Use neuroprosthetic embryonic cell lines for transplantation therapy

 

The invention relates to medicine, in particular to transplantation. Declared immortalitya neuroblastoma cell line of a human embryo, in which SVG cells transfection the plasmid phTH/Neo, with the specified cell line capable of expression of a nucleic acid sequence that encodes tyrosinekinase, and able to produce and secrete dopamine. Also stated transplantable composition comprising cells from a cell line that is encapsulated by a membrane from a gel of alginate impermeable to antibodies. The invention allows to obtain material for transplantation, which does not cause side effects. 2 C. p. F.-ly, 3 tab., 21 Il.

The present invention mainly relates to the creation of methods of treatment of the recipient due to the implant recipient is not genetically related cells. More specifically, in accordance with the present invention offers an immortalized neuroprostanes cell lines of human embryo and methods of treatment of the recipient at the expense of implant recipient or patient of these cell lines.

Organ transplantation has become a successful and widely practiced is ecene in many medical centers. In misery, diseases of many organs can not be eliminated by complete replacement of the entire body for the transplant. For example, lesions of the Central nervous system can't be fixed by replacing the affected tissue to transplant the entire body.

Since the replacement of the affected tissue using stem cell-based therapy whole body is impossible, in the case of many diseases or even for all patients who have certain diseases, attempts were made to develop methods of transplantation of the cells. [San and others, Biomat., Art. Cells. Art. Org., 15:483-496 (1987)]. Parenchymal lesions, which lead to the deficiency of biologically active compounds, can be treated by transplantation of isolated cells or groups of cells that secrete biologically active compound. For example, animals with diabetes were successfully treated with implantation of islets of Langerhans isolated from the pancreas of a donor. [Noel and others, Metabolism, 31:184 (1982)].

Cell transplantation therapy is particularly attractive for the treatment of neurological diseases. Transplantation of whole tissue is not suitable for the treatment of neurological disorders for many reasons. Surgical op is idania ways the nervous system, leading to clinical neurological disorders. Moreover, neurological function often depends on complex intercellular connections that cannot be repaired surgically. In addition, cells of the Central nervous system is extremely sensitive to hypoxia and lack of power. Rapid vascularization of the grafts solid tissues is critical, as the cells within the transplant of whole cloth often do not have sufficient perfusion to maintain viability. [Stenevi and others, Brain Res., 114:1-20 (1976)].

Known neurological syndrome, Parkinson's disease, has already been the object of attempted application of cell transplantation therapy. [Bjorklund and others, Brain Res., 177: 555-560 (1979); Lindvall and others, Science. 247:574-577 (1990); Fried, Restor. Neurol. Neurosci., 3:109-134 (1991)]. Parkinsonism is caused by loss of dopamine-producing neurons in the substantia nigra (black substance) basal nuclei. [Burns and others, N. Engl. J. Med., 312:1418-1421 (1985); wolf and others, Neurobiology, 86:9011-9014 (1989)]. Parkinson's disease is a disorder of unknown etiology that is characterized by certain clinical manifestations and is called idiopathic destruction of these dopamine-producing neurons. Parkinsonism can be caused by various the l-1, 2, 3, 6 tetrahydropyridine. [Burns and others, Proc. Natl. Acad. Sci. USA, 80:4546-4550 (1983) and Bankiewicz and other, Life Sci., 39:7-16 (1985)].

Attempts were made to reverse the clinical manifestations of experimentally induced parkinsonism using transplantation of dopaminergic cells in the striatum (striped body) affected animals. Genetically modified fibroblast (transfection which was produced together with DNA encoding the tyrosine hydroxylase) were successfully transplanted animals with lesions of the dopaminergic pathways. After implantation of producing dopamine of fibroblast improved motor function and behavior of animals. [Wolf and others, Proc. Natl. Acad. Sci. USA, 86:9011-9014 (1989); Fisher and others, Neuron, 6:371-380 (1991)]. Term viability of the graft can be increased, and can even be prolonged clinical improvement when using transplantation of embryonic tissue, when compared with transplantation of cells obtained after birth. [Gaige and Fischer, Neuron, 6:1-12(1991)]. Produced transplant fresh embryonic dopaminergic neurons in the caudal nucleus of the monkey after chemical damage nigrostriatal dopamine system. As a result of transplantation caused by damage to behavioral Russ, tragoudia Parkinson's disease, treated with the help of veins implantation of dopaminergic neurons. [Lindvall etc., Arch. Neurol, 46:615-631(1989); Widner and others, New Engl. J. Med., 327:1556-1563 (1992)]. The transplanted cells were obtained abortions. Before abortion was performed screening women for antibodies of various diseases caused by viruses. After implant surgery patients experienced improvement of neurological function. However, they had to undergo immuno-depressive therapy.

Recent studies show that trophic factors that are highlighted in the supporting cells of the Central nervous system (e.g., astrocytes and oligodendrocytes) are critical to term viability of neurons in cell culture. [O'malley and others, Exp. Neurol., 112:40-48 (1991)]. It was shown that implanted the fibroblast, which have been genetically modified to expression (expression) growth factor nerve, and extend the viability of cholinergic neurons of the basal forebrain after damage to the edge - set of the brain, which causes the destruction of acetylcholine neurons in the basal fore brain, which manifests itself in Alzheimer's disease. [Rosenberg and others, Science, 242 disorders have yielded encouraging results, there are still many significant problems. Supply of fetal tissue for cell transplants are extremely limited. To ensure maximum viability selection of fresh embryonic cells should be performed immediately before transplantation. This requires approval of the procedure of implantation with certain abortions. Even in this case, in the United States there is a wide range of embryonic tissue. In addition, gestational age of the embryo from which the cells affect the pot life of the transplant. [Gaige and Fischer, see above]. The requirement of obtaining embryonic tissue only certain gestational ages adds certain restrictions on the availability of embryonic cells for transplantation. In addition, ethical considerations induce some potential recipients of grafts reluctant to go for the procedure, which is injected fresh embryonic stem cells.

Because fetal tissue obtained from fresh abortion, there is a significant risk of infectious contamination. Despite the fact that women undergoing abortions that will give embryonic tissue, are screened for many infections, some the e screening. As a result, if widely used, the fresh grafts of embryonic cells is likely to be the cause of many infectious complications.

The use of immortalized cell lines allows us to overcome many of the difficulties associated with the required number (grafts) and infections. Message immortalized neuroprosthetic cell lines of human embryo made by Mayer and others, Proc. Natl. Acad. Sci. USA. 82:1257-1262 (1985), and in U.S. patent No. 4707448. It should be borne in mind that an immortalized cell lines, by their very nature are prone to the formation of tumors after transplantation in vivo. As a result, therapeutic intracerebral transplantation of immortalized cells have a high risk of intracranial tumors, and even tumors with benign histology have a poor prognosis when present in the cranial vault.

In addition to the risk of tumors, transplants are not genetically related cells also have the risk of immunological rejection of the transplant and intracerebral inflammation. [Widner and Brandin, Brain Res. Rev., 13:287-324 (1988). All grafts are not genetically related cells possess is transplantat, must be the rate of long-term maintenance immunosuppression, which even in the absence of transplanted immortalized cells has a high risk of infections and malignant complications.

Transplantation of immortalized cells only increases the risk of such complications.

