Cultures of cells immunoreactive for glial fibrillary acidic protein (GFAP), as well as for the intermediate filament marker nestin were grown in a medium including epidermal growth factor (EGF) and serum. The cultured cells had the morphology of astroglial cells. The cells can be proliferated in adherent or suspension cultures. Depending on the culture conditions, the cells can be induced to differentiate to neurons or glial cells. The cultures can be expanded over a large number of passages during several months, and survive, express an astroglial phenotype and integrate well after transplantation into both neonatal and adult rat forebrain.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides glial precursor cultures of GFAP.sup.+ nestin.sup.+ cells with the potential to differentiate to neurons or glial cells, depending on the culture conditions chosen. These cells can be expanded using proliferation-inducing growth factor. Cells in cultures that have been expanded extensively express similar phenotypes to those passaged fewer times. Alternatively, these cells can be induced to make a significant number of neurons when placed under non-proliferating and serum-free conditions. These neurons show regional characteristics from their origin of isolation and will express those markers even after long-term culture. Moreover, these cells can make a significant number of astrocytes when placed under proliferating serum free conditions.

In one embodiment, cell cultures (for example, mouse or human) can be established from cells that have been isolated from the medial ganglionic eminence (MGE) and lateral ganglionic eminence (LGE). The cultured cells display glial morphology and both glial fibrillary acidic protein (GFAP) and nestin immunoreactivity. In this embodiment, the cell cultures contain cells that express the radial glial marker, RC-2. The cells are at least bi-potential and can make both non-mitotic GFAP.sup.+ astrocytes or non-mitotic beta-tubulin III.sup.+ neurons, depending on the culture conditions.

In another embodiment, EGF-stimulated and serum-containing long-term cultures of cells from the embryonic mouse lateral ganglionic eminence (LGE) can be expanded over many passages during several months, survive, express an astroglial phenotype and integrate well after transplantation into both neonatal and adult rat forebrain. Cells propagated in such cultures are interesting to compare and contrast with central nervous system (CNS) neural stem cells grown as neurospheres in EGF-stimulated cultures and are useful for studies of astroglial development and migration, and for use in trials with ex vivo gene transfer.

The invention provides a composition of a GFAP.sup.+ nestin.sup.+ cell in a culture medium supplemented with serum and at least one proliferation-inducing growth factor (for example, epidermal growth factor (EGF) and/or basic fibroblast growth factor (FGF-2) capable of undergoing neuronal differentiation.

The invention provides a method for the in vitro proliferation of neural cells, to produce large numbers of glial precursor cells available for transplantation that are capable of differentiating into neurons and into glial cells. The method includes the steps of (a) obtaining neural tissue from a mammal (e.g., from fetal tissue) (b) dissociating the neural tissue to obtain a cell suspension (c) culturing the cell in a culture medium containing a serum and a proliferation-inducing growth factor, (d) passaging the proliferated cultured cells. Proliferation and perpetuation of the GFAP.sup.+ nestin.sup.+ cells can be carried out either in suspension cultures or by allowing cells to adhere to a fixed substrate such as tissue coated plastic, polylysine, or laminin.

The invention also provides a method for the in vitro differentiation of the proliferated GFAP.sup.+ nestin.sup.+ cells to form neurons and glia. The invention also provides a method for making regionally specified neurons.

The invention provides a method for the in vivo transplantation of GFAP.sup.+ nesting cells, which includes implanting the GFAP.sup.+ nestin.sup.+ cells that have been proliferated in vitro. Thus, the invention provides a means for generating large numbers of undifferentiated and differentiated neural cells for neurotransplantation into a host to treat neurodegenerative disease, neurological trauma, stroke, or in other diseases of the nervous system involving neuronal and glial cell loss or where normal function needs to be restored such as in metabolic or storage diseases. The invention also provides for methods of treating neurodegenerative disease and neurological trauma.

The invention provides a method for the transfection of GFAP.sup.+ nestin.sup.+ with vectors which can express the gene products for growth factors, growth factor receptors, and peptide neurotransmitters, or express enzymes, which are involved in the synthesis of neurotransmitters, including those for amino acids, biogenic amines and neuropeptides, and for the transplantation of these transfected cells into regions of neurodegeneration.

The invention provides a method of generating large numbers of neural cells for screening putative therapeutic agents targeted at the nervous system and for models of CNS development, function, and dysfunction. The invention also provides a method for the screening of potential neurologically therapeutic pharmaceuticals using GFAP.sup.+ nestin.sup.+ cells that have been proliferated in vitro. The invention further provides a cDNA library prepared from a GFAP.sup.+ nestin.sup.+ cell

DETAILED DESCRIPTION OF THE INVENTION

GFAP.sup.+ Nestin.sup.+ cells. The invention provides "NS4" cells. An NS4 cell is an undifferentiated neural cell that can be induced to proliferate using the methods of the present invention. The NS4 cell is capable of self-maintenance, such that with each cell division, at least one daughter cell will also be a NS4 cell. A NS4 cell has a glial morphology and is immunoreactive for both glial fibrillary acidic protein (GFAP) and nestin.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein specifically expressed by astrocytes and glial cells of the central nervous system and by Schwann cells, the glial cells of the peripheral nervous system (Jessen et al., 13 J. Neurocytology 923-934 (1984) and Fields et al., 8 J. Neuroimmunol. 311-330 (1989)). Anti-GFAP antibodies are commercially available (e.g., a rabbit monoclonal antibody raised against GFAP is available from DAKO).

Nestin is an intermediate filament protein found in many types of undifferentiated CNS cells. During neurogenesis and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g. neurofilaments and glial fibrillary acidic protein (GFAP). The nestin marker was characterized by Lendahl et. al., 60 Cell 58.5-595 (1990). Antibodies are available to identify; nestin, including the rat antibody. Rat401.

The co-expression of GFAP and nestin is compatible with the NS4 cells being a population of astroglial cell precursors. Dividing cells cultured from neonatal rat cerebral cortex, with a typical morphology of type I astroglial cells, co-express GFAP and nestin (Gallo et al., 15 J. Neurosci 394-406 (1995)). Also reactive astrocytes surrounding an ischemic or mechanical lesion site co-express GFAP and nestin (Lin et al., 2 Neurobiol. Dis. 79-85 (1995)). The properties of GFAP.sup.+ nestin.sup.+NS4 cells, which can be grown long-term and repeatedly passaged cultures, are also interesting in the light of recent publications describing the existence of GFAP.sup.+ cells in the ependymal or subependymal zones (Doetsch et al., 97 Cell 703-16 (1999), Johansson et al., 96 Cell 25-34 (1999)) which are the areas containing actively dividing neural stem and precursor cells.

