MXPA97009748A - Use of neuro-derived fetal cellular lines for transplant therapy - Google Patents

Abstract

The present invention relates in general to methods for the treatment of a host, by implanting genetically unrelated cells, in the host. More particularly, the present invention provides human fetal neuro-derived cell lines, implantation of these human fetal neurodegenerative cells immortalized in the host.

Description

USE OF NEURO-DERIVED FETAL CELLULAR LINES FOR TRANSPLANT THERAPY BACKGROUND OF THE INVENTION The present invention relates in general to methods for the treatment of a host, by implanting genetically unrelated cells in the host. More particularly, the present invention provides immortalized human fetal neuro-derived cell lines, and methods for treating a host by implanting these cell lines in the host or in the patient. Organ transplantation has become a means of success and widely practiced for the treatment of a variety of diseases. Heart, kidney and even liver transplants are almost routine in many medical centers. Unfortunately, the disorders of many organs are not susceptible to treatment with whole organ transplants. For example, lesions of the central nervous system can not be treated by whole organ transplants to replace damaged tissue. Because it is not possible to replace the injured tissue by a whole-organ transplant therapy for many diseases, or even for all patients with the appropriate diseases, attempts have been made to develop methods to transplant cells. Sun and collaborators, Biomart. , Art. Cells. Art. Org .. 15: 483-496 (1987). Parenchymal lesions that result in a deficiency of a biologically active compound can be treated by transplantation of the isolated cells or groups of cells that secrete the biologically active compound. For example, diabetic animals have been successfully treated by implanting the islets of Langerhans separated from donor pancreas. Noel et al., Metabolism, 31; 184 (1982). Cell transplantation therapy is particularly attractive for the treatment of neurological diseases. The transplantation of solid tissue is especially inappropriate for neurological diseases for several reasons. Exposure to open brain surgery, as required for the transplantation of solid tissue, can cause irreparable damage to the trajectories of the nervous system, resulting in clinical neurological deficits. Also, neurological function often depends on complex intercellular connections that can not be surgically established. In addition, the. Central nervous system cells are exquisitely sensitive to anoxia and nutrient deprivation. A rapid vascularization of solid tissue transplants is critical, as the cells inside solid tissue transplants often lack sufficient perfusion to maintain viability. Stenevi et al., Brain Res. 114: 1-20 (1976). A common neurological syndrome, parkinsonism has been the subject of attempts at cell transplant therapy. Bjdrklund et al., Brain Res., 177: 555-560 (1979); Lindvall et al., Science, 247: 574-577 (1990); Freed, Restor. Neurol. Neurosci., 3: 109-134 (1991). Parkinsonism is caused by a loss of neurons that produce dopamine in the substantia nigra of the basal ganglia. Burns et al., N. Encrl. J. Med., 312: 1418-1421 (1985); Wolff et al., Neurobiolocry. 86: 9011-9014 (1989). Parkinson's disease, a disease of unknown etiology characterized by the clinical manifestations of parkinsonism, is caused by the idiopathic destruction of these dopamine-producing neurons. Parkinsonism can be caused by a variety of drugs, v. gr., those antipsychotics, or chemical agents, v. gr. , l-methyl-4-phenyl-1,2,6-tetrahydropyridine. Burns et al., Proc. Nati Acad. Sci. USA, 80: 4546-4550 (1983) and Bankiewicz et al., Life Sci., 39: 7-16 (1986). Attempts have been made to reverse the clinical manifestations of experimentally induced parkinsonism, by means of the transplantation of dopaminergic cells in the striatum of the affected animals. Genetically modified fibroblasts (transfected with DNA encoding tyrosine hydroxylase) have been successfully transplanted into animals that have lesions of dopaminergic trajectories. The motor function and the behavior of the animals improved immediately after the implantation of the dopamine-producing fibroblasts. Wolff et al., Proc. Nati Acad. Sci. USA, 86: 9011-9014 (1989); Fisher et al., Neuron, 6: 371-380 (1991). Survival to the graft can be improved and, consequently, clinical improvement can be prolonged, by transplantation of the fetal tissue, comparing with the cells obtained immediately after birth. Gage and Fisher, Neuron, 6: 1-12 (1991). Fresh fetal dopaminergic neurons have been transplanted into the caudate nucleus of monkeys following chemical injury to the nigrostriatal dopamine system. After the transplant, deficits in the behavior induced by the lesion improved. Bankiewicz et al., J. Neurosurcr .., 72: 231-244 (1990) and Taylor et al., Procr. Brain Res., 82: 543-559 (1990). Human beings suffering from parkinsonism have been treated by striatal implantation of dopaminergic neurons. Lindvall et al., Arch. Neurol. , 46: 615-631 (1989); idner and collaborators, New Enql. J. Med., 327: 1556-1563 (1992). The transplanted cells were obtained from abortions. Before the abortions, the women were screened for antibodies to several viruses that cause the disease. Following the surgery, the treated patients exhibited an improvement in neurological function. However, patients required maintenance immunosuppressive therapy. Recent research indicates that trophic factors released from the support cells of the central nervous system (eg, astrocytes and oligodendrocytes) are critical for the survival of neurons in a cell culture .. O'Malley et al., Exp. Neurol. , 112: 40-48 (1991). Implanted fibroblasts that were genetically altered to express nerve growth factor have been shown to improve the survival of cholinergic neurons of the basal anterocerebral after injury to fimbria-fornix, which causes the transfer of acetylcholine neurons in the anterior anterocerebral basal, as seen in Alzheimer's disease. Rosenberg et al., Science, 242: 1575-1577 (1988). Although previous attempts at cell transplantation therapy for neurogenic disorders have provided encouraging results, several significant problems remain. The supply of fetal tissue for cellular transplantation is very limited. To ensure maximum viability, fetal cells should be harvested fresh before transplantation. This requires a coordination of the implantation procedure with elective abortions. Even then, fetal tissue has not been widely available in the United States. Also, the gestational age of the fetus, from which the cells are obtained, influences graft survival. Gage and Fisher, supra. Obtaining fetal tissue from only certain gestational ages adds additional limitations to the availability of fetal cells for transplantation. In addition, ethical considerations mean that some potential transplant recipients refuse to undergo the procedure when fresh fetal cells are implanted. Because fetal tissue is obtained from fresh abortions, there is a significant risk of infectious contamination. Although women who suffer abortions that supply fetal tissue are tracked by a variety of infections, some infections, for example, HIV, may not be clinically detectable and, therefore, are not identified during the screening process. Therefore, if they are widely practiced, the transplants of fresh fetal cells could possibly cause many infectious sequelae. The use of immortalized cell lines could overcome many of these difficulties of availability and infection. A human fetal neuro-derived cell line immortalized in 'Major et al., Proc. Nati Acad. Sci. USA, 82: 1257-1262 (1985), and in U.S. Patent Number 4,707,448. In addition, immortalized cell lines, by their nature, are predisposed to cause tumor formation following transplantation in vivo. Therefore, intracerebral therapeutic transplants of immortalized cells carry a high risk of causing intracranial tumors, even tumors that have a benign histology can carry a poor prognosis when they are present within the calvary. In addition to the risk of tumor formation, genetically unrelated cell transplants also carry the risk of immune rejection of the graft and intracerebral inflammation. idner and Brundin, Brain Res. Rev., 13: 287-324 (1988). All genetically unrelated cell transplants carry this risk. Therefore, patients treated by intracerebral cell transplantation have required a long-term maintenance immunosuppression that, even in the absence of transplanted immortalized cells, carries a high risk of infectious and malignant complications. Transplanting immortalized cells only amplifies the risk of these complications. What is urgently needed in this field are methods to therapeutically implant immortalized human fetal neuro-derived cells, and cell lines suitable for this use. Ideally, the methods would not result in tumor formation, nor would they cause intense inflammation immediately after transplantation. Desirably, the methods could employ cells derived from cell line, in such a way that the risk of infectious contamination and limited cellular availability would be minimized. In a very surprising way, the present invention satisfies these and other related needs. SUMMARY OF THE INVENTION The present invention provides methods for the treatment of a host, which comprise implanting cells from a human neuro-derived fetal cell line immortalized in the host. In general, the cell line will be derived from human fetal astrocytes, such as the SVG cell line. The cells will often be implanted in the central nervous system of the host. The cells can be encapsulated by membranes that are impervious to host antibodies. In some embodiments of the invention, the cells can be transfected with a nucleic acid sequence encoding a peptide. The peptides will generally be enzymes, such as tyrosine hydroxylase, or growth factors, such as nerve growth factor. The peptide may also be an antigen associated with the disease. The cells can be implanted for treatment or prophylaxis purposes. In some cases, the cells can be removed immediately after implantation. In the further embodiments, the present invention provides an immortalized human fetal neuro-derived cell line, which comprises a heterologous nucleic acid sequence, wherein the cell line is capable of expressing the heterologous nucleic acid sequence. Particularly preferred cell lines are capable of expressing a nucleic acid encoding tyrosine hydroxylase. In the most preferred aspects, the cell lines of the present invention are capable of expressing serotonin. In a related embodiment, the present invention provides a transplantable composition comprising the cell lines of the invention, with a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 demonstrates the morphology of SVG cells in vitro. Figure 2 illustrates the immunoperoxidase staining of an antibody to the SV40 T protein in SVG cells. Figure 3 demonstrates needle tracking in the basal ganglia with low amplification. Figure 4 illustrates a high amplification view of needle scanning in the basal ganglia. Figure 5 demonstrates another high amplification view of a needle scan in the basal ganglia.