Therefore, urgently needed therapeutic methods of implantation immortalized cells of a human embryo, and cell lines that are suitable for such use. Ideally, such methods should not lead to the formation of tumors or cause severe inflammation after transplantation. It is desirable that such methods could be used cells derived from cell lines to minimize the risk of infectious contamination and the limited availability of cells. Surprisingly the present invention can satisfy these and other related needs.

In accordance with the present invention provides methods of treatment of the recipient, which provides for implantation in the recipient cells immortalizing neuroprostanes cell line of a human embryo. Usually cell line is derived from act in the Central nervous system of the recipient. Cells can be encapsulated by membranes that are permeable to antibodies of the recipient.

In accordance with some variations of the present invention, transfection of cells can be produced with a sequence of nucleic acid that encodes the peptide. Peptides are usually enzymes, such as tyrosine hydroxylase, or growth factors such as growth factor nerve. The peptide may also be associated with a disease antigen. Implantation of cells can be done for treatment or for prevention. In some cases, the cells can be removed after implantation.

In accordance with additional options of the present invention, it is proposed immortalitya neuroblastoma cell line of a human embryo, which contains xenogenic nucleic acid sequence, and cell line is able to exercise (to Express) xenogenic nucleic acid sequence. Particularly preferred cell line is able to demonstrate a nucleic acid which encodes tyrosine hydroxylase. In more preferred aspects, the cell line in accordance with the present invention can be serotonin.

In which contains cell line in accordance with the present invention, as well as pharmaceutically acceptable carrier.

In Fig.1 shows the morphology of SVG cells in vitro.

In Fig.2 shows staining immunoperoxidase antibody protein SV40 T in SVG cells.

In Fig.3 shows the trace of the needle in the basal nuclei at low magnification.

In Fig.4 shows the trace of the needle in the basal nuclei at high magnification.

In Fig.5 shows another view of the track of the needle in the basal nuclei at high magnification.

In Fig.6 shows the socket SVG cells on the wall of the lateral ventricle.

In Fig.7 shows the implanted SVG cells on the wall of the lateral ventricle stained with the antibody for glial fibrillar acidic protein.

In Fig.8 shows a cross-section of in vivo implanted SVG cells stained with the antibody anti-T-protein.

In Fig.9 shows the T1the weighted MRI (with gadolinium enhancement) the brain of a monkey at 6 months after implantation.

In Fig.10 shows the growth of a neuron tyrosine hydroxylase in the implanted layer SVG cells in vivo.

In Fig.11 schematically shows the formation of a plasmid used in the formation of SVG-T cell lines.

In Fig.12 shows immunohistochemical staining of tyrosine-hydroxylase stable phTH/Neo transfectant.

In Fig.13 shows Western blotting of odnesena chromatogram, obtained by HPLC analysis (HPLC), cell culture supernatant SVG-TH. Two peaks, with retention times of 25, 65 and 37, 1 minute, correspond to the retention times for serotonin and 5-hydroxyindoleacetic acid, a breakdown product of serotonin. This was confirmed by immunohistochemical staining SVG-TH cells to serotonin.

In Fig.15 shows an electron micrograph SVG-TH cells.

In Fig.16A shows immunohistochemical staining of cells from cocultivation hNT/SVG-TH, after approximately 72 hours. Small flat cells represent the SVG-TH cells. In Fig.16B shows immunohistochemical staining of cells from cocultivation PC12/SVG-TH, after approximately 72 hours. Cell PC 12 shown near the center of the photograph, with neural processes coming from her neighbor SVG-TH cells. Similar results were obtained for SVG cells.

In Fig.17 shows a graph of the distribution of the number of TONS of positive cells for dopaminergic cells, seeded in the amount of 100,000 cells in the chamber with holes migration (transwell), SVG coculture without it. Shows the number of TONS of positive cells medium rat brain remaining in the chamber transwell (Y-axis), when these cells were cultivated the results.

In Fig.18 shows the effect of the SVG-TH transplantation for Parkinson's disease model rats. Functional impairment is indicated by the number of spins per hour models in rats. Shows the number of spins per hour after the rat was provocative entered apomorphine (control and infected), before and after 4 weeks after implantation SVG-TH cells in the affected striatum of rats (results shown are blacked out squares, blackened by almasiani, light boxes and light almasiani). Also shows the results of two SVG transplant (blackened triangles and light squares).

In Fig.19, 20 and 21 shows the improvements in the treatment of Parkinson's disease in the graft SVG-TH cells in the caudate nucleus and in putamen three rhesus monkeys (N, N and T) who received MRTR to create symptoms of Parkinson's disease.

The present invention mainly relates to immortalized cell lines of human embryo derived from cells of the Central nervous system, and to methods of using these cell lines for the treatment of disorders of the Central nervous system. In particular, cell lines and methods in accordance with the present invention can be used when cured is ing

In accordance with the first variant of the present invention, it is proposed methods of treatment of the patient suffering from a disorder of the Central nervous system, or ways to relieve symptoms of such disorders, due to the implantation of immortalized cell lines of human embryo-derived cells of the Central nervous system. After implantation of these cells into the Central nervous system was not a manifestation of phenomena such as graft rejection, intensive intracerebral inflammation and tumors. In addition, the cells showed the ability to cause migration of the neuron and the extension of neuritis. This suggests that the operation of cells produce trophic factors that stimulate neural responses (reactions).

Implantation immortalized human stem cells derived from cells of the Central nervous system, can cure many diseases. For example, can be conducted in the treatment of Parkinson's disease due to the implantation of such cells in the basal nucleus of the affected recipient. Trophic factors secreted by the implanted cells can slow down the loss of dopaminergic neurons and even cause the reg is practical to improve the condition of patients, suffering from Parkinson's disease.

In accordance with additional options of implementing the present invention may be produced by transfection of cardiac cells with nucleic acid which encodes a neurologically suitable polypeptide. The term "neurological suitable polypeptide" generally refers to a peptide or protein which catalyzes a reaction with the tissues of the Central nervous system. Such peptides can be natural neural peptides, proteins or enzymes, and the fragments of the peptide or protein, which have therapeutic activity within the Central nervous system. Additional examples include neural growth factors, and enzymes used to catalyze the generation of important neurochemicals or their intermediaries. In accordance with a particularly preferred aspect of the present invention, transfection of cells produced from a nucleic acid which encodes tyrosine hydroxylase. Tyrosine hydroxylase is an enzyme that converts tyrosine into L-DOPA, which is also a speed limiter produce dopamine. As a result, the expression (expression) tyrosine hydroxylase by means of implanted cells posvalue, implanted cells can increase the concentration of dopamine in the black substance, as well as to limit or reverse the effect of the loss of dopaminergic neurons.