NS4 cells can be obtained from embryonic neural tissue. The neural tissue can be obtained from any animal that has neural tissue such as insects, fish, reptiles, birds, amphibians, mammals and the like. The preferred source neural tissue is from mammals, preferably rodents and primates, and most preferably, mice (see, EXAMPLE 1) and humans (see, EXAMPLE 2).

When NS4 cells are obtained from a heterologous donor, the donor may be euthanized, and the neural tissue and specific area of interest removed using a sterile procedure. Areas of interest are any area from which NS4 cells can be obtained that can serve to restore function to a degenerated area of the host's nervous system, particularly the host's CNS. Suitable areas include the medial ganglionic eminence and the lateral ganglionic eminence. Autologous neural tissue can be obtained by biopsy, or from patients undergoing neurosurgery in which neural tissue is removed, for example, during epilepsy surgery, temporal lobectomies and hippocampectomies. Human heterologous NS4 cells can be derived from embryonic or fetal tissue following elective abortion (EXAMPLE 2), or from a post-natal, juvenile or adult organ donor.

The present invention provides an alternative way to generate enriched populations of astroglial cells from different regions. The NS4 cells resemble type I astroglial cells both in vitro and following implantation. An interesting migration pattern was observed in the neonatal recipients, with cells migrating along the internal capsule into the globus pallidus and some other adjacent structures, whereas, when the grafts were placed into adult recipients, they remained mostly around the injection site, with only limited migration into the adjacent striatum (see, EXAMPLE 7).

While neurons and glia were present in the dissociated cell culture, after several passages, the cultures are severely deficient of cells possessing neuronal morphologies or expressing neuronal markers. These cultures are highly enriched in cells having GFAP and nestin immunoreactivity and expressing glial morphology. The cell cultures also contain cells expressing the radial glial marker RC2.

NS4 cells can be maintained in vitro in long-term cultures. Cells from the embryonic mouse lateral ganglionic eminence (LGE) were grown in attached, epidermal growth factor (EGF) stimulated and 10% serum-containing cultures, with around 90% GFAP.sup.+ nestin.sup.+ cells, over repeated passages during several months. After a gradual decline in division rate during the first 6-8 passages, the cultures thereafter propagated readily again, with a stable and high growth rate, for at least seven months. Cells grew as attached GFAP.sup.+ nestin.sup.+ cells with an astroglial-like morphology (see EXAMPLE 4).

The cultured mouse NS4 cells were also positive for the mouse-specific neural antibodies M2 (Lagenaur & Schachner, 15 J. Supramol. Struct. Cell Biochem. 335-46 (1981)) and M6 (Lagenaur et al., 23 J. Neurobiol. 71-88 (1992)). Although M2 has been found to label both glial and neuronal cell surfaces, in cerebellar monolayer cultures and in cerebellar tissue sections (Lagenaur & Schachner, 15 J. Supramol. Struct. Cell Biochem. 335-46 (1981)), several in vivo studies have characterized the M2 antibody as a reliable marker for astroglial cells (Zhou & Lund, 317 J. Comp. Neurol. 145-55 (1992)). M6 is a neuronal cell surface glycoprotein with unknown function (Mi et al., 106 Dev. Brain Res. 145-54 (1998); Lagenaur et al., 23 J. Neurobiol. 71-88 (1992); Hankin & Lund, 263 J. Comp. Neurol. 45 5-66 (1987); Wictorin et al., 3 Eur. J. Neurosci. 86-101 (1991)). Several in vivo studies have shown that M6 can also be expressed on glial cells (Mi et al., 106 Dev. Brain Res. 145-54 (1998)). The M6-immunoreactivity is observed in the majority of the cultured NS4 cells and therefore does not indicate that the cells have certain neuronal characteristics. The M2 and M6 staining patterns were clearly similar to those of GFAP and nestin, with the vast majority of the cells immunopositive for all of these four markers, at both early and late passages.

In addition, some NS4 cells show immunoreactivity for the radial glia marker RC-2. RC2 is a monoclonal antibody that specifically recognizes a radial glial cell antigen that is expressed at varying amounts during CNS development. There is a high level of expression during embryonic brain development, lower levels in early postnatal transitional glia, and none in astrocytes after the second postnatal week. Hunter et al., 33 J. Neurobiol. 459-472 (1997); Mission et al., 44 Dev. Brain Res. 95-108 (1988).

NS4 cells were negative when stained for neuronal markers, such as, beta-III tubulin or NeuN. Antibodies recognizing, beta-tubulin isotype III (beta-III-tubulin) are commercially available (for example, mouse monoclonal antibodies from Sigma Chemicals, St. Louis Mo.). Antibody to Neuro-Specific Nuclear Protein (NeuN) reacts with most neuronal cell types throughout the nervous system, is available from Chemicon (Temecula Calif.). The antibody is neuron-specific; no staining of glia is observed. Other neuronal markers include the homeobox-related murine gene MEIS2 labels the lateral somitic compartment and derivatives during early mouse embryogenesis and later becomes a marker for the dorso-ectodermal region, overlying cells of the paraxial mesoderm. MEIS2 is also highly expressed in specific areas of the developing central nervous system from embryonic day 9 (E9) onward. In later developmental stages, a strong expression is detectable in differentiating nuclei and regions of the forebrain, midbrain, hindbrain, and spinal cord. (see, Toresson et al., 126 (6) Development 13 17-1326 (1999)). Another neuronal marker is the DLX homeobox gene, which is expressed in distinct regions of the embryonic forebrain, including the striatum, neocortex and retina (see, Eisenstat et al., 414(2) J. Comp. Neurol. 2 17-37 (1999))

NS4 cells survive transplantation into neonatal or adult animal striatum, with astroglia-like properties for the implanted cells, and good integration and migration, especially in the neonatal recipients. Transplantation of astroglial cells is today a widely used method for in vivo studies of astroglial cells during development and in regeneration, often with the cells grafted as a component of primary tissue, with a mix of different precursor cells. To determine specific properties of the astroglial cells, it is however interesting to be able to acquire relatively-pure cell populations.