Figure 6 demonstrates a nest of SVG cells on the wall of the lateral ventricle. Figure 7 illustrates SVG cells implanted on the wall of the lateral ventricle stained with an antibody to the glial fibrillary acidic protein. Figure 8 demonstrates a live section of implanted SVG cells stained with an antibody against the T protein. Figure 9 demonstrates a T1-weighted MRI (with gadolinium enhancement) of a monkey brain, 6 months following implantation. Figure 10 demonstrates the growth of a tyrosine hydroxylase neuron on a layer of SVG cells implanted in vivo. Figure 11 shows a schematic representation of the construction of the phTH / Neo plasmid used in the construction of the SVG-TH cell lines. Figure 12 shows the immunohistochemical staining with tyrosine hydroxylase of a stable phTH / Neo transfectant. Figure 13 shows the Western blot of a TH positive clone (1B1B) confirming the immunohistochemistry shown in Figure 12. Figure 14 shows a chromatogram of the high pressure liquid chromatography analysis of the SVG-TH cell culture supernatant . Two s, at the retention times of 25.65 and 37.1 minutes, corresponded to the retention times for serotonin and 5-hydroxy-indoleacetic acid, a product of the decomposition of serotonin, respectively. This was confirmed by immunohistochemical staining of SVG-TH cells for serotonin. Figure 15 shows an electron micrograph of SVG-TH cells. Figure 16A shows the immunohistochemical staining of cells from a coculture of hNT / SVG-TH, after about 72 hours. The small flat single cells are the SVG-TH cells. Figure 16B shows the immunohistochemical staining of cells from a co-culture of PC12 / SVG-TH, after approximately 92 hours. A PC12 cell is shown near the center of the photograph, the neuronal processes extending from it to the nearby SVG-TH cells. Similar results were obtained for SVG cells. Figure 17 shows a graph of the distribution of positive cell counts for tyrosine hiroxylase, for dopaminergic cells coated in 100,000 cells per transcavity chamber with and without SVG coculture. The number of rat mesencephalic cells positive for remaining tyrosine hydroxylase per transcavity chamber (Y axis) is shown, when these cells were cultured for 96 hours in the absence and in the presence of SVG cells. The co-culture with SVG-TH cells produced identical results. Figure 18 shows the effect of the SVG-TH transplant in rat models of parkinsonism. The functional deficit is indicated by the number of rotations per hour in the model rats. The number of rotations per hour is shown after the rat was stimulated with apomorphine, before, and for 4 weeks following the grafting of SVG-TH cells into the injured striatum of the rats (shown as solid squares, solid diamonds, open squares, open diamonds and solid squares with a clear point). The results are also shown for two SVG transplants (shown as solid triangles and open squares with a black dot). Figures 19, 20 and 21 show the time course for the improvement in the Parkinson's Disability Scores, the grafting of SVG-TH cells in the caudate and the putamen of three Rhesus monkeys (H002, H005 and T022) that had treated with MPTP to induce the symptoms of Parkinson's disease. DESCRIPTION OF SPECIFIC MODALITIES The present invention relates generally to immortalized human fetal cell lines derived from cells of the central nervous system, and to methods for using these cell lines in the treatment of disorders of the central nervous system. In particular, the cell lines and methods of the present invention can be used in the treatment of disorders caused by lesions in the central nervous system., such as parkinsonism. I. Methods of Treatment In one embodiment, the present invention provides methods for the treatment of a host suffering from a central nervous system disorder, or for alleviating the symptoms of that disorder, by implantation of immortalized human fetal cells derived from of cells of the central nervous system. No graft rejection, intense intracerebral inflammation, or tumor formation has been demonstrated following the implantation of these cells in the central nervous system. In addition, it has been shown that cells induce the migration of neurons and the extension of neurites. This shows that the cells are functioning to produce trophic factors that stimulate neuronal responses. The implantation of immortalized human fetal cells derived from cells of the central nervous system provides a means for the treatment of many diseases. For example, Parkinson's disease can be treated by implanting these cells in the basal ganglia of an affected host. The trophic factors produced by the implanted cells can inhibit the transfer of dopaminergic neurons, and even induce the regeneration of dopaminergic neurons. The greater population of dopaminergic neurons can provide a clinical improvement to people suffering from parkinsonism. In the additional embodiments, the implanted cells can be transfected into a nucleic acid encoding a neurologically relevant polypeptide. The term "neurologically relevant peptide" refers generally to a peptide or a protein that catalyzes a reaction within the tissues of the central nervous system. These peptides can be naturally occurring peptides, proteins or neural enzymes, or they can be peptide or protein fragments having a therapeutic activity within the central nervous system. Examples include neural growth factors, and enzymes used to catalyze the production of important neurochemicals, or their intermediates. In particularly preferred aspects, the cells will be transfected with a nucleic acid encoding tyrosine hydroxylase. Tyrosine hydroxylase is the enzyme that converts tyrosine to L-DOPA, which is also the speed limiting step in the production of dopamine. Accordingly, the expression of tyrosine hydroxylase by the implanted cells allows these cells to produce and secrete dopamine. Therefore, in addition to promoting neuronal regeneration, implanted cells can increase the concentration of dopamine in the substantia nigra, and limit or reverse the effect of the loss of dopaminergic neurons. The methods of the present invention can also be used to treat other neurogenic disorders, such as Huntington's chorea, Alzheimer's disease, or multiple sclerosis. Since immortalized human fetal neuro-derived cells are compatible with the central nervous system (CNS), these cells can also be transfected with DNA sequences that encode physiologically active peptides, and can be implanted in the central nervous system to effect the treatment of other disorders. For example, in Huntington's chorea and amyotrophic lateral sclerosis, the peptide can block excitatory neurotransmitters, such as glutamate. When applied to the treatment of multiple sclerosis, for example, the peptide would typically be a trophic myelinating stimulator, such as platelet-derived growth factor, or a ciliary trophic factor that can block the transfer of oligodendrocytes. Since these diseases are more widespread than local lesions, alternative implantation methods may be desirable. For example, the cells can be implanted on a surface exposed to the cerebrospinal fluid. Next to the expression and secretion, the peptide will be washed over the entire surface of the brain by the natural circulation of the cerebrospinal fluid. Suitable sites for implantation include the lateral ventricles, the lumbar thecal region, and the like. In Alzheimer's disease, cells can be transfected to produce nerve growth factor, to support the neurons of the anterobrain, as described by Rosenberg et al., Science, 242: 1575-1578 (1988), incorporated herein as reference. The methods of the present invention can also be used to treat hosts by implanting cells at extraneural sites. This embodiment of the present invention is particularly useful for the prophylactic treatment of a host. • Immortalized human fetal neuro-derived cells can be transfected with DNA encoding an antigen associated with the disease, eg, HIV gpl20 pellipeptides, which encompass the main HIV neutralizing domain, as described, for example, in the Patent of the United States of America Number 5,166,050. The cells can then express and secrete the antigen encoded by the transfected DNA. The antigen can be continuously secreted by the implanted cells, and can cause a strong immune response. After an adequate interval of time to completely immunize the host, the cells can be removed.