Methods in accordance with the present invention can also be used to treat other neurological disorders, such as Huntington's chorea, Alzheimer's disease or multiple sclerosis. Immortalized neuroprostanes embryonic human cells are compatible with the Central nervous system (CNS); transfection of these cells can be produced with a DNA sequence encoding a physiologically active peptides, for implantation in the Central nervous system to effect the treatment of various disorders. For example, in the case horei Huntington's and amyotrophic lateral sclerosis, the peptide can block the excitatory neurotransmitters, such as glutamine. In the case of the use for the treatment of, for example, multiple scleroses, the peptide is typically trophic stimulator myelination, such as derived from platelet growth factor or ciliary trophic factor that can block the death of oligodendrocyte. Because these diseases are more common than local lesions, desire, the ability, affected by cerebrospinal fluid. After manifestations of expression and secretion of the peptide is washed away with the entire surface of the brain due to the natural circulation of cerebrospinal fluid. Desirable locations for implantation are the lateral ventricle, the lumbar area of the shell, etc. In the case of Alzheimer's disease can be produced by transfection of cells for the production of growth factor nerve to maintain neurons in the basal forebrain, as described by Rosenberg and others in the publication: Science, 242:1575-1578 (1988), which is included in the description of the application as references.

Methods in accordance with the present invention can be also used for the treatment of recipients by using the implantation of cells in extranervosae location. This variant of implementation of the present invention are particularly useful for prophylactic treatment of the recipient. Transfection of immortalized neuroprosthetic human stem cells can be produced with DNA coding associated with the disease antigen, for example, HIV (HIV) gp120 polypeptides that surround the main neutralizing region HIV (HIV), as described, for example, in U.S. patent No. 5166050. Cells can then be implanted cells and can elicit a powerful immune response. After an adequate time frame for full immunization of recipient cells can be deleted.

Used in the present description, the term "treatment recipient" includes prevention, palliative and curative intervention in the disease process. Used in the present description, the term "treatment" generally refers to therapeutic methods to mitigate or eliminate the symptoms of certain diseases, for which it was intended treatment. In the present description, the term "recipient" refers to any warm-blooded mammal such as a human, primates, rodents, etc. that receive a certain treatment. Usually used here, the term "recipient" or "patient" refers to humans.

Methods in accordance with the present invention can be used to treat a variety of diseases and symptoms. Usually the disease is a neurological disorder such as Parkinson's disease (including Parkinson's disease), Alzheimer's disease, Huntington's chorea, multiple sclerosis, amyotrophic lateral sclerosis, Gaucher disease, disease, and Tay-Sachs, neuropathy, brain tumors, etc., Methods in accordance with the present invention IU the present invention can be used for immunization of recipients against infectious diseases, such as caused by viruses, bacteria, protozoan, as described above. Transfection of immortalized neuroprosthetic human stem cells can be produced with a DNA that encodes a physiologically active peptides or peptides that contain immunological epitopes. Methods in accordance with the present invention can be used for implantation of peptide producing cells and continuously supplying the recipient in vivo other types of peptides, such as growth hormone.

II. Cell line

For the implementation in practice of the above described methods of treatment the present invention also provides cell lines that are suitable for transplantation to a recipient or patient.

Typically, the cells are implanted using methods in accordance with the present invention, are immortalized neural-derived embryonic human cells. The term "neuroprostanes" means that before immortality cells had the phenotype of neurological cells or represented embryonic cell undergoing differentiation to obtain cells neurological type. The types of neurological cells include astrocytes, epithelial cells nervously the lines can usually be carried out in accordance with the following procedures. The selection of embryonic cells can be produced by selective abortion. Women, from which after an abortion receive the embryos should be subjected to serological screening of various infectious diseases, including human immunodeficiency virus, hepatitis b virus, hepatitis C virus, cytomegalovirus and herpes viruses Type 1 and 2. Gestational age of the embryo is usually equal to 9-11 weeks (7-9 weeks after conception). The age of the embryos may be confirmed by ultrasound examination. Extraction of embryos can be produced with ultrasonic location to minimize the risk of injury of the brain of the embryo.

After extraction is the identification and dissection (separation) of the brain of the embryo derived from abortion. Cells can be prepared as follows. Brain tissue is sucked through the needle 19 caliber and washed twice minimum primary tool Needle (E-MEM, Gibco, New York, N. Y.). Cells plated on Petri dishes with the processing of poly-lysine (0, 1 mg/ml for 5 minutes). Cells grow on the E-MEM with the addition of 20% fetal beef serum, 75 μg/ml streptomycin, 75 units/ml penicillin, 1% dextrose and 2 µg/ml Fungizone (Gibco). Before immortality cells incubated with temperaturesare and other methods preparation of cells.

Cells that must be implanted using methods in accordance with the present invention, can be immortality using various types of equipment. Usually produce the immortality of cells as follows. Cell culture usually produce precursor cells of neural and glial cells, and neurons, as described by Meyer and VACANTA in the publication: J. Neuropath and Exp. Neurol, 48:425-436 (1989), which is incorporated into this description by reference. With regular feeding brain cells have a pot life of several months, but have low cell proliferation. Transformation of cells produced by transfection with a mutant with a deletion of SV40. The mutant DNA is not the beginning of replication (ori) and could not reproduce. However, transfection of DNA gives the cells an unlimited growth potential, as described by Gluzman in the publication: Cell 23:175-182 (1981). After growth of embryonic cell cultures for 3 weeks, can be made tranfaglia cells with 100 μg/tube plasmid DNA (RMC) containing the SV40 ori-mutant, using the technique of deposition of calcium phosphate as described by Graham and others, Virol, 52:456-467 (1973). Alternatively, transfection of cells can be produced the publication: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (1988), which is incorporated into this description by reference. After transfection, the growth of crops is carried out at weekly feeding. After a few weeks the proliferation of glial cells in certain areas of the Petri dish becomes obvious. This is followed by the migration of cells by using trypsinization (0,025%) in the new culture. Transformed cells can be identified by fluorescent analysis of antibodies, which detect SV40 T protein, which manifested transformed cells (Fig.2). The passages of the cells is carried out every 10 days until the detected increase in the number of positive cells in the T-protein.

Transformed cells exhibit the phenotype of continuous cell lines. In particular, cells grow to high density saturation with a generation time of 18 hours. However, cells do not exhibit a transformed phenotype, or independent from, the anchor substrate growth, which is characteristic for namutoni SV40 transformed cells. The morphology of the cells also does not change during the establishment of cell lines. Foci (centers) cells are usually not detected. Particularly useful cells are cells from SVG cellular USA No 4707448, included in this description as a reference (Fig.1). Hereinafter the terms "SVG cells" or "cell line SVG" means a cell or cell line A. T. C. S. CRL 8621. Derivatives involve subclan, replica or genetically modified mutant cell lines A. T. C. S. CRL 8621.

Alternatively, cells can be immortality using other types of equipment that are well known in themselves. For example, can be used immortality by Epstein-Barr, as described in U.S. patent No 4464465 included in the present description by reference. Especially useful mutant virus, Epstein-Barr, which do not start replication OriP and OriLyt. Another useful method of immortalization is overexpression of the cellular gene to control growth, such as C-myc, as described by Bartlett and others in the publication: Proc. Natl. Acad. Sci. USA. 85:3255-3259 (1988), included in the present description by reference. Usually suitable for implantation of the transformed cells are dependent on the anchor substrate, do not grow in soft agar and do not form foci (centers).

Can then be defined histological origin of transformed cells. Characteristically, astrorum acidic protein (GFAP). On the other hand, oligodendroglial cells are cells which produce myelin; they can be identified by their synthesis of galactocerebroside (gal), which is a component of myelin.