Contrast between NS4 cells of the invention and CNS neural stem cells. The NS4 cells of the invention are similar to and yet different from CNS neural stem cells. Neurobiologists have used various terms interchangeably to describe the undifferentiated cells of the CNS. Terms such as "stem cell", "precursor cell" and "progenitor cell" are commonly used in the scientific literature to describe different types of undifferentiated neural cells, with differing characteristics and fates. One approach to obtain CNS neural stem cells is to trophic factor-stimulate and grow neural stem (or progenitor) cells in the form of neurospheres (see, U.S. Pat. Nos. 5,750,376 and 5,851,832, to Weiss et al. U.S. Pat. No. 5,753,506, to Johe, U.S. Pat. No. 5,968,829, to Carpenter (all incorporated herein by reference), Weiss et al., 19 Trends Neurosci. 387-93 (1996); Reynolds et al, 12 J. Neurosci. 4565-74 (1992), Reynolds & Weiss, 255 Science 1707-10 (1992); Reynolds & Weiss, 175 Dev. Biol. 1-13 (1996)). Indeed, such precursor cells derived from, for instance, the embryonic or adult rodent or human forebrain, can be grown and multiplied as non-attached neurospheres over long periods, in a serum-free medium including EGF. In vitro, cells in the neurospheres differentiate into neurons, astrocytes or oligodendrocytes, when plated onto an adhesive substrate.

Cells isolated from the embryonic (or adult) mouse striatum can proliferate in response to EGF-stimulation and grow in a medium without serum, as non-attached clusters or spheres of clonally derived undifferentiated progenitor/stem cells, but with a potential to differentiate into neurons, astrocytes or oligodendrocytes. EGF-expanded neurospheres are nestin.sup.+ but GFAP.sup.-. Upon transplantation into the CNS, the neurospheres give rise to neuronal, glial, and nonneural cells and are capable of differentiating into various neurons such as hippocampal neurons of the granule cell layer and olfactory interneurons (Hammang et al., 147 Exp. Neurol. 84-95 (1997); Winkler et al., 11 Mol. Cell. Neurosci. 99-116 (1998)); Fricker et al., 19 J. Neurosci. 5990-6005 (1999), Svendsen et al., 148 Exp. Neurol. 135-46 (1997), and Vescovi et al., 156 Exp. Neurol. 71-83 (1999)). NS4 cells appear to be more restricted and retain their regional specification and, for example, cells differentiated from precursors derived from the LGE and propagated for multiple passages expressed striatal neuronal markers such as MEIS2 and DLX1 and did not express markers of cortical or medial ganglionic eminence neuronal precursors (see EXAMPLE 5).

Culture conditions. NS4 cells can be proliferated using the methods described herein. Cells can be obtained from donor tissue by dissociation of individual cells from the connecting extracellular matrix of the tissue (see, EXAMPLES 1-2). Tissue from a particular neural region is removed from the brain using a sterile procedure, and the cells are dissociated using any method known in the art including treatment with enzymes such as trypsin, collagenase and the like, or by using physical methods of dissociation such as with a blunt instrument or homogenizer. Dissociation of fetal cells can be carried out in tissue culture medium. Dissociation of juvenile and adult cells can be carried out in 0.1% trypsin and 0.05% DNase in DMEM. Dissociated cells are centrifuged at low speed, between 200 and 2000 rpm, usually between 400 and 800 rpm, and then resuspended in a culture medium. The neural cells can be cultured in suspension or on a fixed substrate. Dissociated cell suspensions are seeded in any receptacle capable of sustaining cells, particularly culture flasks, culture plates or roller bottles, and more particularly in small culture flasks such as 25 cm.sup.2 culture flasks. Cells cultured in suspension are resuspended at approximately 5.times.10.sup.4 to 2.times.10.sup.5 cells/ml (for example, 1.times.10.sup.5 cells/ml). Cells plated on a fixed substrate are plated at approximately 2-3.times.10.sup.3 10 cells/cm.sup.2 (for example, 2.5.times.10.sup.3 cells/cm.sup.2).

The dissociated neural cells can be placed into any known culture medium capable of supporting cell growth, including HEM, DMEM, RPMI, F-12, and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and proteins such as transferrin and the like. The culture medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like. The culture medium may contain serum derived from bovine, equine, chicken and the like.

In one embodiment, the invention provides a culture medium for the proliferation of NS4 cells. The medium is a defined culture medium containing a mixture of DMEM/F12, supplemented with N2 (Gibco), and fetal calf serum. This culture medium is referred to as "NS4 Complete Medium" and is described in detail in EXAMPLE 1.

Conditions for culturing should be close to physiological conditions. The pH of the culture medium should be close to physiological pH. (for example, between pH 6-8, between about pH 7 to 7.8, or at pH 7.4). Physiological temperatures range between about 30.degree. C. to 40.degree. C. NS4 cells can be cultured at temperatures between about 32.degree. C. to about 38.degree. C. (or example, between about 35.degree. C. to about 37.degree. C.).

The culture medium is supplemented with at least one proliferation-inducing ("mitogenic") growth factor. A "growth factor" is protein, peptide or other molecule having a growth, proliferation-inducing, differentiation-inducing, or trophic effect on NS4 cells. "Proliferation-inducing growth factors" are trophic factor that allows NS4 cells to proliferate, including any molecule that binds to a receptor on the surface of the cell to exert a trophic, or growth-inducing effect on the cell. Proliferation-inducing growth factors include EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor alpha (TGF.alpha.), and combinations thereof. EGF is a known proliferation-inducing growth factor for astroglial cells (Simpson et al., 8 J. Neurosci. Res. 453-62 (1982)), and is also used in media for the propagation of CNS neural stem cells (Reynolds & Weiss, 255 Science 1707-10 (1992)). The combination of the EGF-containing neurosphere growth medium with the addition of serum gives rise to readily propagating attached cultures with high proportions of GFAP.sup.+ cells.

Growth factors are usually added to the culture medium at concentrations ranging between about 1 fg/ml to 1 mg/ml. Concentrations between about 1 to 100 ng/ml are usually sufficient. Simple titration assays can easily be performed to determine the optimal concentration of a particular growth factor.