As used herein, "treating a host" includes prophylactic, palliative, and curative intervention in a disease process. Accordingly, the term "treatment", as used herein, typically refers to therapeutic methods to reduce or eliminate the symptoms of the particular disorder for which the treatment is sought. The term "host", as used herein, generally refers to any warm-blooded mammal, such as humans, non-human primates, rodents and the like, which is to be the recipient of the particular treatment. Typically, the terms "host" and "patient" are used interchangeably herein to refer to a human subject. A wide variety of diseases and syndromes can be treated by the methods of the present invention. In general, the disease will be a neurological disease, such as parkinsonism (including Parkinson's disease), Alzheimer's disease, Huntington's chorea, multisclerosis, amyotrophic lateral sclerosis, Gaucher's disease, Tay-Sachs disease, neuropathies, brain tumors, and Similar. The methods of the present invention can also be employed in the treatment of non-neurological diseases. For example, the methods of the present invention can be employed to immunize hosts against infectious diseases, such as viruses, bacteria, protozoa and the like, as described above. Immortalized human fetal neuro-derived cells can be transfected by DNA encoding physiologically active peptides, or peptides containing immunological epitopes. The methods of the present invention can be employed to implant the peptide producing cells, and to provide continuous in vivo delivery of other types of peptides, such as growth hormone, to the host. II. Cell Lines A. General In order to practice the treatment methods described above, the present invention also provides suitable cell lines to be transplanted to a host or a patient. In general, the cells implanted by the methods of the present invention are immortalized human fetal neuro-derived cells. "Neuro-derived" means that, before immortalization, the cells had a neurological cell phenotype, or were an embryonic cell compromised for differentiation to a neurological cell type. Neurological cell types include neurons, astrocytes, oligodendrocytes, epithelial cells of the choroid plexus, and the like. The preparation of the immortalized fetal cell lines can be performed in general in accordance with the following procedures. Fetal cells can be collected immediately after elective abortion. Women who donate fetuses following abortion will typically be screened serologically for a variety of infectious diseases, including human immunodeficiency virus, Hepatitis B virus, Hepatitis C virus, cytomegalovirus, and Herpes virus types 1 and 2. Fetuses They will generally be 9 to 11 weeks gestational age (7 to 9 weeks after conception). Fetal age can be confirmed by ultrasound. The fetuses can be removed under ultrasound guidance to minimize trauma to the fetal brain. Following the extraction, the fetal brain is identified and dissected from the abortion. The cells can be prepared as follows. The brain tissue is aspirated through a 19 gauge needle, and washed twice in Eagle's minimal essential medium (E-MEM, Gibco, New York, N.Y.). The cells are coated on culture dishes treated with poly-D-lysine (0.1 milligrams / milliliter for 5 minutes). Cells are grown on E-MEM supplemented with 20 percent fetal bovine serum, 75 micrograms / milliliter streptomycin, 75 units / milliliter of penicillin, 1 percent dextrose, and 2 micrograms / milliliter of fungizone (Gibco). Before immortalization, the cells are incubated at 37 ° C in a humidified environment with 5 percent C02. An expert in this field will recognize that other methods for the preparation of the cells can also be employed. The cells to be implanted by the methods of the present invention can be immortalized by a variety of techniques. Typically, the cells will be immortalized as follows. Cell cultures will generally produce neuronal and glial progenitor cells, as well as neurons, as described by Major and Vacante, J. Neuropath. and Exp. Neurol. , 48: 425-436 (1989), incorporated herein by reference. With regular feedback, brain cells will survive for several months, but will show little cell proliferation. The cells are transformed by transfection with a SV40 deletion mutant. The DNA of the mutant lacks a replication origin (ori-) and can not multiply. However, transfection of the DNA will transform the cells for unlimited growth potential, as described by Gluzman, Cell. 23: 175-182 (1981). After growth of fetal cell cultures for 3 weeks, the cells can be transfected with 100 μg / vial of plasmid DNA (pMK16) containing the OR40 mutant, using the calcium phosphate precipitation technique as described by Graham and collaborators, Virol .. 52: 456-467 (1973). Alternatively, the cells can be transfected by electroincorporation, or other well-known techniques, as described in Sambrook et al., Molecular Cloninq. A Laboratorv Manual. Cold Spring Harbor Press, 1988, incorporated herein by reference. Following the transfection, the crops are grown with weekly feedback. After several weeks, the proliferation of glial cells in areas separated from the plates becomes evident. Then the cells are transferred by trypsinization (0.025 percent) to new cultures. Transformed cells can be identified by fluorescence antibody assays to detect the SV40 T protein, which is expressed by the transformed cells (Figure 2). The cells are passaged every 10 days until an increase in the number of T-cell positive cells is detected. The transformed cells will exhibit the phenotype of a continuous cell line. In a specific manner, the cells will grow to a high saturation density with a generation time of 18 hours. However, the cells do not show the transformed phenotype or anchorage-independent growth, which is characteristic of non-mutant SV40 transformed cells. The cell morphology is not altered during the course of the establishment of the cell line. In general no foci of cells are detected. Particularly useful cells include those from the SVG cell line deposited at the American Type Culture Collection, Rockville MD (ATCC CRL 8621), which is described in U.S. Patent No. 4,707,448, incorporated herein by reference (FIG. 1) . Hereinafter, "SVG cells" or "SVG cell line", mean cells or a cell line derived from cell line A.T.C.C. CRL 8621. The derivatives mean a subclone, a replica, or a genetically altered mutant of the A.T.C.C. cell line. CRL 8621. Alternatively, cells can be immortalized by other techniques that are well known in the art. For example, immortalization can be employed by Epstein-Barr virus, as described in U.S. Patent No. 4,464,465, incorporated herein by reference. Epstein-Barr virus mutants lacking the origins of replication OriP and OriLyt are particularly useful. Another useful method of immortalization is the over-expression of a cellular gene for growth control, such as c-myc, as described by Bartlett et al., Proc. Nati Acad. Sci. USA, 85: 3255-3259 (1988), incorporated herein by reference. In general terms, the transformed cells suitable for implantation will depend on the anchor, will not grow on soft agar, and will not exhibit foci formation. The histological origin of the transformed cells can then be determined. Characteristically, astroglial cells can be recognized by the presence of an intermediate filament composed of glial fibrillary acidic protein, PAFG. Oligodendroglial cells, on the other hand, are myelin-producing cells, and can be identified by their synthesis of a galactocerebroside, gal C, which is a component of myelin. Following the transformation, the cells will be prepared for implantation. The cells are suspended in a physiologically compatible vehicle, such as a cell culture medium (eg, Eagle's minimal essential medium) or phosphate-buffered serum. The cell density is generally about 10 4 to 10 7 cells / ml.li.li.tro. The cell suspension is gently oscillated before implantation. The volume of the cell suspension to be implanted will vary depending on the implantation site, the treatment goal, and the cell density in the solution. Typically, the amount of cells transplanted to the patient or the host will be a "therapeutically effective amount". As used herein, a "therapeutically effective amount" refers to the number of transplanted cells that are required to effect the treatment of the particular disorder for which the treatment is sought. For example, where the treatment is for parkinsonism, transplantation of a therapeutically effective amount of cells will typically result in a reduction in the amount and / or severity of symptoms associated with that disorder, eg, stiffness, akinesia and gait disorder. . In parkinsonism treatment, typically 5 microliters to 60 microliters of cell suspension will be administered in each injection to achieve this effective amount. Several injections can be used in each guest. The skilled persons will understand how to determine the appropriate cell dosages. In alternative preferred embodiments of the present invention, cells that are useful for transplantation can be transfected with, and are capable of expressing, a heterologous nucleic acid sequence encoding a neurologically relevant peptide. The term "heterologous", as used to describe nucleic acids herein, refers in general to a sequence that, as a whole, does not occur naturally within the cell line transfected with that sequence. Accordingly, the heterologous sequence may comprise a segment that is entirely foreign to the cell line, or alternatively may comprise a native segment that is incorporated into the cell line in a non-native manner, eg, linked to a promoter / enhancer sequence. non-native, linked to a native promoter that is not typically associated with the segment, or provided in multiple copies where the cell line usually provides one or no copies.