After the transformation, the cells are prepared for implantation. Cells are suspended in a physiologically compatible carrier, such as a cell culture medium (e.g., the minimum basic medium Needle) or with phosphate-saline buffer solution. Cell density typically ranges from approximately 104up to 107cells/ml Prior to implantation of the cell suspension gently mix (shake). The amount of implanted cell suspension depends on the location of implantation, treatment goals and the density of cells in suspension. Usually the number of transplanted patient or recipient cells constitutes a "therapeutically effective amount". Used herein, the term " therapeutically effective amount refers to the number of transplanted cells, which is required for effective treatment of a particular disease for which is provided by the treatment. For example, in the treatment of Parkinson's disease, transplantation is therapeutically effective amount of cells usually of printie and abnormal gait. In the treatment of parkinsonism with each injection is usually administered from 5 to 60 μl of cell suspension to obtain the specified effective amount. Each recipient can be made of different injections. Experts know how to determine the appropriate cell dosage.

In accordance with an alternative preferred variant implementation of the present invention, transfection of cells suitable for transplantation can be performed with xenogenic nucleic acid sequence (with the possibility of its expression), which encodes neurologically suitable peptide. The term "xenogeneic" used here to describe nucleic acids, refers mainly to the sequence, which as a whole is not naturally found in the cell lines, transfection of which are produced with this sequence. Thus, xenogenic sequence may contain a fragment that is completely foreign to the cell line, or alternatively, can contain innate segment, which is integrated into the cell line is not peculiar way, for example, has a relationship with is not peculiar to the sequence of the promoter/amplifier, is connected with projectsam copies while cell line usually gives one copy or not giving them at all.

Typically, the sequence of nucleic acids operable associated with the promoter of transcription and transcription terminator. The DNA segment operable connected, when it is placed in a state of functional communication with another segment of DNA. For example, the promoter or the amplifier is operable linked to a sequence encoding, if it stimulates the transcription of the sequence; DNA for a signal sequence operable associated with the encoding the polypeptide DNA, if it is manifested as preprotein that participates in the secretion of the polypeptide. Typically operable linked DNA sequences are contiguous and, in the case of the signal sequence are contiguous and are in the phase of reading. However, the amplifiers need not be contiguous with the coding sequence, a transcription of which they control. The coupling is carried out using ligation (ligation) to the desired limited locations or adapters or linkers introduced instead. The DNA sequence may also be connected with the amplifier transcription. The expression of the DNA in the implanted cells can be obratiti vectors manifestations, such as plasmid vectors rd, pHyg and pRSVneo, vectors monkey virus 40 vectors, bovine papillomavirus, or vectors, virus, Epstein-Barr, as described by Sambruna and others in the publication: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press (1988), which was previously included in the present description by reference. Vectors can be introduced into cells using standard methods, such as electroporation, transfection by calcium phosphate, transfection of polybrene (polybrene), etc.

The peptide encoded by nucleic acid, usually can be directly therapeutic compound, such as an inhibitor of motion in the treatment horei Huntington. Alternatively, the peptide encoded by nucleic acid may be selected to add or replace deficient production of endogenous peptide tissues of the recipient, and this deficit is the cause of the symptoms of certain diseases. In this case, cell lines act as an artificial source peptide. Alternatively, the peptide may be an enzyme that catalyzes the production of therapeutically or neurologically relevant compounds. And in this case, such compounds can be exogenous with respect to the system of the recipient or mo is ipient provides additional ways to develop connections. For example, in accordance with a preferred variant of the present invention, transfection of immortalized neuroprosthetic embryonic cell lines man with a nucleic acid which encodes the enzyme tyrosine hydroxylase. Tyrosine hydroxylase catalyzes the synthesis of dopamine from tyrosine. Dopamine has shown its effectiveness in the treatment of parkinsonism.

In accordance with a particularly preferred variant implementation of the present invention, termed neuroprostanes embryonic cell lines, transfection was performed with the tyrosine hydroxylase is the SVG cell line, for example, obtained from the SVG cell line deposited in the American type collection of cultures, Rockville MD, (A. T. C. C. CRL 8621), which is described in U.S. patent No 4707448 included in this description as a reference (Fig.1). Such cell lines are referred to here SVG-TH cell lines. In accordance with another preferred aspect of the present invention, transfection of SVG cell line is made with phTH/Neo-plasmids.

Noclegowa acid may also encode a trophic factor such as growth factor, nerve, inhibiting growth factor or titlerow and to produce and secrete dopamine, cell lines in accordance with the present invention is particularly useful in the treatment of disorders of the Central nervous system that are associated with the loss of dopaminergic cells in the Central nervous system of the recipient, such as parkinsonism. Surprisingly it was also found that cell lines in accordance with the present invention is also able to produce more neurotransmitters. In accordance with a particularly preferred variant of the present invention, for example, cell lines in accordance with the present invention is also capable of expression (manifestation) of serotonin. Serotonin is involved in cases of clinical depression patients. In particular, it was found that elevated levels of serotonin in the tissues of the Central nervous system lead to relief of symptoms of depression, and serotonin are the basis for the manufacture of a number of antidepressants, such as Prozac. Therefore, cell lines in accordance with the present invention may also be particularly useful in methods of treatment of disorders associated with reduced levels of serotonin in the Central nervous system such as depression. Usually these methods are similar or substantially similar methods, kotoroya line in accordance with the present invention have the dual effect of relief of symptoms of the disorder due to the secretion of dopamine and nerve regeneration, and treatment of depression associated with the disorder, due to the secretion of serotonin.

III. Implantation of cell lines with

Typically, cells from cell lines in accordance with the present invention can be implanted into the brain parenchyma, in the space containing cerebrospinal fluid, such as space subroutines shell or ventricle, or extraneural. Used herein, the term "extraneural" is intended to designate areas of the recipient that are not within the Central nervous system or peripheral nervous tissue, such as the celiac ganglion or sciatic nerve. "Extremevalue" field can contain peripheral nerves. The term "Central nervous system" is intended to refer to all structures within the dura mater (Dura).

When implanting the cells in the brain normally using stereotactic methods, as described in the publication Leksell and Jernberg, Acta Neurochir., 52:1-7(1980), and in the publication Leksell etc., J. Neurosurg., 66:626-629 (1987), which is incorporated into this description by reference. Localization of target areas usually involves pre-implantation MRI, as described in the publication Leksell etc., J. Aut using pre-implanted MRI.

Prior to implantation assess the viability of the cells, as described in the publication Brandina and others, Brain Res., 331:251-259 (1985), which is incorporated into this description by reference. Briefly, an aliquot of sample suspension cells (1-4 μl) are mixed on a glass slide with 10 μl of a mixture of acridine orange pigment and bromide etida (3.4 mg/ ml each component in 0.9% saline; Sigma). The suspension is transferred into hemocytometer and produce visual counting of viable and non viable cells using a fluorescent microscope with EPI - illumination at 390 nm in combination with TRANS-illumination with white light for visualization grid camera count. Acridine orange pigment colors in green living cell nuclei, while the bromide Atid penetrates dead cells and causes their orange-red fluorescence.

Cell suspension must usually contain more than 98% viable cells.