The biological effects of growth and trophic factors are generally mediated through binding to cell surface receptors. The receptors for a number of these factors have been identified and antibodies and molecular probes for specific receptors are available. NS4 cells can be analyzed for the presence of growth factor receptors at all stages of differentiation. In many cases, the identification of a particular receptor provides guidance for the strategy to use in further differentiating the cells along specific developmental pathways with the addition of exogenous growth or trophic factors.

Generally, after about 3-10 days in vitro, and more particularly after about 6-7 days in vitro, the proliferating NS4 cells are fed every 2-7 days (for example, every 2-4 days by aspirating the medium, and adding fresh "NS4 Complete Medium" containing a proliferation-inducing growth factor to the culture flask).

The NS4 cell culture can be easily passaged to reinitiate proliferation. After 6-7 days in vitro, the culture flasks are shaken well and NS4 cells The NS4 cells are then transferred to a 50 ml centrifuge tube and centrifuged at low speed. The medium is aspirated, the NS4 cells are resuspended in a small amount of "NS4 Complete Medium" with growth factor The cells are then counted and replated at the desired density to reinitiate proliferation. This procedure can be repeated weekly to result in a logarithmic increase in the number of viable cells at each passage. The procedure is continued until the desired number of NS4 cells is obtained.

NS4 cells and NS4 cell progeny can be cryopreserved by any method known in the art until they are needed. (See, e.g., U.S. Pat. No. 5,071,741, PCT International patent applications WO.dagger.93/14191, WO.dagger.95/07611, WO.dagger.96/27287, WO.dagger.96/29862, and WO.dagger.98/14058, Karlsson et al., 65 Biophysical J. 2524-2536 (1993)). The NS4 cells can be suspended in an isotonic solution, preferably a cell culture medium, containing a particular cryopreservant. Such cryopreservants include dimethyl sulfoxide (DMSO), glycerol and the like. These cryopreservants are used at a concentration of 5-15% (for example, 8-10%). Cells are frozen gradually to a temperature of -10.degree. C. to -150.degree. C. (for example, -20.degree. C. to -100.degree. C., or -70.degree. C. to -80.degree. C.).

Differentiation of NS4 Cells. Depending on the culture conditions, NS4 cells can be differentiated into neurons and glial cells.

N54 cells can be differentiated into neurons by culturing the N54 cells on a fixed substrate in a culture medium that is free of the proliferation-inducing growth factor and serum. After removal of the proliferation-inducing growth factor and the serum, the N54 cells begin to differentiate into neurons. At this stage the culture medium may contain serum such as 0.5-1.0% fetal bovine serum (FBS). However, if defined conditions are required, serum is not used. Within 2-3 days, many of the N54 cell progeny begin to lose immunoreactivity for GFAP and nestin and begin to express antigens specific for neurons (e.g., .beta.-tubulin III). Under the same conditions, N54 cells can be differentiated into mature astrocytes by culturing the cells on a fixed substrate in a culture medium that is free or deficient of serum. After removal of the serum, the cells flatten, and begin to differentiate into glia. Cells exhibit the astroglial morphology and lose immunoreactivity for nestin and begin to express GFAP in a fibrillary pattern characteristic for astrocytes.

Differentiation of the NS4 cells can also be induced by any method known in the art which activates the cascade of biological events which lead to growth, which include the liberation of inositol triphosphate and intracellular Ca.sup.2+, liberation of diacyl glycerol and the activation of protein kinase C and other cellular kinases, and the like. Treatment with phorbol esters, differentiation-inducing growth factors and other chemical signals can induce differentiation. Instead of proliferation-inducing growth factors for the proliferation of NS4 cells (see above), differentiation-inducing growth factors can be added to the culture medium to influence differentiation of the NS4 cells. Differentiation inducing growth factors include NGF, platelet-derived growth factor (PDGF), thyrotropin releasing hormone (TRH), transforming growth factor betas (TGF), insulin-like growth factor (IGF-1) and the like.

Differentiated neuronal and glia cells can be detected using immunocytochemical techniques know in the art. Immunocytochemistry (e.g. dual-label immunofluorescence and immunoperoxidase methods) uses antibodies that detect cell proteins to distinguish the cellular characteristics or phenotypic properties of neurons from glia. Cellular markers for neurons include NSE, NF, .beta.-tubulin, MAP-2 and NeuN. Cellular markers for glia include GFAP (an identifier of astrocytes), RC-2 (an identifier of radial glia) and M2.

Immunocytochemistry can also be used to identify neurons, by detecting the expression of neurotransmitters or the expression of enzymes responsible for neurotransmitter synthesis. For the identification of neurons, antibodies can be used that detect the presence of acetylcholine (ACh), dopamine, epinephrine, norepinephrine, histamine, serotonin or 5-hydroxytryptamine (5-HT), neuropeptides such as substance P, adrenocorticotrophic hormone, vasopressin or anti-diuretic hormone, oxytocin, somatostatin, angiotensin II, neurotensin, and bombesin, hypothalamic releasing hormones such as TRH and luteinizing releasing hormone, gastrointestinal peptides such as vasoactive intestinal peptide (VIP) and cholecystokinin (CCK) and CCK-like peptide, opioid peptides such as endorphins and enkephalins, prostaglandins, amino acids such as GABA, glycine, glutamate, cysteine, taurine and aspartate, and dipeptides such as carnosine. Antibodies to neurotransmitter-synthesizing enzymes can also be used such as glutamic acid decarboxylase (GAD) which is involved in the synthesis of GABA, choline acetyltransferase (ChAT) for ACh synthesis, dopa decarboxylase (DDC) for dopamine, dopamine-.beta.-hydroxylase (DBH) for norepinephrine, and amino acid decarboxylase for 5-HT. Antibodies to enzymes that are involved in the deactivation of neurotransmitters may also be useful such as acetyl cholinesterase (AChE) which deactivates ACh. Antibodies to enzymes involved in the reuptake of neurotransmitters into neuronal terminals such as monoamine oxidase and catechol-o-methyl transferase for dopamine, for 5-HT, and GABA transferase for GABA may also identify neurons. Other markers for neurons include antibodies to neurotransmitter receptors such as the AChE nicotinic and muscarinic receptors, adrenergic receptors, the dopamine receptor, and the like. Cells that contain a high level of melanin, such as those found in the substantia nigra, could be identified using an antibody to melanin.