In general, the nucleic acid sequence will be operably linked to a transcription promoter and a transcription terminator. A DNA segment is operably linked when placed in a functional relationship with another segment of DNA. For example, a promoter or enhancer is operably linked to a coding sequence, if it stimulates the transcription of the sequence; the DNA for a signal sequence is operably linked to the DNA encoding a polypeptide, if it is expressed as a preprotein that participates in the secretion of the polypeptide. In general, the operably linked DNA sequences are contiguous, and in the case of a signal sequence, they are both contiguous and in reading phase. However, enhancers do not need to be contiguous with the coding sequences whose transcription they control. The link is made by ligation in the convenient restriction sites or in the adapters or linkers inserted in their place. The DNA sequence can also be linked to a transcription enhancer. The expression of DNA in implanted cells can be constitutive or inducible. A variety of expression vectors having these characteristics can carry the DNA for transfection of the cells, such as the plasmid vectors pTK2, pHyg and pRSVneo, simian virus vectors 40, bovine papillomavirus vectors, or virus vectors. Epstein-Barr, as described in Sambrook et al., Molecular Clonincr. A Laboratorv Manual, Cold Spring Harbor Press, 1988, formerly incorporated herein by reference. Vectors can be introduced into cells by conventional methods, such as electroincorporation, calcium phosphate mediated transfection, polybrene transfection, and the like. The peptide encoded by the nucleic acid can generally be a directly therapeutic compound, such as a movement inhibitor in the treatment of Huntington's chorea. Alternatively, the peptide encoded by the nucleic acid can be selected to complement or replace the deficient production of the peptide by the host's endogenous tissues, whose deficiency is a cause of the symptoms of a particular disorder. In this case, the cell lines act as an artificial source of the peptide. Alternatively, the peptide can be an enzyme that catalyzes the production of a therapeutic, or of neurologically relevant compounds. Again, these compounds can be exogenous to the host system, or they can be an endogenous compound, whose path of synthesis is impaired in another way. In the latter case, the production of the peptide within the central nervous system of the host provides complementary trajectories for the production of the compound. For example, in a preferred embodiment, immortalized human fetal neuro-derived cell lines are transfected with a nucleic acid encoding a tyrosine hydroxylase enzyme. Tyrosine hydroxylase catalyzes the synthesis of dopamine from tyrosine. It has been shown that dopamine is effective in the treatment of parkinsonism. In particularly preferred aspects, the immortalized fetal neuro-derived cell lines that are transfected with a nucleic acid encoding tyrosine hydroxylase will be an SVG cell line, v. gr. , those of the SVG cell line deposited in the American Type Culture Collection, Rockville MD, (A.T.C.C. CRL 8621), which is described in United States Patent No. 4,707,448, incorporated herein by reference (Figure 1). These cell lines are referred to herein as SVG-TH cell lines. In still more preferred aspects, the SVG cell line is transfected with a phTH / Neo plasmid. The nucleic acid can also encode a trophic factor, such as a nerve growth factor, a growth inhibitory factor, or a cytokine useful in the treatment of brain tumors. Due to its ability to improve neural regeneration and to produce and secrete dopamine, the cell lines of the present invention are particularly useful in the treatment of central nervous system disorders that are associated with the loss of dopaminergic cells in the central nervous system of the brain. host, such as parkinsonism. Surprisingly, it has also been discovered that the cell lines of the present invention are also capable of producing additional neurotransmitters. In a particularly preferred embodiment, for example, the cell lines of the present invention can also express serotonin. Serotonin has been implicated in presentations of clinical depression in human subjects. Specifically, the increase in serotonin levels in the tissues of the central nervous system has been found to alleviate the symptoms of depression, and form the basis of a number of antidepressant treatments, for example, Prozac As such, the cell lines of the present invention may also be particularly useful in methods for the treatment of disorders associated with reduced levels of serotonin in the central nervous system, such as depression. Typically, these methods are the same or substantially similar to the methods described herein for the treatment of other disorders of the nervous system. For those suffering from parkinsonism, the cell lines of the present invention, therefore, have a dual benefit of alleviating the symptoms of the disorder through the secretion of dopamine and neural regeneration, as well as treating the depression associated with the disorder through the secretion of serotonin. III. Implantation of Cell Lines Typically, the cells of the cell lines of the present invention can be implanted into the parenchyma of the brain, in a space containing cerebrospinal fluid, such as the subarachnoid space or the ventricles, or extraneurally. As used herein, the term "extraneurally" indicates regions of the host that are not within the central nervous system or peripheral nervous tissue, such as the celiac ganglion or the sciatic nerve. The "extraneural" regions may contain peripheral nerves. "Central nervous system" means that it includes all the structures that are inside the hard mother. When cells are implanted in the brain, stereotactic methods, as described in Leksell and Jernberg, Neurochir Acta, will generally be employed. , 52: 1-7 (1980), and Leksell et al., J. Neurosurg. , 66: 626-629 (1987), both of which are incorporated herein by reference. The location of the objective regions will generally include a preimplantation MRI as described in Leksell et al., J. Neurol. Neurosurq. Psvchiatrv, 48: 14-18 (1985), incorporated herein by reference. The objective coordinates will be determined from the preimplantation MRI.

Before implantation, the viability of the cells can be evaluated as described by Brundin et al., Brain Res., 331: 251-259 (1985), incorporated herein by reference. Said briefly, sample aliquots of the cell suspension (from 1 to 4 microliters) are mixed on a glass slide with 10 microliters of a mixture of acridine orange and ethidium bromide (3.4 microliters / milliliter of each component in 0.9 percent serum, Sigma). The suspension is transferred to a hemocytometer, and viable and non-viable cells are counted visually using a fluorescence microscope under epi illumination at 390 nanometers, combined with trans white light illumination to visualize the grid of the counting chamber. The acridine orange stains the living nuclei with green, while the ethidium bromide will enter the dead cells, resulting in an orange-red fluorescence. Cell suspensions should generally contain more than about 98 percent viable cells. Injections will generally be made with sterilized 10 microliter Hamilton syringes, which have 23-27 gauge needles. The syringe, loaded with cells, is mounted directly on the head of a stereotaxic frame. The needle of the injection is lowered to the predetermined coordinates through small holes drilled in the skull, 40 to 50 microliters of suspension are deposited at the speed of approximately 1 to 2 microliters per minute, and another 2 to 5 are passed through. minutes for diffusion before a slow retraction of the needle. Often two deposits will be made separated by 1 to 3 millimeters, along the same penetration of the needle, and can easily be made up to 5 deposits scattered over the objective area in the same operation. The injection can be done manually or by an infusion pump. At the end of the surgery following the retraction of the needle, the host is removed from the frame, and the wound is sutured. Prophylactic antibiotics or immunosuppressive therapy may be administered, as necessary. For the treatment of more generalized neurogenic disorders, the cells can be transfected to express a therapeutic compound, or they can be implanted in the ventricles or in the lumbar teak. Since the therapeutic compound is secreted by the cells, the natural circulation of the cerebrospinal fluid washes the therapeutic compound throughout the central nervous system, providing a means of generalized treatment. Implantation in the ventricles can be performed by an open procedure, as described in Madrazo et al., New Enql. J. Med. 316: 831-834 (1987), or Penn et al., Neurosurqery, 22: 999-1004 (1988), both of which are incorporated herein by reference. The implantation of the cells in the lumbar teak is carried out more conveniently by conventional procedures similar to the instillation of radiographic contrast medium or antitumor drug by means of a lumbar puncture. In some cases, it may be desirable to implant cells extraneurally in accordance with the present invention. The cells can be implanted percutaneously through a needle or an endoscope, or through an open procedure. The skilled person will readily appreciate the most appropriate method for implanting the cells for particular applications. The cells can be encapsulated in membranes before implantation. The encapsulation provides a barrier to the host's immune system, and inhibits graft rejection and inflammation. Various methods of cell encapsulation can be employed. In some cases, the cells will be encapsulated individually. In other cases, many cells can be encapsulated within the same membrane. When the cells are removed immediately after implantation, the relatively large size of a structure that encapsulates many cells within a single membrane provides a convenient means to recover the implanted cells. Various methods of cell encapsulation are well known in the art, such as those described in European Patent Publication Number 301,777, or in the Patents of the United States of North America Numbers 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350; or 5,089,272, each of which is incorporated herein by reference. A method of cell encapsulation is as follows. The transformed cells are mixed with sodium alginate (an extract of plianionic marine algae), and extruded in a solution of divalent cations, for example, calcium chloride, which forms complex with sodium alginate to form a gel, giving as a result the formation of gelled granules or droplets containing the cells. The gel granules are incubated with a concentration (of 0.03 to 0. 