The injections are usually produced using sterilized syringes Hamilton 10 μl with needle gauge 23-27. The syringe filled with the cells placed directly over the head of a stereotactic frame. The needle injection is lowered to the specified coordinates is expecting additional 2-5 minutes before the implementation of the diffusion slow before removing the needle. Often produce two separate input suspension with an interval of 1-3 mm with the same penetration of the needle, and in one and the same operation can be made up to 5 inputs suspension is distributed over a zone of the target. Injection can be performed manually or by using infusion pump. After removing the needle upon completion of the surgery, the recipient is removed from the frame and the wound sutured. If necessary, can be introduced prophylactic antibiotics or immunosuppressive therapy.

For the treatment of neurological disorders of a more General nature, may be produced by transfection of cells for the expression (manifestation) of therapeutic compounds, and implantation of cells produced in the ventricles or lumbar shell. Since the secretion of therapeutic compounds is carried out by cells, the natural circulation of cerebrospinal fluid washes therapeutic compound across the Central nervous system, ensures that the total treatment. Implantation in the ventricles can be made using the open procedure, as described in the publication of Madrazo and others, New Engl. J. Med., 316:831-834 (1987) or in the publication Penn and others, Neurosurgerv. 22:999-1004 (1988), naibolee conveniently carried out using standard procedures, installation is the same as radiographic contrast media or the introduction of anticancer drugs using puncture the shell.

In some cases, in accordance with the present invention, it is desirable to implant cells extraneural. Cells can be implanted with the introduction through the skin with a needle or endoscope, and also when using the open procedure. Specialists can easily find the most suitable method of implantation of cells for a specific application.

Before implantation, the cells can be encapsulated by membranes. Encapsulation creates a barrier to the immune system of the recipient, inhibits graft rejection and prevents inflammation. Can be used in different ways encapsulation. In some cases, using individual encapsulation of cells. In other cases, many cells capsulebuy in the same membrane. In the case of subsequent removal of the cells after implantation, the relatively large size of the capsules for a variety of cells comfortable for the return of implanted cells. Well-known different ways encapsulation of cells, such as described in European patent application No 301777 or Pat the ways encapsulation is as follows. Transformed cells are mixed with sodium alginate (polyanionic algae extract) and ekstragiruyut in a solution of divalent cations, such as calcium chloride, which forms complexes with sodium alginate in the form of a gel, which leads to the formation of granules or droplets of the gel, in which the enclosed cells. Granules of the gel incubated polyominoes acid with high molecular weight (MW 60-500103and with a high concentration (0,03-0,1% w/v (weight percent)), such as poly-S-lysine, in a short period of time (3-20 min) for the formation of the membrane. The inside of the formed capsule is purified by treatment with sodium citrate. Single membrane around cells is highly permeable (MW cutoff 200-400103). Containing a single cell, the membrane is incubated in physiological solution for 1-3 hours for o diffusion from the capsule captured of sodium alginate and give a capsule of balance. Received depleted alginate capsule is introduced into the reaction polyominoes acid with low molecular weight (MW 10-30103), such as poly-L-lysine (PLL), or chitosan (W slice 40-80103). After that make the cultivation of cells encapsulated in double membrane, in the environment of E-MEM for two to three weeks, as described previously.

Despite the fact that it was made specific reference to capsules of sodium alginate, experts can easily understand that it can be used any other non-toxic water-soluble substance which can form a gel in the form of retaining the shape of the mass in the changing conditions of the environment into which it is placed. Such forming gel materials usually contain several chemical compounds, which are easily ionized with the formation of anionic or cationic groups, so that the surface layers may be cross-linking with the formation constant of the membrane by interaction with oppositely charged polymers. Many polysaccharide of the rubber, both natural and synthetic, can be cross-stitching using polymers containing positively charged reactive groups such as amino groups. Capable of cross-stitching biocompatible polymers that can react with the rubber mixture of sodium alginate, include polylysine and other polyami the Oh of the acid, having a desirable molecular weight. The preferred polymeric material is poly-L-lysine (PLL), but can also be used chitosan and polyacrylate. Molecular weight usually lie in the range of 104up to 106.

Further, the present invention illustrada examples. These examples are only illustrative and do not limit the present invention.

Example 1. Preparation of SVG cells

This example describes the preparation of SVG cells (A. T. C. C. CRL 8621), for implantation makaka RH. Were screened for infection with Mycoplasma, HIV-1, hepatitis b virus, simian virus 40, simplex herpes virus and JC virus.

The growth of SVG cells continued to merge. Cell growth is dependent on the anchoring substrate. The formation of foci (centers) does not occur and the morphology of the cells is homogeneous. Cells are removed from the cups of tissue culture by fermentation of 0.05% trypsin in 0.01 M EDTA (buffer Versene) in balanced salt solution Khanka. The collection of cells produced by centrifugation, washing 3 times and re-suspendirovanie in phosphate-saline buffer solution. The final cell density was 106cells/ml Cell suspension stored until the TRANS is the Isan implantation SVG cells in the basal nucleus of six rhesus monkeys. Implantation were performed stereotactic method without surgical complications.

Animals were originally shot of ketamine and maintained in a state of anaesthesia using gas isoptera during surgery. Animals were placed in a stereotaxic frame (Kopf) and guidelines for implantation were found using stereotaxic coordinates. The superior sagittal sinus was opened to find the middle line. On the skull were labeled above the caudate nuclei and putamen on both sides. The coordinates were as follows: AR was +24 mm front 0. Lateral coordinates were 5 mm from the midline to the nucleus caudate nuclei (caudate) and 10 mm from the midline to putamen.

Were made 5 trepanation holes. One was done over the superior sagittal sinus, the other two over the caudate nuclei and two over palamanui. Were used two different techniques.

1. Were used syringes Hamilton 10 μl with needle gauge 26 or syringes Hamilton 50 ál with needle gauge 23. SVG cells were transplanted on the right side of the brain. Using syringes at putamen were carried out two enter. One entry was made in the side of putamen, and the other in the middle. Needles from to the first implantation needle was withdrawn at a rate of 1 mm per minute to 3 mm, and then conducted a second injection of 10 µl of the cell suspension. After the second implantation needle was withdrawn at a rate of 1 mm per minute. The second implantation was carried out in the opposite of putamen with the same coordinates and using the same technique.

After the implementation of the injection of putamen produced implantation of the same cell suspension in the caudate nucleus. Were two injections into the caudate nucleus, the lateral and median aspect. The depth of injection was 15 mm, if you enter 10 µl. The needle was withdrawn at a rate of 1 mm per minute to 3 mm, and then conducted a second injection of 10 µl of the cell suspension. In putamen was transplantability SVG cells without transfection, and in the caudate nucleus was transplantability SVG cells with transfection of the gene tyrosine hydroxylase. The cell concentration was 2106cells in 1 ml

2. In addition to implantation using syringes with needles were designed cannula with blue intake tubes and needles 22 gauge. The tubes were connected to a tuberculin syringe with a capacity of 1 cm using connectors with zero dead volume. After introduction of the target needle before the infusion was maintained for 15 minutes. After that he joined infusion the minutes with a flow rate of 0.2 µl/min was increased flow rate of 0.4 μl/min and the infusion was continued for 100 minutes. At the end of the infusion needle was kept in place for 30 minutes to extract. Then the needle is slowly withdrawn from the brain.

The wound was filtered and then was closed in anatomical layers. Animals were awakened from anesthesia and were transferred to their home cages after 20 minutes after surgery.

