I: R1 is selected from hydrogen, cyano, C1-3alogenoalkinyl, SO2NRbRc, C0-2alkyl(O)NRbRc, C1-4alkylNBbRc, SO2Ri, C(O)ORa, CH(OH)Rj and C(O)Rj; R2 and R4 are independently selected from hydrogen, halogeno, cyano, NO2, C1-4alkyl, C1-3ahalogenoalkyl, ORa, C(O)NRbRc, SO2Ri, and C(O)ORa; or R1 and R2 together with atoms, to which they are bound, are bound with formation of 5- or 6-member heterocyclic ring, which contains one S, any of the hydrogen atoms of group CH2 in said heterocyclic ring can be substituted by oxo, hydroxy, and sulphur atom in said heterocyclic ring is probably oxydised to -SO2-; R3 and R5 represent hydrogen; R6 represents tetrahydropyran; R7 is selected from hydrogen, C1-3alkyl, cyano and C1-3halogenoalkyl; R8 represents hydrogen; Ra is selected from C1-3alkyl and C1-3halogenoakryl. Other radicals are given in formula of invention.

EFFECT: compounds can be applied in manufacturing medication for prevention and/or treatment of predemential states, moderate cognitive failure and type II diabetes, Alzheimer's disease and Parkinson disease, as well as bone-associated malfunctions.

40 cl, 3 dwg, 1 tbl, 122 ex

FIELD: medicine.

SUBSTANCE: what is offered is a monoclonal antibody which binds with Aβ Globulomer containing CDRs of a heavy and light chain. There are described: composition for diagnosing Alzheimer's disease, and also a composition and an antibody-based vaccine for preventing or treating Alzheimer's disease. What is disclosed is application of the antibody for preparing a drug for the intended application stated above. There are described: versions of a diagnostic technique for Alzheimer's disease, an antibody-based diagnostic kit for Alzheimer's disease.

EFFECT: use of the invention provides new Aβ Globulomer antibodies which are detected more selectively than common antibodies.

16 cl, 10 dwg, 5 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to application of neramexane or its pharmaceutically acceptable salt in a combination with a glutamate release inhibitor for treating neurodegenerative disorders, and also emotional instability and pseudobulbar affect associated with neurodegenerative disorders. The glutamate release inhibitor is selected from riluzole and its pharmaceutically acceptable salts. The neurodegenerative disorder represents a motor neuron disease.

EFFECT: therapy with the combined neramexane and riluzole provides relieved progression of the neurodegenerative disease which surpasses a relief effect induced by therapy with riluzole only.

35 cl, 4 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel cyclohexylamine derivatives of formula (I), having inhibiting properties towards at least one monoamine transporter, such as serotonin transporter, dopamine transporter or norepinephrine transporter, or a combination of two or more transporters. The compounds can be used to treat and/or prevent central nervous system disorders such as pain, depression, anxiety, schizophrenia, sleep disorder etc. In formula (I) , n equals 0 or 1; s equals 1, 2 or 3, m equals a whole number from 0 to 12; Ar is

or where Y and Z are (i) both halogen; or (ii) one of Y and Z is CF3 or OCF3 and the other is hydrogen; Y1, Z1, Y2 and Z2 each independently denotes H or a halogen; each X independently denotes H, halogen, CF3, OR5, (C1-C4)alkyl, optionally substituted with halogen or OH, or NR6R7; each R1 and R2 independently denotes H or (C1-C6)alkyl; and each R3 and R4 independently denotes H or (C1-C9)alkyl optionally substituted with OH; where each R5 independently denotes H, (C1-C4)alkyl or phenyl; and each R6 and R7 independently denotes H or (C1-C4)alkyl; where at least two of R1, R2, R3, R4 and X together with atoms to which they are bonded are optionally bonded to form a 5-6-member ring, where the 5-6-member ring is selected from: a) R3 and R4 together with a nitrogen atom to which they are bonded optionally form a pyrrolidine, piperidine, piperazine or morpholine ring, which is optionally substituted with (C1-C4)alkyl; b) when R3 is H or lower alkyl, X and R4 together with atoms to which they are bonded optionally form a 1,3-oxazine ring; c) two X substitutes together with a carbon atom to which they are bonded optionally form a 1,3-dioxolane ring; and d) when R1 and R3 denote hydrogen, R2 and R4 together with atoms to which they are bonded optionally form a 5- or 6-member saturated heterocyclic ring containing one nitrogen atom.