1 percent by weight / volume) of high molecular weight (molecular weight of 60 to 500 x 10 of polyamide, such as poly-L-lysine, for a short period of time (from 3 to 20 minutes) to form a The interior of the formed capsule is liquefied again by its treatment with sodium citrate.The only membrane around the cells is highly permeable (molecular weight cut-off of 200-400 x 3 10). The single membrane capsule containing the cell is incubated in saline for 1 to 3 hours to allow the trapped sodium alginate to diffuse out of the capsule, and expand the capsule to a state of equilibrium. The resulting alginate-poor capsule is reacted with a low molecular weight polyamino acid (molecular weight of 10-30 x 10), such as a poly-L-lysine (PLL) or 3-chitosan (deacetylated chitin, molecular weight 240). x 10), to produce a less permeable, interacting membrane (molecular weight cutoff of 40-80 x 10). The double membrane encapsulated cells are then cultured in E-MEM for two to three weeks, as described above. Although reference has been made specifically to sodium alginate granules, it will be appreciated by those skilled in the art that any non-toxic water-soluble substance that can be gelled to form a shape-retaining mass can be employed by a change in the conditions of the medium in which it is placed. This gelling material generally comprises various chemical fractions which are easily ionized to form anionic or cationic groups, such that the surface layers can be crosslinked to form a permanent membrane upon exposure to oppositely charged polymers. Most polysaccharide gums, both natural and synthetic, can be crosslinked by polymers containing positively charged reactive groups, such as amino groups. The crosslinking biocompatible polymers that can be reacted with the sodium alginate gum include polylysine and other polyamino acids. The degree of permeability of the formed membrane can be controlled by a careful selection of a polyamino acid having the desired molecular weight. Poly-L-lysine (PLL) is the preferred polymeric material, but others include chitosan and polyacrylate. Molecular weights typically range from about 10 4 to about 106. The present invention is further illustrated by the following examples. These examples are merely to illustrate aspects of the present invention, and are not intended to be limitations of this invention. Example 1 - Preparation of SVG Cells This example describes the preparation of SVG cells (A.T.C.C. CRL 8621) to be implanted in Rhesus monkeys. The cells were screened for infection with mycoplasma, HIV-1, Hepatitis B virus, Simian virus 40, Herpes simplex virus, cytomegalovirus and JC virus. SVG cells were cultured until confluency. Cell growth depended on anchorage. There was no formation of foci, and the morphology of the cells was homogeneous. The cells were removed from the tissue culture dishes by digestion with 0.05% trypsin in 0.01M ethylenediaminetetraacetic acid (Versene pH Regulator) in a Hank's balanced salt solution. Cells were harvested by centrifugation, washed 3 times and resuspended in phosphate-buffered serum. The final cell density was 10 cells / milliliter. The cell suspension was stored at 4 ° C until transplantation.

Example 2 - Implantation of SVG Cells This Example describes the implantation of SVG cells in the basal ganglia of six Rhesus monkeys. The implantations were performed by stereotaxic methods without surgical complications. The animals were initially anesthetized with ketamine and kept anesthetized with isofluorine gas during the course of the surgery. The animals were placed in the stereotaxic frame (Kopf), and the brands for the implantation were established through the stereotactic coordinates. The superior sagittal sinus was exposed in order to establish the midline. The marks were placed on the skull on the caudato and the putamen on both sides. The coordinates were as follows: AP was +24 millimeters opposite 0. The lateral coordinates were 5 millimeters from the median line for the caudato nucleus, and 10 millimeters from the median line for the putamen. Five holes were drilled. One was made on the superior sagittal sinus, two on the caudatos and two on the putámenes. Two different implantation techniques were used. 1. Hamilton 10-microliter syringes were used with a 26-gauge needle, or Hamilton 50-microliter syringes with 23-gauge needles. On the right side of the brain, SVG cells were transplanted. Using the syringes, two deposits were made in the putamen. One deposit was in the lateral putamen, and the second was in the middle putamen. The needles were lowered to 18 millimeters from the cortex, and then 10 microliters of the cell suspension were implanted using the Kopf microinjector. After the first implantation, the needle was removed at 1 millimeter minute up to 3 millimeters, and then the second injection of 10 microliters of the cell suspension was followed. After the second injection, the needle was removed at 1 millimeter per minute. A second implantation was made in the opposite putamen in the same coordinates and with the same technique. After injecting the putamen, implantation was performed in the caudato nucleus with the same cell suspension. Two injections were made in the caudato, in the lateral and medial aspects. The depth of the injection was 15 millimeters, and 10 microliters were implanted. The syringe was removed at 1 millimeter per minute up to 3 millimeters, and then the second injection of 10 microliters of the cell suspension was performed. The non-transfected SVG cells were transplanted into the putamen, and the SVG cells transfected with the tyrosine hydroxylase gene were transplanted into caudate. The concentration of the cells was 2 x 10 cells per milliliter. 2. In addition to using implantation with syringes with needles, blue slit tubing cannulae connected with 22 gauge waters were constructed. The tubing was connected to tuberculin syringes of 1 cubic centimeter, using connectors of dead volume 0. Next to the insertion in the objective, the needle was allowed to remain for 15 minutes before infusion. Then a Harvard infusion pump containing the cell suspension was ripped off at 0.2 microliters per minute. After infusing for 15 minutes at 0.2 microliters per minute, the velocity was increased to 0.4 microliters per minute, and continued for 100 minutes. After finishing the infusion, the needles were left in place for 30 minutes before retiring. Then the needles were removed very slowly from the brain. The wound was rinsed and then closed in anatomical layers. The animals were awakened from anesthesia, and transferred to their cages 20 minutes after surgery. Example 3 - SVG Cell Grafting in Monkeys This example demonstrates the successful grafting of SVG cells implanted in two of the monkeys sacrificed one month after implantation. The transplanted cells were histologically healthy. There was no evidence of inflammation or tumor formation. The brain tissue in the region of the implantations was examined as follows: For histopathological studies, the animals were sacrificed by an overdose of pentobarbital (460 milligrams intravenous), and perfused through the ascending aorta with 15 milliliters of ice-cold phosphate-regulated serum (SRF), followed by 10 percent formalin. The brains were quickly removed, cut into 6-millimeter coronal slices, and then fixed for 30 minutes in the same fixative. The tissue slices were rinsed for 48 hours in 30 percent sucrose in phosphate-buffered serum, and then frozen rapidly at -70 ° C. The tissue was cut into coronal sections of 40 microns in a freezing microtome, and series of sections were collected in phosphate-regulated serum. Sections were processed for immunohistochemistry with antibodies against tyrosine hydroxylase, glial fibrillary acidic protein, and T protein. Sections adjacent to those examined by TH-IR were stained with hematoxylin and eosin. Some tissue blocks containing the implant were processed into paraffin sections of 5 microns, and stained as described above. Figure 3 illustrates the needle footprint in a basal ganglia of one of the monkeys at a low power. The higher power views of the needle footprint (Figures 4 and 5) demonstrate viable SVG cells in the footprint. Cells are easily identified by large nuclei that contain multiple nucleoli, as exhibited by SVG cells in vitro. The morphology of the implant cells is surprisingly different from the morphology of the surrounding cells. No inflammatory cells or tumor formation were identified. Identical tests were performed on monkeys slaughtered at nine months after transplantation. The graft was identified, and no evidence of inflammatory cells or tumor formation was discovered, indicating that the cells had been grafted and had not been rejected by the host. Example 4 - Evaluation by MRI of Grafted SVG cells This example describes the evaluation with cerebral MRI one month after the implantation of the four remaining monkeys. There was no evidence of tumor formation present in any of the monkeys. Following the induction of anesthesia, the monkeys were placed in a standard MRI frame. Were the weighted images T? and 2 without contrast, and the images weighted T. with gadolinium, using a Tesla 1.5 magnet (Sigma). The scans did not reveal any evidence of tumor or nodule formation (Figure 9). Example 5 This example demonstrates the functioning of SVG cells transplanted into the central nervous system. The neurons of the host migrated to the implanted cells, the neuronal dopaminergic bodies, and the dopaminergic processes from the origin of the host were extended to the implanted cells. Two of the surviving monkeys that received SVG cell implants, as described in Example 2 above, were sacrificed as described. The brains were removed intact as described above, and sectioned. Each section was placed on gelatin-coated slides. The representative sections were stained with hematoxylin and eosin to characterize the anatomy (Figure 6). The implanted cells exhibited a characteristic SVG morphology with large nuclei that had multiple nucleoli. Adjacent sections were stained with either monoclonal antibody for glial fibrillary acidic protein (PAFG), SV40 T protein, or tyrosine hydroxylase. Then the sections were counter-stained with hematoxylin alone. Figure 7 illustrates an adjacent section stained with antibody to glial fibrillary acidic protein, a cytoplasmic protein of the astrocytic lineage. The astrocytic origin is demonstrated by the cytoplasmic dense dyeing. The origin of the cells is also illustrated in Figure 8, which clearly shows the implanted cells stained with antibody against the T protein.