Example 3. Engraftment SVG cells in monkeys

This example demonstrates the successful engraftment of implanted SVG cells in two monkeys that were killed one month after implantation. The transplanted cells were histologically healthy. Signs of inflammation or cancer was not.

Brain tissue in the area of implantation was investigated as follows:

To conduct histopathologically studies, animals were euthanized by cordozo of pentobarbital (460 mg intravenously), by perfusion through the ascending aorta 15 ml of phosphate-saline buffer solution (PBS), and then 10% formalin. The brain was removed very quickly, cut into 6 mm coronal layers and subjected to a secondary fixation for 30 minutes in the same fixative. The fabric layers were washed for 48 hours in 30% sucrose solution in phosphate-saline buffer solution (PBS), and then quickly frozen, pre; series of sections were stored in phosphate-saline buffer solution (PBS). Was performed immunohistochemical processing sections with antibodies against tyrosine hydroxylase, glial fibrillar acidic protein, T-protein. Sections adjacent to those that were analyzed for TH-IR, were stained with hematoxylin and eosin. Some blocks of tissue, which contained the implant, were treated with 5 µm paraffin sections were stained as indicated above.

In Fig.3 shows the trace of the needle in the basal nuclei of one of the monkeys at low magnification. In Fig.4 and 5 shows the traces of the needle in the basal nuclei at high magnification; the track you can see a viable SVG cells. Cells can be easily identified due to the large nucleus containing many nuclei in SVG cells in vitro. The morphology of the cells implant surprisingly different from the morphology of the surrounding cells. Inflammatory cells or the characteristics of the tumors were not found.

Similar tests were conducted on monkeys, dead 9 months after transplantation. Was identified transplant, and inflammatory cells or signs of cancer were not detected, indicating that the engrafted cells and terebralia MRI evaluation, conducted 1 month after implantation for the other four monkeys. None of the monkeys there were no signs of cancer.

After the introduction of anesthesia monkeys were placed in a standard MRI frame. Were made weighted image T1and T2without contrast, and T1weighted image with gadolinium using a 1.5 Tesla magnet (Signa). In the scan results revealed no traces of cancer or nodules (Fig.9).

Example 5

This example illustrates the functioning of the transplanted SVG cells within the Central nervous system. Neurons recipient migrated in the direction of the implanted cells, neural dopaminergic bodies, and dopaminergics the processes of the recipient were distributed on the implanted cells.

The two remaining living monkeys who received the SVG cells in accordance with Example 2 were euthanized as described. The brain was extracted is not damaged as described and divided into slices.

Each slice was placed on the floor gelatin slides. Representative sections were stained with hematoxylin and eosin to identify the anatomy (Fig.6). Implanted cells have a characteristic body to glial fibrillar acidic protein (GFAP), SV40 T protein or the tyrosine hydroxylase. Then the slices received contrasting coloration only using hematoxylin. In Fig.7 shows an adjacent slice stained with antibody to GFAP, which is a cytoplasmic protein astrocytes pedigree. Astrocyte origin demonstrates dense cytoplasmic staining. The start cell is also shown in Fig.8, where clearly visible implanted cells stained with the antibody anti-T-protein.

Cell transplant in the caudate nuclei and putamen were viable and easily identified by staining with an antibody anti-T-protein, as described above. SVG cells were also identified on the walls of the lateral ventricles of all monkeys. Dopaminergic neurons are the neural processes in the direction of the implanted cells. (Of Fig.10 shows the neuron tyrosine hydroxylase, stained with antibody antimyosin hydroxylase in the layer SVG cells in vivo). Dopaminergic neural body was also present in the field of cardiac SVG cells. Strengthening neuritis and the presence of neural tel indicate that SVG cells produce neurotropic factors that cause the neuron migration and distribution of neural crest.

N and or knot.

Example 6. Capsulate SVG cells

This example describes the individual capsulate SVG cells and preparation of cells for implantation. Capsulate cells were produced in the sediment centrifugation of sodium alginate.

SVG cells grow to confluence in Petri dishes. Removal of cells from the Petri dishes was performed using 0.05% trypsin and 1 mm EDTA in phosphate-saline buffer solution Dulbecco (PBS). Was established cell suspension in PBS with the addition of MgCl2, l2, 0.1% glucose and 5% fetal beef serum. The cells were collected by centrifugation, were washed 2 times in solution suspension, as described previously, and tsentrifugirovanie to obtain a precipitate.

The cell sediment at the bottom of the centrifuge tubes were again transferred in suspension in 5 ml of 1.5% (w/v) solution of sodium alginate (Keltone LV, Keico Ltd, Chicago, Illinois). Alginate cell suspension was extruded in 50 ml of 1.5% (w/v) solution of CaCl2. Spherical droplets of the suspension were formed with the help of the generator of droplets with air jet syringe pump. Then with the help of this device suspension cells sodium alginate was extruded through a needle 22 gauge located inside the protective tube (inner diameter 3 mm)crystals using a syringe pump with a flow rate of 20 cm3/hour), drops off due to the efforts of the cut created by the rapidly flowing air stream. The needle tip is kept at a distance of 8 cm above the surface of the solution of CaCl2, the formation of uniform spherical droplets of the gel with a diameter of about 300-1000 microns.

The size and shape of the sample with melirovanie the microspheres is investigated using preprofile magnifier (Model M8 Wild Heerbrugg), equipped with a calibrated eyepiece. After migration, the gel beads of calcium alginate containing immobilized cells in 50 ml plastic centrifuge tube with conical bottom, the granules are washed with 30 ml of a solution of 0.1% (w/v) CHES and 1.1% CaCl2. The volume of supernatant was decreased after each washing by using a vacuum aspirator. A semi-permeable capsule membrane was formed by using the reaction drops of the gel with water to 0.05% (w/v) PLL solution (M/v PLL=22.000) for 8 minutes. After adding PLL solution centrifugation tube was closed by the cap and manually turned over from one end to another in the course of the reaction so that the capsules are not sticking to each other. The obtained microcapsules with a diameter of about 300-1000 μm were washed with 30 ml of a solution of 0.1% (w/v) CHES and 1.1% (w/v) CaCl2and 30 ml aliquatum isotonic. Capsular esni layer on the capsules. The inside of the microcapsules is purified by treatment with 30 ml of 0.05 M sodium citrate for 6 minutes. The obtained microcapsules with a diameter of 400-1400 μm are washed several times in saline solution to remove residual citrate, and then divided into five 1 ml aliquatum. Each aliquat incubated in 10 ml of DMEM medium at 25 cm3flask culture at 37 ° With the incubator series isotemp WITH 4002(model 413D, Fisher Scientific Co., Nepean, Ontario).

Example 7. Genetic engineering SVG cells for the expression of tyrosine hydroxylase

This example describes the transfection of SVG cells with nucleic acid that encodes a tyrosine hydroxylase. SVG cells that carry the expression of tyrosine hydroxylase, were identified in culture after transfection.