EFFECT: high efficiency of using the compounds.

29 cl, 36 dwg, 11 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there is introduced a pharmaceutical composition containing a polyunsaturated fatty acid selected from omega-3 fatty acid or omega-6 fatty acid, uridine and choline salt.

EFFECT: intellectual improvement ensured by a synergic effect of the ingredients of the composition to increase the concentration of brain phospholipids.

19 cl, 12 ex, 9 tbl, 10 dwg

FIELD: medicine.

SUBSTANCE: standard treatment regimen additionally includes gliatilin which is injected intramuscularly 2 ml daily in the therapeutic course 10 injections, and combined with transcranial micropolarisation. Transcranial micropolarisation is performed every second day at exposition 20 minutes on the first session with exposure time to be increased by 5 minutes on each following session; the therapeutic course is 5 procedures. Electrodes are applied on a frontoanterior area (an anode), on a mastoid process (cathode), on an occipital region (anode) of a hemisphere with the same name with involving thereafter an opposite hemisphere in the end of treatment.

EFFECT: improved values of a visual memory, a audio-voice memory, attention, thought processes, emotional sphere that allows reducing considerably hospitalisation length and providing higher clinical effectiveness.

10 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: what is offered is application of idebenone for preparing a drug for transmucosal introductions for treating mitochondral, neurological and neuromotor diseases, and the drug is introduced through a nasal mucosa, an oral mucosa or a colon mucosa.

EFFECT: composition for transmucosal introduction enables to avoid a considerable first-pass metabolism of idebenone, to provide introduction in a lower dose, reduced risk of side effects and higher compliance.

8 cl, 2 dwg, 7 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention discloses a compound of formula I

, in which radicals and groups are described in the claims. Said compounds are 5-hydroxytryptamine-6 (5-HT6) ligands and can be used to treat central nervous system disorders associated with or influenced by the 5-HT6 receptor. The invention also relates to a pharmaceutical composition and a method of treating said disorders.

EFFECT: high efficiency of using said derivatives.

19 cl, 13 tbl, 204 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine. Described is preparative form for transcutaneous introduction, which makes possible stable introduction of medication against dementia for a long period of time. Preparative form for transcutaneous introduction of medication against dementia, which is used as plaster on skin, contains, at least, one adhesive layer, intermediate membrane and layer, which is reservoir of medication, successively, from the side, which is stuck on skin, where layer, which is reservoir of medication, contains, at least, one medication against dementia, aminated polymer, polyatomic alcohol and one or several esters of carboxylic acids.

EFFECT: intermediate membrane makes possible controlled penetration of medication against dementia towards skin, adhesive layer makes it possible to stick preparative form for transcutaneous introduction on skin and is permeable for medication against dementia.

9 cl, 4 dwg, 1 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to compounds of formula (I) given below or pharmaceutically acceptable salts thereof:

[where: each of X, Y, Z and W independently denotes a methane group which optionally contains substitutes selected from a group of substitutes α, or a nitrogen atom (except when all elements X, Y, Z and W denote a methane group which optionally contain substitutes selected from the group of substitutes α); A denotes -(C(R3)(R4))m1-; B denotes -O-; D denotes -C(O)-; m1 equals 0; Q denotes a methane group or a nitrogen atom; R denotes a group of formula (II)

, where R6 denotes a lower alkyl group; R7 and R8, together with the nitrogen atom with which they are bonded, form a 5-6-member nitrogen-containing aliphatic heterocyclic group; and where the group of substitutes α includes the following substitutes. Group of substitutes α: halogen atom, hydroxyl group, lower alkyl group, alkoxyl group (said group can be substituted with a cycloalkyl group), amino group, mono- or disubstituted lower alkylamino group, aryl group (said group can be substituted with a halogen atom, a -SO2CH3 group), aryloxy group (said group can be substituted with a halogen atom), heteroaryl group, where 'heteroaryl group' denotes a 5- or 6-member monocyclic saturated or unsaturated group containing 1-2 heteroatoms selected from an oxygen atom or a nitrogen atom (said group can be substituted with an alkoxyl group, alkyl group). The invention also relates to a histamine 3 receptor antagonist, histamine 3 receptor inverse agonist, a prophylactic or medicinal agent, as well as a pharmaceutical composition.

EFFECT: obtaining novel biologically active compounds having histamine H3 receptor antagonist or inverse agonist action.

15 cl, 57 ex, 1 tbl

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