The cells grafted into caudate and putamen were viable and were easily identified by the antibody against the T protein as described above. SVG cells were also identified on the wall of the lateral ventricles of all monkeys. The dopaminergic neurons exhibited an overgrowth of neurites towards the implanted cells (Figure 10 demonstrates a tyrosine hydroxylase neuron stained with tyrosine hydroxylase antibody in a layer of SVG cells in vivo). Dopaminergic neuronal bodies were also present in the region of the implanted SVG cells. The overgrowth of neurites, and the presence of neuronal bodies, indicate that SVG cells produced neurotransmitters caused by the migration of neurons and the extension of neuronal processes. No evidence of inflammation, graft rejection, tumor or nodule formation was found in any of the sections. Example 6 - Encapsulation of SVG Cells This example describes the individual encapsulation of SVG cells, and the preparation of cells for implantation. The cells are encapsulated in a granule of sodium alginate. SVG cells are grown to confluence in petri dishes. The cells are removed from the petri dishes with 0.05 percent trypsin and 1 mM ethylenediamine tetraacetic acid in Dulbecco's phosphate-regulated serum (SRF). Cells are suspended in phosphate-buffered serum supplemented with MgCl_, CaCl2, 0.1 percent glucose, and 5 percent fetal bovine serum. The cells are harvested by centrifugation, washed twice in the suspension solution as described above, and centrifuged to a pellet. The remaining cell pellet at the bottom of the centrifuge tube is resuspended in 5 milliliters of a 1.5 percent (w / v) sodium alginate solution ® (Keltone LV by Kelco, Ltd., Chicago, Illinois). The alginate cell suspension is extruded in 50 milliliters of a 1.5% (w / v) CaCl solution. Spherical droplets of the suspension are formed by a droplet generator of air jet syringe pump. With this device, the sodium alginate cell suspension is extruded through a 22-gauge needle located inside a shed tube (3 millimeters in internal diameter), through which air flows at a controlled rate (9 liters per minute). When the droplets of liquid are forced out of the end of the needle by the syringe pump (at 20 cubic centimeters per hour), the droplets are pulled outward by the tearing forces established by the rapidly flowing air stream. The tip of the needle is maintained 8 centimeters above the surface of the CaCl solution, to ensure that uniform spherical gel droplets with a diameter of approximately 300 to 1,000 microns are formed. A sample of the gelled microgranules is examined for its size and the consistency of its shape, using a dissecting microscope (Wild Heerbrugg, Model M8) adapted with a calibrated eyepiece. After transferring the calcium alginate gel granules, which contain the immobilized cells, to a 50 milliliter plastic centrifuge tube with a conical bottom, the granules are washed with 30 milliliters of each of the 0.1 CHES solutions. percent (weight / volume) and CaCl2 at 1.1 percent (weight / volume). The volume of the supernatant is reduced after each wash, using a vacuum aspirator. A semipermeable capsule membrane is formed by reacting the gel droplets with a 0.05 percent (w / v) aqueous PLL solution (M / v PLL = 22,000) for 8 minutes. After the addition of the PLL solution, the centrifuge tube is manually capped and oscillated from end to end for the duration of the reaction, to prevent the capsules from sticking together. The resulting microcapsules, 300 to 1,000 microns in diameter, are washed with 30 milliliters of each of the 0.1% CHES and 1.1% CaCl2 solutions, and with two aliquots of 30 milliliters of isotonic serum. The encapsulated cells are contacted with 30 milliliters of the sodium alginate solution at 0.03 percent (w / v) for 4 minutes, forming an outer layer on the capsule. The interior of the microcapsules is blended with 30 milliliters of a 0.05 M sodium citrate solution for 6 minutes. Microcapsules of 400 to 1,400 microns in diameter are washed several times in serum to remove excess citrate, and then divided into aliquots of 1 milliliter. Each aliquot is incubated in 10 milliliters of DMEM medium in a 25 cm 3 culture flask. 37 ° C in a 400 series C02 isotemperature incubator, (Model 413D, Fisher Scientific Co., Nepean, Ontario) Example 7- Genetic Engineering of SVG Cells for Expressing Tyrosine Hydroxylase This example describes the transfection of SVG cells with a nucleic acid encoding tyrosine hydroxylase. expressed tyrosine hydroxylase in the cultures following the transfection The SVG cell line was transfected with a nucleic acid encoding the enzyme tyrosine hydroxylase (TH) The plasmid phTH-63 has the type 2 cDNA for the tyrosine hydroxylase cloned in the EcoRI site of the Bluescript KS vector The tyrosine hydroxylase cDNA was cloned into two different eukaryotic expression vectors, pcDNA / Neo and pRSFV / Neo (both available from Invitrogen, Corp., San Diego, Calif.) A HindIII fragment / PhTH-63 BamHI, which contains the tyrosine hydroxylase cDNA, was cloned into the HindIII / HindI site of pcDNA / Neo, resulting in the phtH / Neo plasmid. similarly, a HindIII / Spel fragment of phTH-63, containing the tyrosine hydroxylase cDNA was cloned into the HindIII / Spel site of pRC / RSV, resulting in the plasmid pRSV / hTH / Neo. As seen in Figure 11, phTH / Neo consists of the immediate early cytomegalovirus promoter upstream of the tyrosine hydroxylase cDNA, on a plasmid conferring resistance to neomycin. The construction of pRSV-hTH / Neo consists of RSV LTR upstream of the tyrosine hydroxylase cDNA on a plasmid conferring resistance to neomycin. Separate cultures of SVG cells were established, and each was transfected either with phTH / Neo or with pRSV-hTH / Neo. Following transfection, the cells were cultured in a medium containing geneticin at 500 micrograms / milliliter for two months. Seven clones that were stably resistant to geneticin were established from the phTH / Neo transfection. Both transfectants were able to produce tyrosine hydroxylase; however, stable long-term clones were not established using the pRSV-hTH / Neo construct, due to the weak expression of the neomycin resistance marker in this plasmid. Figure 12 shows the immunohistochemical staining of tyrosine hydroxylase from one of the stable phTH / Neo transfectants. The clones were from 30 percent to 60 percent positive for tyrosine hydroxylase. One clone (1B1B) that was 40 to 60 percent positive for tyrosine hydroxylase was expanded, and the Western blot was made to confirm immunohistochemistry. As seen in Figure 13, when Western blot was probed with a polyclonal antibody to tyrosine hydroxylase, a band was detected that migrated at approximately 60 Kd, consistent with the size of tyrosine hydroxylase type 2. Clone 1B1B Subsequently designated as SVG-TH. To determine if there was biologically active tyrosine hydroxylase in the SVG-TH cells, and to determine if there was L-dopa secretion by the cells, a high pressure liquid chromatography analysis was performed in the cell culture supernatant. The cells were incubated with 1 mM biopterin (BH.), A cofactor necessary for the function of tyrosine hydroxylase, before collecting the cell culture supernatant for high pressure liquid chromatography analysis. Controls included the supernatant of the SVG-TH cell cultures incubated in the absence of biopterin, as well as the culture supernatant of the SVG cell line of origin, incubated with or without biopterin. The results are shown in the following Table 1. TABLE 1 Production of L-Dopa in the Supernatant of SVG? in Cell Cultures of SVG-TH Incubated with and without BH4 L-Dopa SVG Cells SVG-TH Cells Without BH Not detectable Not detectable With BH4 (1 μM) Not detectable 4-5 pmol / ml / min As shown in Table 1, no L-dopa could be detected from the parental SVG cell culture, with or without biopterin, and it could not be detected in the SVG-TH cell culture that was incubated without biopterin. However, when the SVG-TH cells were incubated with biopterin, approximately 4 to 6 grams / milliliter / minute of L-dopa was produced in the cell culture supernatant. This confirmed that the tyrosine hydroxylase seen in the immunohistochemistry and Western blots was biologically active. In an unexpected manner, two other prominent s were also seen in the high-pressure liquid chromatography analysis of the supernatant, from the SVG-TH cell culture (Figure 14), independently of the addition of biopterin to the media. These two s were not seen in the parental SVG cell line. Using a series of standards, it was determined that one of the two s represented serotonin, and the second represented 5-hydroxyindoleacetic