Was produced by transfection of the SVG cell line with a nucleic acid that encodes the enzyme tyrosine hydroxylase (TH). Plasmids phTH-63 is of type 2 cDNA for tyrosine hydroxylase, cloned into the EcoRl site of the vector KS Bluescript. TN cDNA was cloned in two different eukaryotic expression vector, pcDNA/Neo and pRSV/ Neo (which can be purchased from Invitrogen Corp., San Diego, California). Hindin/BamHl fragment phTH-63, which contains TONS cDNA, was cloned into the HindIII/Hind 1 website pcDNA/Neo in HindIII/Spel site of pRc/RSV, resulting plasmid pRSV-hTH/Neo. As shown in Fig.11, phTH/Neo consists of the nearest early CMV promoter from above TN cDNA, plasmid, which tells resistance to neomycin. Design pRSV-hTH/Neo consists of RSV LTR from above TN cDNA, plasmid, which tells resistance to neomycin.

Were created a separate culture SVG cells, transfection of each of which was performed with phTH/Neo or pRSV-hTH/Neo. After transfection cells were grown in medium containing 500 μg/ml of geneticin, within two months. When phTH/Neo transfection were obtained 7 clones that stably resistant to geneticin. Both transfectant were able to work out a TON, but using design pRSV-hTH/Neo were obtained with long-term stability clones, due to the weak expression of the marker neomycin resistance in the plasmid. Immunohistochemical staining of tyrosine-hydroxylase one of the stable transfectants phTH/Neo can be seen in Fig.12. The clones are in the range from 30 to 60% positive TN. Was held clonal expansion of one of the clones (1V 1V), which was 40-60 TON-positive; to confirm immunohistochemistry was performed Western blotting. As shown in Fig.13, when the 60 Kd, coinciding with the size of the type 2 TN. In subsequent clone 1B 1B was identified as SVG-TH.

To detect the presence of biologically active T SVG-TH cells and to determine whether the secretion of L-DOPA in the cells, the analysis was performed on cell culture supernatant using HPLC. Prior to selection of cell culture supernatant to HPLC cells were incubated in 1 mm biopterin (BH4); biopterin is cofactor necessary for the function of VT. A control sample contained supernatant SVG-TH cell cultures incubated in the absence biopterin, and the supernatant from cultures of the parent SVG cell line, incubated with biopterin or without him. The results are shown in table.1.

As shown in the table.1, L-DOPA is not found in the parent SVG cell culture, as with biopterin, and without it, and is not found in the SVG-TH cell culture without biopterin. However, in the case when the SVG-TH cells incubated with Bioperine, the supernatant of the cell culture produced approximately 4-6 PG/ml/min L-DOPA. This confirms that TN, which can be seen by immunohistochemistry and Western-blotting, is biologically active.

Suddenly DV is .14), regardless supplements biopterin on Wednesday. These two peaks are not visible in the parent SVG cell line. By some standards, it was determined that one of the two peaks displays the serotonin and the other pic shows 5-hydroxyindoleacetic acid (HIAA), the breakdown product of serotonin.

To confirm the presence of serotonin in these cells was carried out immunohistochemistry SVG-TH cells using polyclonal antibodies to serotonin. SVG-TH cells were positive for serotonin as when immunoablative, and when carrying out HPLC. Production of serotonin in these cell lines is unique to cells of glial origin, as there are no reports that they can produce serotonin.

SVG-TH cells were analyzed using immunohistochemical methods, using the same panel of antibodies that were used for the analysis of SVG cells. Comparative results are given in table.2.

In the study in the electron microscope (EM) SVG-TH cells reveal a pronounced expansion of the granular ER that is not visible in the parent SVG cell line (Fig.15). Again were easily identified bordered pits, mitochondria and ribosomes the TCI were also tested for their ability to enhance neuritis and viability of primary neurons and neural cell lines.

A. Tocultivate hNT cell line

As described previously, in the experiments of cocultivation with SVG and SVG-TH cells was used cell line, derived from teratocarcinoma person. This cell line derived from a parent cell line teratocarcinoma by treatment of the parent cell line retinoic acid and combination antimitoticescoy substances. After processing the parent cell line differentiated into post-mitotic neurons. [Andrews P. U., Called retinoic acid neural differentiation of cloned embryonic cell line carcinoma human In Vitro, Dev.BioL, 103:285-293 (1984)]. These cells ending hNT neurons, which were used to conduct the experiments described here cocultivation retain many of the phenotypic quality of the neurons, including the expression of neurofilament and secretion of neurotransmitters. To save these cells they should be placed in a Petri dish on laminin or Matrigel and must eat in air-conditioned environment.

In three separate experiments, cells SVG, SVG-TH and Cos were inoculated in 6 cups with holes that are not covered extracellular were merged by 30% and in the same wells were seeded 1105hNT cells. The hNT cells were inoculated in the fourth hole, which did not contain any of these cell lines and which was not covered by extracellular matrix. Cultures were fed only D-MEM with 2% fetal beef serum. 48 hours after seeding some of hNT cells attached to the free zones from SVG and SVG-TH cells and attached directly to these cells. It was found that the number of hNT cells is approximately equal to the number of SVG or SVG-TH cells. Various small shoots were observed in hNT cells, coculturing with SVG or SVG-T cells. In cocultivation hNT/Cos, hNT cells were directly attached to Cos cells, however, were not found in areas free from Cos cells. Additionally, only about 1% of Cos cells have attached to them hNT cells and hNT cells was not observed any shoots. In the control Petri dish, in the case of seeding only hNT cells on the untreated surface, it was possible to see only a few attached cells. 72 hours hNT cells rose from Cos cells and in the control Petri dish hNT cells were not found. In contrast, hNT cells in both cocultivated SVG and SVG-TH remained attached and have long processes, which is some OLE cultures and performed immunohistochemistry on T protein to clearly distinguish between the two cell populations (Fig.16A). These cultures remained viable for two weeks, after which cells SVG and SVG-TH merged. Coculture were transferred to new Petri dishes and the same phenomenon was observed again. After two additional weeks, the experiments were finished.

Century Tocultivate PC12

In this set of experiments was performed cocultivated cells PC12 cells SVG, SVG-TH or Cos; PC12 cells were cultivated also in isolation. In hNT experiment cells SVG, SVG-TH or Cos were inoculated in 6 cups with holes (1105cells per well) in untreated plastic dishes. After 48 hours of PC12 cells were inoculated in all three cell lines and isolated in untreated plastic dishes. 48 hours after implementation of cocultivation cells SVG or SVG-TH cells, PC12 attached to the free zones from SVG and SVG-TH, and also directly to the cells SVG and SVG-TH. The PC12 cells released naranya appendages that for 92 hours came into contact with surrounding cells SVG and SVG-TH. Some of the cultures were fixed in acetone and methanol and was performed immunohistochemistry on T protein to separate the two cell populations (Fig.16B). After 17 days of culture were grown and the experiment was finished. In the skali processes.

In a separate set of experiments, PC12 cells were inoculated separately on Petri dishes coated with poly-lysine, and then ate not air-conditioned environment, or the environment, air-conditioned from cell cultures SVG or SVG-TH. After 72 hours of PC12 cells, which were fed with conditioned environment, released naranya processes, while PC12 cells, which were fed reconditionnement environment did not change their morphology.