In situ hybridization histochemistry can also be performed, using cDNA or RNA probes specific for the peptide neurotransmitter or the neurotransmitter synthesizing enzyme mRNAs. These techniques can be combined with immunocytochemical methods to enhance the identification of specific phenotypes. If necessary, the antibodies and molecular probes discussed above can be applied to Western and Northern blot procedures respectively to aid in cell identification.

Transplantation of NS4 Cells. Transplantation of new cells into the damaged CNS has the potential to repair damaged neural pathways and provide neurotransmitters, thereby restoring neurological function. However, the absence of suitable cells for transplantation purposes has prevented the full potential of this procedure from being met. "Suitable" cells are cells that meet the following criteria: (1) can be obtained in large numbers; (2) can be proliferated in vitro to allow insertion of genetic material, if necessary; (3) capable of surviving indefinitely but stop growing after transplantation to the brain; (4) are non-immunogenic, preferably obtained from a patient's own tissue or from a compatible donor; (5) are able to from normal neural connections and respond to neural physiological signals (Bjorklund, 14(8) Trends Neurosci. 319-322 (1991). The NS4 cells obtainable from embryonic or adult CNS tissue, which are able to divide over extended times when maintained in vitro using the culture conditions described herein, meet all of the desirable requirements of cells suitable for neural transplantation purposes and are a particularly suitable cell line as the cells have not been immortalized and are not of tumorigenic origin. The use of NS4 cells in the treatment of neurological disorders and CNS damage can be demonstrated by the use of animal models.

NS4 cells can be administered to any animal with abnormal neurological or neurodegenerative symptoms obtained in any manner, including those obtained as a result of mechanical, chemical, or electrolytic lesions, as a result of aspiration of neural areas, or as a result of aging processes. Lesions in non-human animal models can be obtained with 6-hydroxy-dopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), ibotenic acid, and the like.

NS4 cells can be prepared from donor tissue that is xenogeneic to the host. For xenografts to be successful, some method of reducing or eliminating the immune response to the implanted tissue is usually employed. Thus NS4 cell recipients can be immunosuppressed, either through the use of immunosuppressive drugs such as cyclosporin, or through local immunosuppression strategies employing locally applied immunosuppressants. Local immunosuppression is disclosed by Gruber, 54 Transplantation 1-11 (1992). U.S. Pat. No. 5,026,365 discloses encapsulation methods suitable for local immunosuppression.

As an alternative to employing immunosuppression techniques, methods of gene replacement or knockout using homologous recombination in embryonic stem cells, taught by Smithies et al., 317 Nature 230-234 (1985), and extended to gene replacement or knockout in cell lines (Zheng et al., 88 Proc. Natl. Acad. Sci. 8067-8071 (1991)), can be applied to NS4 cells for the ablation of major histocompatibility complex (MHC) genes. NS4 cells lacking MHC expression allows for the grafting of enriched neural cell populations across allogeneic, and perhaps even xenogeneic, histocompatibility barriers without the need to immunosuppress the recipient. General reviews and citations for the use of recombinant methods to reduce antigenicity of donor cells are also disclosed by Gruber, 54 Transplantation 1-11(1992). Exemplary approaches to the reduction of immunogenicity of transplants by surface modification are disclosed by PCT International patent application WO 92/04033 and PCT/US99/24630. Alternatively the immunogenicity of the graft may be reduced by preparing NS4 cells from a transgenic animal that has altered or deleted MHC antigens.

NS4 cells can be encapsulated and used to deliver factors to the host, according to known encapsulation technologies, including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350, herein incorporated by reference) and macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and PCT International patent applications WO 92/19195 and WO 95/05452, each incorporated herein by reference). If the cells are encapsulated, we prefer macroencapsulation, as described in U.S. Pat. Nos. 5,284,761; 5,158,881; 4,976,859; 4,968,733; 5,800,828 and PCT International patent application WO 95/05452, each incorporated herein by reference. Cell number in the devices can be varied; preferably each device contains between 10.sup.3-10.sup.9 cells (for example, 10.sup.5 to 10.sup.7 cells). Many macroencapsulation devices can be implanted in the host; we prefer between one to 10 devices.

NS4 cells prepared from tissue that is allogeneic to that of the recipient can be tested for use by the well-known methods of tissue typing, to closely match the histocompatibility type of the recipient.

NS4 cells can sometimes be prepared from the recipient's own nervous system (e.g., in the case of tumor removal biopsies). In such instances the NS4 cells can be generated from dissociated tissue and proliferated in vitro using the methods described above. Upon suitable expansion of cell numbers, the NS4 cells may be harvested, genetically modified if necessary, and readied for direct injection into the recipient's CNS.

Transplantation can be done bilaterally, or, in the case of a patient suffering from Parkinson's Disease, contralateral to the most affected side. Surgery is performed in a manner in which particular brain regions may be located, such as in relation to skull sutures, particularly with a stereotaxic guide. NS4 cells are delivered throughout any affected neural area, in particular to the basal ganglia, the caudate, the putamen, the nucleus basalis or the substantia nigra. Cells are administered to the particular region using any method which maintains the integrity of surrounding areas of the brain, such as by injection cannula. Injection methods are exemplified by those used by Duncan et al., 17 J. Neurocytology 351-361 (1988), and scaled up and modified for use in humans. Methods taught by Gage et al., supra, for the injection of cell suspensions such as fibroblasts into the CNS can also be used for injection of NS4 cells. Additional approaches and methods may be found in Neural Grafting in the Mammalian CNS, Bjorklund & Stenevi, eds. (1985).

NS4 cells administered to the particular neural region can form a neural graft, so that the cells form normal connections with neighboring neurons, maintaining contact with transplanted or existing glial cells, and providing a trophic influence for the neurons. Thus the transplanted NS4 cells re-establish the neuronal networks which have been damaged due to disease and aging.