acid (5-HIAA), the decomposition product of serotonin. To confirm the presence of serotonin in these cells, the immunohistochemistry of the SVG-TH cells was done using a polyclonal antibody for serotonin. The SVG-TH cells were positive for serotonin by immunostaining, as well as by high pressure liquid chromatography. The production of serotonin by these cell lines is unique for cells of glial origin, which has not been reported to produce serotonin. The SVG-TH cells were characterized by immunohistochemical methods, using the same panel of antibodies that was used to characterize the SVG cells. The comparative results are shown in the following Table 2. TABLE 2 SVG SVG-TH Vimentina + + PAFG weakly + MHC Class I + + MHC Class II Thy 1.1 + + T + + protein Serotonin + l-dopa (CLAR) + NSE Neurofilament An EM study of SVG-TH cells revealed a remarkable dilatation of the endoplasmic reticulum that is not seen in the parental SVG cell line (Figure 15). Again the coated holes, mitochondria and ribosomes were easily identified. Example 8 - Promotion of Growth Out of Neurites, by SVG-TH Cells As with previous SVG cells, SVG-TH cells were also tested for their ability to promote neurite outgrowth, and the survival of primary neurons or of neuronal cell lines. A. Coculture of the hNT Cell Line A previously described cell line derived from a human teratocarcinoma was used in cocultivation experiments with SVG and SVG-TH cells. This cell line is derived from the parental teratocarcinoma cell line, by treating the parental cell line with retinoic acid and a combination of antimitotic agents. On treatment, the parental cell line differentiates into post-mitotic neurons. Andrews, P.W., Retinoic Acid Neuronal Induces Differentiation of a Cloned Human Embryonal Carcinoma Cell Line In Vitro, Dev. Biol. (1984) 103: 285-293. These cells, termed hNT neurons, which were used in the cocultivation experiments described herein, retain many of the phenotypic qualities of neurons, including neurofilament expression and neurotransmitter secretion. The maintenance of these cells requires that they cover on plates covered with laminin or matrigel, and that they are fed with conditioned medium. In three separate experiments, the SVG, SVG-TH or Cos cells were coated in 6-well dishes, which had not been coated with any extracellular matrix (1x10 5). cells per cavity). Forty-eight hours later, the SVG, SVG-TH or Cos cells were coated, were 30 percent confluent, and 1x10 5 hNT cells were coated in the same wells. The hNT cells were also coated in a fourth cavity that did not have any of the three previous cell lines, and that was not coated with an extracellular matrix. The cultures were fed only with D-MEM with 2 percent fetal calf serum. Twenty-four hours after the coating, some of the hNT cells had bound to the areas that had no SVG and SVG-TH cells, and had also bound directly to these cells. Almost equal numbers of hNT cells and SVG or SVG-TH cells appeared. Numerous small processes were seen on hNT cells co-cultured with SVG or SVG-TH cells. In the cocultivation of hNT / Cos cells, the hNT cells had bound directly to the Cos cells, but they were not found in the areas that were empty of Cos cells. Additionally, only about 1% of the Cos cells had hNT cells adhered to them, and no processes were seen on the hNT cells. In the control dish, where the hNT cells were coated alone on an untreated surface, only a rare bound cell was seen. At seventy-two hours, the hNT cells had been lifted from the Cos cells, and no hNT cells were found in the control dish. In contrast, the hNT cells from the cocultivation of both SVG and SVG-TH had remained attached, and sent long processes that now made contact with the surrounding SVG / SVG-TH cells (Figure 16A). Some of the cultures were fixed in acetone / ethanol, and immunohistochemistry was performed for the T protein, in order to unambiguously distinguish the two cell populations (Figure 16A). These cultures remained viable for two weeks, after which SVG and SVG-TH cells became confluent. The cocultivations were changed to new dishes, and the same phenomena were seen again. After two more weeks, the experiments were finished. B. Cocultivation of PC12 In this set of experiments, PC12 cells were co-cultured with SVG, SVG-TH or Cos cells, or coated alone. As in the hNT experiment, the SVG, SVG-TH or Cos cells were coated on plates of 6 cavities at 1x10 / cavity, on an untreated plastic plate. Forty-eight hours later, the PC12 cells were coated on the three cell lines, as well as coated on the untreated plastic alone. Forty-eight hours after co-cultivation with SVG or SVG-TH cells, PC12 cells had bound to areas that had no cells, as well as directly to SVG and SVG-TH cells. The PC12 cells had extended the neuritic processes that, at ninety-two hours, had made contact with the surrounding SVG and SVG-TH cells. Some of the cultures were fixed in acetone and methanol, and immunohistochemistry was performed for the T protein, in order to distinguish the two cell populations (Figure 16B). After seventeen days, the cultures grew to overgrow, and the experiment was finished. In contrast, PC12 cells co-cultured with Cos cells, or co-cultivated in isolation, failed to extend any processes. In a separate set of experiments, PC12 cells were coated alone on poly-d-lysine-coated dishes, and then fed with non-conditioned medium, or conditioned medium from the SVG or SVG-TH cell cultures. After seventy-two hours, PC12 cells fed conditioned medium had developed neuritic processes, whereas those fed with non-conditioned medium did not change their morphology. C. Primary Cultures of Fetal Rat Mesencephalic Neurons To determine if SVG and / or SVG-TH cells could also support the survival of primary neurons, the mesencephalon of fetal rat E13 was dissected, dissociated, and plated in triplicate on plates. of six cavities. After 24 hours, a Costar transcavity chamber was placed in the cavities, and SVG or 5 SVG-TH cells were passed into the transcavity chamber (1x10 cells). A set of mesencephalic cultures was not cocultivated with any cell to act as a negative control. After seven days, the trans-cavity chamber with the cells was removed, and the mesencephalic cultures of the cavities were fixed in acetone and methanol, and stained by immunohistochemistry for tyrosine hydroxylase, in order to determine the number of surviving mesencephalic neurons. As seen in Figure 17, these mesencephalic cultures that were cocultivated with the SVG cells, had a survival of the tyrosine hydroxylase neurons two to three times higher, in relation to the control dish. Similar results were found with the SVG-TH cell line. No difference was seen in the morphology of the neurons positive for tyrosine hydroxylase neither in the control nor in the coculture dishes. Example 9 - Grafting and Identification of the SVG and SVG-TH Cells in the Rodent Striatum To determine whether SVG or SVG-TH cell grafts could be transplanted into the striatum, and then unambiguously identified after the transplant, grafted 5xl05 cells, either SVG or SVG-TH, into the striatum of Sprague-Dawley rats using a stereo-toxic head frame for the procedure. Ten animals were grafted with the SVG cells, and ten were grafted with the SVG-TH cells. Three days or seven days after the transplant, the animals were sacrificed, and the brain was processed for immunohistochemistry. Five animals of both groups were perfused systemically with 4 percent paraformaldehyde at the time of euthanasia. Sections of the brains of animals fixed with paraformaldehyde were used for immunohistochemical staining with polyclonal antibodies, while brain sections not fixed for immunohistochemical staining with monoclonal antibodies were used. The SVG and SVG-TH cells grafted onto the striatum could be differentiated in an unambiguous way from the surrounding parenchyma, based on the staining for the SVG-40 T protein, which is only found in the grafted cells. Furthermore, the grafted cells expressed the same antigens in vivo as those expressed in vitro, as confirmed by immunohistochemical staining. These include vimentin, serotonin, human MHC class I, protein T and tyrosine hydroxylase. Similar to what is seen in vitro, only 40 percent of the SVG-TH cells were positive for tyrosine hydroxylase in vivo. The surrounding parenchyma of the host was also immunostained for vimentin and tyrosine hydroxylase as well. The SVG-TH cells remained PAFG-, whereas the surrounding parenchyma of the host clearly has PAFG + astrocytes. Staining for class I rat MHC stained the surrounding parenchymal blood vessels and the occasional vessel of the host seen in the graft, but failed to stain the