C. Primary cultures of fetal middle cerebral neurons of the rat

To determine the ability to maintain cells SVG and/or SVG-TH viability of primary neurons was performed dissection of the mid-brain of embryos of rats E13, his Department and the seed in three sets 6 Petri dishes. After 48 hours in the wells was installed camera with hole transfer Costar, and was made the passage SVG or SVG-TH cells in a cell with a hole transfer (1105cells). One set of the cultures of the middle brain had not been cocultivation with any cells and was used as a negative control. After 7 days the camera with a hole transfer from the cells was removed and culture medium of the brain in the wells were fixed in acetone and methanol, and OCD were the middle of neurons of the brain. As shown in Fig.17, the culture medium of the brain, which has been cocultivation with SVG cells are 2-3 times more surviving neurons, tyrosine hydroxylase, than in the control seeding. Similar results were obtained for the SVG-TH cells. There was no difference in the morphology of positive neurons tyrosine hydroxylase between the control seeding and planting with Skulltula.

Example 9. Transplantation and identification of SVG and SVG-TH cells in the striatum of rodents

To determine whether transplantation SVG or SVG-TH cells in the striatum (striped body) rodents and subsequent unambiguous identification of the results of transplantation, transplantation was performed 5105cells SVG or SVG-TH in the striatum of rats Sprague Dawley, using a stereotactic frame. Ten animals were transplanted cells SVG, and ten other cells SVG-TH. After 3 or 7 days after transplantation, animals were euthanized and their brains processed for carrying out immunohistochemistry. Five animals of two groups was performed systematic perfusion of 4% paraformaldehyde at killing. The slices of the brain fixed by paraformaldehyde animals were used for immunohistochemical staining of polillo is anyone staining monoclonal antibodies. Unambiguous differentiation of transplanted SVG or SVG-TH cells from the surrounding parenchyma can be carried out on the basis of staining SV40 T protein, which was detected only in the transplanted cells. Moreover, the transplanted cells had expression of the same antigens in vivo and in vitro, as evidenced by immunohistochemical staining. These antigens include vimentin, serotonin, MTL human class I, T-protein and TN. Similar to the observed in vitro situation in vivo, where only 40% SVG-TH cells were TH-positive. The surrounding parenchyma recipient was also immunoalkaline for vimentin and TN. SVG-TH cells remain GFAP-, while the surrounding parenchyma recipient was clearly GFAP+astrocytes. There is staining for rat MHC class I others parenchymal blood vessels and occasional vessels of the recipient in a transplant, however, staining of transplanted cells not as it was expected. While studying under an electron microscope SVG-TH transplanted cells, it was found that the transplanted cells retained the characteristics of the expanded granular endoplasmic reticulum and edged bubbles, as shown in Fig.15. Similar results were procedimental SVG and SVG-TH cells in the damaged 6-hydroxydopamine striatum of rats Sprague Dawley

After confirming identification SVG and SVG-TH cells in the striatum, the next task was to confirm the possibility of correcting these cells functional deficits in animal models with Parkinson's disease.

Seven rats Sprague Dawley were subjected to unilateral chemical damage of the dark matter using 6-hydroxydopamine, using a stereotactic frame for direction drugs to the appropriate anatomical location (site). 5 weeks after injury, animals received provocative apomorphine for assessing the degree of denervation. As shown in Fig.18, all 7 animals had a core speed of 400 revolutions/hour or more. 6 weeks after the injury of five animals in the affected striatum were transplanted SVG-T cells (approximately 5105cells). Two animals in the affected striatum were transplanted SVG cells. At weekly intervals after transplantation, only for 4 weeks, animals were provocative entered apomorphine (control and infected) to determine changes in their main activity. As shown in Fig.18, after 1 week after transplantation was observed creatures who from this, animals transplanted SVG cells were minor changes that could be expected, given that these rats were completely denervated and unable to give appendages dopaminergic neurons, and SVG cells were unable to secrete dopamine. However, over the next three weeks the animals transplanted with SVG-TH cells gradually returned to pre-transplantation rotational behavior, as shown in Fig.18.

Example 11. Characterization of transplanted cells one month after transplantation

Seven rats Sprague Dawley damage using 6-hydroxydopamine described in Example 10, were killed after 1 month after transplantation. Three animals with SVG-TH transplanted cells and one animal with SVG transplanted cells produced systematic perfusion of 4% paraformaldehyde at killing. Three other animals when killing wasn't commit perfusion. The slices of the brain fixed by paraformaldehyde animals were used for immunohistochemical staining of polyclonal antibodies, while the unfixed brain sections were used for immunohistochemical staining of monoclonal antibodies is Kim staining SV40 T protein vimentin, serotonin and TN. However, the transplant was significantly smaller grafts, which were observed after 3 and 7 days after transplantation. Observed immunohistochemical staining in transplant large amounts of T protein, serotonin and vimentin; however, immunoablative TN could be observed in the graft and in the surrounding denervated striatum. The graft can be further identified by the absence of staining for rat Thy 1.1, and the antigen has a strong expression in the surrounding parenchyma recipient. In the parenchyma of the recipient are observed labeled astrocytes around the graft. Staining of sections of rat CD4 and CD8 positive cells were identified in the graft and around it, indicating that immunological rejection of the graft. The above data indicate that cells represent a xenograft in the CNS of rodents, while the survival of cells in the Central nervous system of primates to nine months reflects the fact that these cells represent the allograft in the system.

Example 12. The influence of transplanted SVG-TH cells on the rate of disability in Parkinson's disease

Were created in this experiment RH 1-methyl-4-phenyl-1, 2, 3, 6 tetrahydropyridine (MRTR). In Fig.19, 20 and 21 shows the effects of transplantation SVG-TH cells in the caudate nucleus and in putamen three rhesus monkeys that received MRTR to create symptoms of Parkinson's disease. Before transplantation monkeys received injections MRTR to reach the value of 10 indicator of disability in Parkinson's disease (the highest level), which was maintained for several months after the injection.

When carrying out the procedure of transplantation 750,000 cells in a volume of 10 ál were placed in each of the four sites in the caudate nucleus and in putamen each animal. The chart was made of the rate of disability in Parkinson's disease in relation to the time after surgery (days). Within one week all three macaques showed improvement, which is reflected by the decrease in disability (table.3).

Macaque N (results 21 days for which is shown in Fig.19) within 90 days after surgery did not receive the standard medicines for Parkinson's disease (drug Sinemet).

The current rate of disability 2-3 for this animal is similar to the levels that can be expected in patients with Parkinson's disease in normal medicamentosas operation the animal is able to feed itself, is quite active and mobile, responds well to the environment and gaining weight.

As shown in Fig.20 and 21, macaques N and T also showed improvement in their disability, and continue to improve, although they were not observed during such a long period of time.

Thus, transplantation SVG-TH cells leads to a significant improvement in Parkinson's disease caused MRTR damage.

All mentioned in the present description publications, patents and patent applications included as references in this volume, which corresponds to that which would exist if each individual publication, patent or patent application was individually incorporated by reference.

Although for clarity of understanding of the essence of the present invention has been described in detail the preferred embodiment of the invention, it is clear that it specialists in this field can be amended and supplemented, which do not extend, however, beyond the scope of the following claims.

Claims

1. Immortalitya neuroblastoma cell line of the embryo to the forehead, characterized in that the SVG cells transfection with phTH/Neo-plasmid with the receipt of the indicated cell lines, with the specified cell line capable of the expression of specified nucleic acid sequence, and is able to produce and secrete dopamine.

2. Transplantable composition, characterized in that it includes cells from the cell line under item 1 and a pharmaceutically acceptable carrier, when said cells are encapsulated by a membrane from a gel of alginate impermeable to antibodies.

 

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