Survival of the NS4 cell graft in the living host can be examined using various non-invasive scans such as computerized axial tomography (CAT scan or CT scan), nuclear magnetic resonance or magnetic resonance imaging (NMR or MRI), or positron emission tomography (PET) scans. Post-mortem examination of graft survival can be done by removing the neural tissue, and examining the affected region macroscopically and microscopically. Cells can be stained with any stains visible under light or electron microscopic conditions, more particularly with stains that are specific for neurons and glia. Particularly useful are monoclonal antibodies that identify neuronal cell surface markers such as the M6 antibody that identifies mouse neurons. Also useful are antibodies that identify neurotransmitters (such as GABA, TH, ChAT, and substance P) and to enzymes involved in the synthesis of neurotransmitters (such as GAD). Transplanted cells can also be identified by prior incorporation of tracer dyes such as rhodamine-labeled or fluorescein-labeled microspheres, fast blue, bisbenzamide, or retrovirally introduced histochemical markers such as the lacZ gene, which produces, .alpha.-galactosidase.

Functional integration of the graft into the host's neural tissue can be assessed by examining the effectiveness of grafts on restoring various functions, including but not limited to tests for endocrine, motor, cognitive and sensory functions. Motor tests that can be used include those that measure rotational movement away from the degenerated side of the brain, and those that measure slowness of movement, balance, coordination, akinesia or lack of movement, rigidity and tremors. Cognitive tests include various tests of ability to perform everyday tasks, as well as various memory tests, including maze performance.

The ability to expand NS4 cells in vitro for use in transplantation is also useful for ex vivo gene therapy. For instance, rat primary astroglial cells (Lundberg et al., 139 Exp. Neurol. 39-53 (1996) or a human astroglial cell line (Tornatore et al., 5 Cell Transplant 145-63 (1996)) have been transduced with the tyrosine hydroxylase gene and implanted in models of Parkinsonis disease. More recently, astroglial cells for ex vivo gene therapy have also been derived from adult human cortex (Ridet et al., 10 Hum. Gene Ther. 27 1-80 (1999)). Thus, NS4 cells provide an additional way to retrieve and expand astroglial cells for use as vehicles in ex vivo gene therapy trials.

Genetic Modification of NS4 Cells. Although the NS4 cells are non-transformed primary cells, they possess features of a continuous cell line. In the undifferentiated state, the NS4 cells continuously divide and are thus targets for genetic modification. In some embodiments, the genetically modified cells are induced to differentiate into neurons or glia by any of the methods described above.

The term "genetic modification" refers to the stable or transient alteration of the genotype a of a NS4 cell by intentional introduction of exogenous DNA. DNA may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful DNA sequences. The term "genetic modification" as used herein is not meant to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like.

Any useful genetic modification of the cells is within the scope of the present invention. For example, NS4 cells may be modified to produce or increase production of a biologically active substance such as a neurotransmitter or growth factor or the like. In one embodiment the biologically active substance is a transcription factor such as a transcription factor that modulates genetic differentiation, e.g., Nurr-1. In an alternative embodiment the biologically active substance is a non-mitogenic proliferation factor, e.g. v-myc, SV-40 large T or telomerase.

The genetic modification can be performed either by infection with viral vectors (retrovirus, modified herpes viral, herpes-viral, adenovirus, adeno-associated virus, and the like) or transfection using methods known in the art (lipofection, calcium phosphate transfection, DEAE-dextran, electroporation, and the like) (see, Maniatis et al., in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1982). For example, the chimeric gene constructs can contain viral, for example retroviral long terminal repeat (LTR), simian virus 40 (SV40), cytomegalovirus (CMV); or mammalian cell-specific promoters such as tyrosine hydroxylase (TH, a marker for dopamine cells), DBH, phenylethanolamine N-methyltransferase (PNMT), ChAT, GFAP, NSE, the NF proteins (NE-L, NF-M, NF-H, and the like) that direct the expression of the structural genes encoding the desired protein. In addition, the vectors can include a drug selection marker, such as the E. coli aminoglycoside phosphotransferase gene, which when co-infected with the test gene confers resistance to geneticin (G418), a protein synthesis inhibitor.

NS4 cells can be genetically modified using transfection with expression vectors. In one protocol, vector DNA containing the genes are diluted in 0.1.times.TE (1 mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of 40 .mu.g/ml. 22 .mu.l of the DNA is added to 250 .mu.l of 2.times.HBS (280 mM NaCl, 10 mM KCl, 1.5 mM Na.sub.2HPO.sub.4, 12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5 ml plastic tube. 31 .mu.l of 2 M CaCl.sub.2 is added slowly and the mixture is incubated for 30 minutes (min) at room temperature. During this 30 min incubation, the cells are centrifuged at 800 g for 5 min at 4.degree. C. The cells are resuspended in 20 volumes of ice-cold PBS and divided into aliquots of 1.times.10.sup.7 cells, which are again centrifuged. Each aliquot of cells is resuspended in 1 ml of the DNA-CaCl.sub.2 suspension, and incubated for 20 min at room temperature. The cells are then diluted in growth medium and incubated for 6-24 hr at 37.degree. C. in 5%-7% CO.sub.2. The cells are again centrifuged, washed in PBS and returned to 10 ml of growth medium for 48 hr.

NS4 cells can also be genetically modified using calcium phosphate transfection techniques. For standard calcium phosphate transfection, the cells are mechanically dissociated into a single cell suspension and plated on tissue culture-treated dishes at 50% confluence (50,000-75,000 cells/cm.sup.2) and allowed to attach overnight. In one protocol, the modified calcium phosphate transfection procedure is performed as follows: DNA (15-25 .mu.g) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA, pH 7.5) diluted to 440 .mu.L with TE, and 60 .mu.L of 2 M CaCl.sup.2 (pH to 5.8 with 1M HEPES buffer) is added to the DNA/TE buffer. A total of 500 .mu.L of 2.times.HBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na.sub.2HPO.sub.4, 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is added dropwise to this mix. The mixture is allowed to stand at room temperature for 20 mm. The cells are washed briefly with 1.times.HBS and 1 ml of the calcium phosphate precipitated DNA solution is added to each plate, and the cells are incubated at 37.degree. C. for 20 mm. Following this incubation, 10 ml of "NS4 Complete Medium" is added to the cells, and the plates are placed in an incubator (37.degree. C., 9.5% CO.sub.2) for an additional 3-6 hours. The DNA and the medium are removed by aspiration at the end of the incubation period, and the cells are washed 3 times with "NS4 Complete Growth Medium" and then returned to the incubator.