grafted cells, as expected. An electron microscope study of the SVG-TH grafted cells found that the transplanted cells had retained the characteristic distended endoplasmic reticulum and the coated vesicles, as seen in Figure 15. Similar results were obtained for SVG cells, with the exception of that the SVG cells were positive for PAFG and negative for tyrosine hydroxylase. Example 10 - Grafting of the SVG and SVG-TH Cells in the Spraque-Dawley Rat Striatum with 5-Hydroxydopamine Having determined that the SVG and SVG-TH cells could be identified in the striatum, the next object was to determine whether these cells could be correct a functional deficit in an animal model of Parkinson's disease. Certain Sprágue-Dawley rats suffered unilateral chemical injury of the substantia nigra with 6-hydroxydopamine, using a stereotaxic head frame to direct the drug to the appropriate anatomical site. Five weeks after the injury, the animals were stimulated with apomorphine to quantify the degree of denervation. As seen in Figure 18, the seven animals had base line rotation speeds of 400 revolutions per hour or more. Six weeks after the injury, five of the animals had SVG-TH cells (approximately 5x10 5 cells) grafted onto the injured striatum. The remaining two animals had SVG cells grafted onto the striatum. At weekly intervals after the transplant, for a total of four weeks, the animals were stimulated with apomorphine to determine any change in their baseline activity. As seen in Figure 18, one week after transplantation, there was a substantial reduction in the amount of rotational behavior seen in the five animals grafted with the SVG-TH cells. In contrast, animals grafted with SVG cells showed some insignificant changes, as expected, since these rats were completely denervated, lacking the ability to provoke dopaminergic neurons, and SVG cells were unable to secrete dopamine. However, over the course of the next three weeks, the animals grafted with the SVG-TH cells gradually returned to the rotating behavior before transplantation, as seen in Figure 18. Example 11 - Characterization of Grafted Cells One Month After of the Transplant The seven animals injured with 5-hydroxydopamine described in Example 10 above were sacrificed one month after transplantation. Three of the animals grafted with SVG-TH, and one of the animals grafted with SVG, were perfused systemically with 4 percent paraformaldehyde at the time of euthanasia. The remaining three animals were not perfused, and were fixed at the time of euthanasia. Brain sections from paraformaldehyde-fixed animals were used for immunohistochemical staining with polyclonal antibodies, while non-fixed brain sections were used for immunohistochemical staining with monoclonal antibodies. The grafted cells could still be identified one month after transplantation by immunohistochemical staining for the SV40 T protein, vimentin, serotonin and tyrosine hydroxylase. However, the graft was significantly smaller than the grafts seen on day three or on day seven after transplantation. The graft was immunostained for the large T protein, serotonin and vimentin; however, immunostaining of tyrosine hydroxylase could not be identified in the graft or in the surrounding denervated striatum. The graft could further be identified by its lack of staining for rat Thy 1.1, an antigen strongly expressed in the surrounding parenchyma of the host. The host's parenchyma showed remarkable astrocytosis around the graft. When the CD4 and CD8 sections of rats were stained, numerous positive cells were identified in and around the graft, suggesting that the graft was suffering from an immunological rejection. The above data indicate that the cells are a genograft in the central nervous system of the rodent, while the survival of the cells in the central nervous system of the primate for more than nine months, reflects that these cells are allografts in that system. Example 12 - Effects of Grafting SVG-TH Cells on Parkinson's Disability Score An experimental condition that closely resembles Parkinson's disease in humans can be induced in Rhesus monkeys by administering l-methyl-4 phenyl-l, 2,3,6-tetrahydropyridine (MPTP). Figures 19 to 21 show the effect of grafting SVG-TH cells on the caudate and putamen of three rhesus monkeys that had been treated with MPTP to induce the symptoms of Parkinson's disease. Before grafting, the monkeys were treated with a MPTP injections regimen until the Parkinson's Disability Score of 10 (the highest level) was reached, and was maintained for several months following the injections. In the grafting procedure, 750,000 cells were placed in a volume of 10 microliters, in each of the four sites of the caudate and putamen of each animal. The graphs plot the Parkinson's Disability Score against time (in days) following the operation. Within a week, the three monkeys showed an improvement reflected in the decrease of the Disability Score. INCAPACITY SCORE Pre-Op Post-Op H002 10 3 H005 10 7.5 T022 10 5 The monkey H002 (for which 21 days of results are shown in Figure 19) has now been followed for 90 days after grafting, and is not has been treated with Sinemet (a standard medication for Parkinson's) for any time after the operation. The current disability score of 2-3 in this monkey is similar to the level that could be expected in a Parkinson's patient with drug treatment, and represents an improvement over Sinemet treatment in the monkey. In contrast to its condition before the operation, the monkey can feed, is very active and mobile, responds well to its environment and has gained weight. As you can see in Figures 20 and 21, the monkeys H005 and T022 have also shown an improvement in their disability scores, and they continue to improve, although they have not been followed for such a long period of time. Therefore, grafting of SVG-TH cells results in a substantial improvement in the parkinsonian condition induced by MPTP injury. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in the specification, to the same extent as if each publication, patent or individual patent application was specifically and individually indicated as incorporated into the patent. the present as a reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be seen that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (16)

1. NOVELTY OF THE INVENTION Having described the foregoing invention, it is considered as a novelty and, therefore, the content of the following is claimed as property: CLAIMS 1. An immortalized human fetal neuro-derived cell line, comprising a sequence of heterologous nucleic acid, wherein this cell line can express said heterologous nucleic acid sequence.
2. 2. The cell line according to claim 1, characterized in that this cell line is a glial cell line.
3. 3. The cell line according to claim 1, characterized in that this cell line is derived from human fetal astrocytes.
4. 4. The cell line according to claim 1, characterized in that the heterologous nucleic acid sequence encodes a biologically active peptide.
5. 5. The cell line according to claim 3, characterized in that this biologically active peptide is an enzyme.
6. 6. The cell line according to claim 3, characterized in that this biologically active peptide is an antigen associated with the disease.
7. 7. The cell line according to claim 3, characterized in that this biologically active peptide is tyrosine hydroxylase.
8. 8. The cell line according to claim 1, characterized in that the nucleic acid is operably linked to a transcription promoter.
9. 9. The cell line according to claim 7, characterized in that this cell line is an SVG-TH cell line.
10. 10. The cell line according to claim 7, characterized in that this cell line can express the phTH / Neo plasmid.
11. 11. The cell line according to claim 1, characterized in that this cell line can also express serotonin.
12. 12. A transplantable composition, which comprises: cells of the cell line according to claim 1; and a pharmaceutically acceptable vehicle.
13. 13. The transplantable composition according to claim 12, characterized in that these cells are encapsulated with a membrane impermeable to the antibodies.
14. 14. The transplantable composition according to claim 13, characterized in that said membrane is an alginate gel membrane.
15. 15. A method for treating a disease or neurological disorder in a patient in need of such treatment, said method comprising administering to the aforementioned patient, an effective amount of the cell line according to claim 1, in a pharmaceutically vehicle acceptable.
16. 16. The method according to claim 14, characterized in that said disease or neurological disorder is Parkinson's disease.