When the genetic modification is for the production of a biologically active substance, the substance can be one that is useful for the treatment of a given CNS disorder. NS4 cells can be genetically modified to express a biologically active agent, such as growth factors, growth factor receptors, neurotransmitters, neurotransmitter synthesizing genes, neuropeptides, and chromaffin granule amine transporter. For example, it may be desired to genetically modify cells so they secrete a proliferation-inducing growth factor or a differentiation-inducing growth factor. Growth factor products useful in the treatment of CNS disorders include NGF, BDNF, the neurotrophins, CNTF, amphiregulin, FGF-1, FGF-2, EGF, TGF.alpha., TGF, PDGF, JGFs, and the interleukins.

Cells can also be modified to express a certain growth factor receptor (r) including, but not limited to, p75 low affinity NGF receptor, CNTF receptor, the trk family of neurotrophin receptors (trk, trkB, trkC), EGFr, FGFr, and amphiregulin receptors. Cells can be engineered to produce various neurotransmitters or their receptors such as serotonin, L-dopa, dopamine, norepinephrine, epinephrine, tachykinin, substance P, endorphin, enkephalin, histamine, N-methyl D-aspartate, glycine, glutamate, GABA, ACh, and the like. Useful neurotransmitter-synthesizing genes include TH, DDC, DBH, PNMT, GAD, tryptophan hydroxylase, CHAT, and histidine decarboxylase. Genes that encode for various neuropeptides, which may prove useful in the treatment of CNS disorders, include substance-P, neuropeptide-Y, enkephalin, vasopressin, VIP, glucagon, bombesin, CCK, somatostatin, calcitonin gene-related peptide, and the like.

The genetically modified NS4 cells can be implanted for cell therapy or gene therapy into the CNS of a recipient in need of the biologially active molecule produced by the genetically modified cells. Transplantation techniques are detailed below.

Alternatively, the genetically modified NS4 cell can be subjected to various differentiation protocols in vitro prior to implantation. For example, genetically modified NS4 cells may be removed from the culture medium, which allows proliferation and differentiated using any of the protocols, described above. The protocol used depends upon the type of genetically modified cell desired. Once the cells have differentiated, they are again assayed for expression of the desired protein. Cells having the desired phenotype can be isolated and implanted into recipients in need of the protein or biologically active molecule that is expressed by the genetically modified cell.

Methods for screening effects of drugs on NS4 cells. NS4 cell cultures can be used for the screening of potential neurologically therapeutic compositions. These test compositions can be applied to cells in culture at varying dosages, and the response of the cells monitored for various time periods. Physical characteristics of the cells can be analyzed by observing cell and neurite growth with microscopy. The induction of expression of new or increased levels of proteins such as enzymes, receptors and other cell surface molecules, or of neurotransmitters, amino acids, neuropeptides and biogenic amines can be analyzed with any technique known in the art which can identify the alteration of the level of such molecules. These techniques include immunohistochemistry using antibodies against such molecules, or biochemical analysis. Such biochemical analysis includes protein assays, enzymatic assays, receptor binding assays, enzyme-linked immunosorbant assays (ELISA), electrophoretic analysis, analysis with high performance liquid chromatography (HPLC), Western blots, and radioimmune assays (RIA). Nucleic acid analysis such as Northern blots can be used to examine the levels of mRNA coding for these molecules, or for enzymes which synthesize these molecules.

Alternatively, NS4 cells treated with these pharmaceutical compositions can be transplanted into an animal, and their survival, their ability to form neural connections, and their biochemical and immunological characteristics examined.

NS4 cells can be used in methods of determining the effect of a biological agents on neural cells. The term "biological agent" refers to any agent, such as a virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro-drug or other substance that may have an effect on neural cells whether such effect is harmful, beneficial, or otherwise. Biological agents that are beneficial to neural cells are referred to herein as "neurological agents", a term which encompasses any biologically or pharmaceutically active substance that may prove potentially useful for the proliferation, differentiation or functioning of CNS cells or treatment of neurological disease or disorder. For example, the term may encompass certain neurotransmitters, neurotransmitter receptors, growth factors, growth factor receptors, and the like, as well as enzymes used in the synthesis of these agents.

The biological agent can be the biological agent is selected from the group consisting of basic fibroblast growth factor, acid fibroblast growth factor, epidermal growth factor, transforming growth factor .alpha., transforming growth factor .beta., nerve growth factor, insulin like growth factor, platelet derived growth factor, glia-derived neurotrophic factor, brain derived neurotrophic factor, ciliary neurotrophic factor, phorbol 12-myristate 13-acetate, tryophotin, activin, thyrotropin releasing hormone, interleukins, bone morphogenic protein, macrophage inflammatory proteins, heparan sulfate, amphiregulin, retinoic acid, tumor necrosis factor .alpha., fibroblast growth factor receptor, epidermal growth factor receptor. Examples of biological agents include trophic factors such as glial-derived neurotrophic factor (GDNF); regulators of intracellular pathways associated with growth factor activity such as staurosporine, CGP-4 1251, and the like; hormones; various proteins and polypeptides such as interleukins and the Bcl-2 gene product; oligonucleotides such as antisense strands directed, for example, against transcripts for receptors; heparin-like molecules; and a variety of other molecules that have an effect on radial glial cells or CNS neural stem cell.

To determine the effect of a potential biological agent on neural cells from a particular host, a culture of NS4 cells can be obtained from normal neural tissue or, alternatively, from a host afflicted with a CNS disease or disorder. The choice of culture conditions depends upon the particular agent being tested and the effects one wants to achieve. Once the cells are obtained from the desired donor tissue, they are proliferated in vitro in the presence of a proliferation-inducing growth factor.

The ability of various biological agents to increase, decrease or modify in some other way the number and nature of the NS4 cells can be screened on cells proliferated in the presence of EGF or other proliferation-inducing factor by the methods described in EXAMPLE 1-2.

It is possible to screen for biological agents that increase the proliferative ability of NS4 cells which would be useful for generating large numbers of cells for transplantation purposes. It is also possible to screen for biological agents that inhibit NS4 cell proliferation. NS4 cells are plated in the presence of the biological factors of interest and assayed for the degree of proliferation that occurs. The effects of a biological agent or combination of biological agents on the differentiation and survival of NS4 cells and their progeny can be determined.

It is possible to screen NS4 cells which have already been induced to differentiate prior to the screening. It is also possible to determine the effects of the biological agents on the differentiation process by applying them to NS4 cells prior to differentiation. Generally, the biological agent can be solubilized and added to the culture medium at varying concentrations to determine the effect of the agent at each dose. The culture medium may be replenished with the biological agent every couple of days in amounts so as to keep the concentration of the agent somewhat constant.

Changes in proliferation are observed by an increase or decrease in the number of neurospheres that form or an increase or decrease in the size of the neurospheres (which is a reflection of the rate of proliferation as determined by the numbers of NS4 cells per neurosphere). A "regulatory factor" is a biological factor that has a regulatory effect on the proliferation of NS4 cells. For example, a biological factor would be considered a "regulatory factor" if it increases or decreases the number of NS4 cells that proliferate in vitro in response to a proliferation-inducing growth factor (such as EGF). Alternatively, the number of NS4 cells that respond to proliferation-inducing factors may remain the same, but addition of the regulatory factor affects the rate at which the NS4 cells proliferate. A proliferation-inducing growth factor may act as a regulatory factor when used in combination with another proliferation-inducing growth factor.

Other regulatory factors include heparan sulfate, TGF, activin, BMP-2, CNTF, retinoic acid, TNF, MIP-1, MJP-2, NGF, PDGF, interleukins, and the Bcl-2 gene product. Other factors having a regulatory effect on stem cell proliferation include those that interfere with the activation of the c-fos pathway (an intermediate early gene, known to be activated by EGF), including phorbol 12 myristate 13-acetate (PMA; Sigma), which up-regulates the c-fos pathway and staurosporine (Research Biochemical International) and CGP-41251 (Ciba-Geigy), which down regulate c-fos expression and factors, such as tyrphostin (Fallon et al., 11(5) Mol. Cell Biol. 2697-2703 (1991)) and the like, which suppress tyrosine kinase activation induced by the binding of EGF to its receptor.

The regulatory factors are added to the culture medium at a concentration in the range of about 10 pg/ml to 500 ng/ml (preferably, for example, about 1 ng/ml to 100 ng/ml, or more preferably about 10 ng/ml). The regulatory factor retinoic acid is prepared from a 1 mM stock solution and used at a final concentration between about 0.01 .mu.M and 100 .mu.M (preferably, for example, between about 0.05 .mu.M to 5 .mu.M).

The glycosaminoglycan, heparan sulfate, is a ubiquitous component on the surface of mammalian cells known to affect a variety of cellular processes, and which binds to growth factor molecules such as FGF and amphiregulin, thereby promoting the binding of these molecules to their receptors on the surfaces of cells. Heparan sulfate can be added to the culture medium in combination with other biological factors, at a concentration of about 1 ng/ml to 1 mg/ml (preferably, for example, about 0.2 .mu.g/ml to 20 .mu.g/ml, or more preferably about 2 g/ml).

Using these screening methods, one of skill in the art can screen for potential drug side-effects on pre-natal and post-natal CNS cells by testing for the effects of the biological agents on neural cell proliferation and differentiation or the survival and function of differentiated CNS cells. The proliferated NS4 cells are typically plated at a density of about 5-10.times.10.sup.6 cells/ml. if it is desired to test the effect of the biological agent on a particular differentiated cell type or a given make-up of cells, the ratio of neurons to glial cells obtained after differentiation can be manipulated by separating the different types of cells. Astrocytes can be panned out after a binding procedure using the RAN 2 antibody (available from ATCC). Tetanus toxin (available from Boerhinger Jngelheim) can be used to select out neurons. By varying the trophic factors added to the culture medium used during differentiation it is possible to intentionally alter the phenotype ratios. Such trophic factors include EGF, FGF, BDNF, CNTF, TGF, GDNF, and the like. For example, FGF increases the ratio of neurons, and CNTF increases the ratio of oligodendrocytes. Growing the cultures on beds of glial cells obtained from different CNS regions can also affect the course of differentiation.

The effects of the biological agents are identified based upon significant differences relative to control cultures with respect to criteria such as the ratios of expressed phenotypes (neurons, glial cells, or neurotransmitters or other markers), cell viability and alterations in gene expression. Physical characteristics of the cells can be analyzed by observing cell and neurite morphology and growth with microscopy. The induction of expression of new or increased levels of proteins such as enzymes, receptors and other cell surface molecules, or of neurotransmitters, amino acids, neuropeptides and biogenic amines can be analyzed with any technique known in the art which can identify the alteration of the level of such molecules. These techniques include immunohistochemistry using antibodies against such molecules, or biochemical analysis. Such biochemical analysis includes protein assays, enzymatic assays, receptor binding assays, enzyme-linked immunosorbant assays (ELISA), electrophoretic analysis, analysis with high performance liquid chromatography (HPLC), Western blots, and radioimmune assays (RIA). Nucleic acid analysis such as Northern blots and PCR can be used to examine the levels of mRNA coding for these molecules, or for enzymes which synthesize these molecules.

The factors involved in the proliferation of NS4 and the proliferation, differentiation and survival of NS4 cell progeny, and their responses to biological agents can be isolated by constructing cDNA libraries from NS4 cells or NS4 cell progeny at different stages of their development using techniques known in the art. The libraries from cells at one developmental stage are compared with those of cells at different stages of development to determine the sequence of gene expression during development and to reveal the effects of various biological agents or to reveal new biological agents that alter gene expression in CNS cells. When the libraries are prepared from dysfunctional tissue, genetic factors may be identified that play a role in the cause of dysfunction by comparing the libraries from the dysfunctional tissue with those from normal tissue. This information can be used in the design of therapies to treat the disorders. Additionally, probes cant be identified for use in the diagnosis of various genetic disorders or for use in identifying neural cells at a particular stage in development.

Electrophysiological analysis can be used to determine the effects of biological agents on neuronal characteristics such as resting membrane potential, evoked potentials, direction and ionic nature of current flow and the dynamics of ion channels. These measurements can be made using any technique known in the art, including extracellular single unit voltage recording, intracellular voltage recording, voltage clamping and patch clamping. Voltage sensitive dyes and ion sensitive electrodes may also be used.
 

Claim 1 of 27 Claims

1. An in vitro cell culture of RC-2.sup.+ cells, wherein a) one or more cells in the culture have the capacity to differentiate into neurons; b) the cell culture divides in a culture medium containing at least one proliferation-inducing growth factor; and c) one or more cells in the culture differentiate into neurons upon withdrawal of the proliferation-inducing growth factor.

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