BACKGROUND OF THE INVENTION Until recently, it was established dogma that neurons in the brain did not divide and therefore that loss of neurons was an irreversible process. The traditional view was that the adult brain in mammals was structurally stable and that neurogenesis and synapse formation occur only during development (P. Rakic, Science 227: 1054 (1985); P. Rakic, Nature Neurosci, 1: 645 (1998); E. Gould, A. J.

Reeves, M. S. A. Graziano, C. G. Gross, Science 286: 548 (1999)). In 1992, Reynolds and Weiss showed that cells isolated from adult rodent brain, from the subependymal zone in the striatum, could be cultured in vitro and would divide and differentiate into neurons, astrocytes and oligodendrocytes (B. A. Reynolds and S. Weiss, Science 255: 1707 (1992)). These cells were described as stem cells on the basis that they were thought to be pluripotent (i. e. that all cell types in the brain could be derived from these stem cells if treated in an appropriate manner).

The usual method of isolating neural stem cells in vitro is to dissect out a region of the fetal or adult brain known to contain dividing cells in vivo. The tissue is disaggregated and the dissociated cells are exposed to a high concentration of mitogens such as fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) (B. A. Reynolds and S. Weiss, Dev. Biol. 175: 1 (1996)) in either a defined or supplemented medium on a matrix as a substrate for binding. The cells proliferate, and subsequently they can be either induced to differentiate by withdrawing the

mitogens or by exposing the cells to another factor that induces some of the cells to develop into different lineages. The phenotypes of cells are then determined by staining with antibodies directed against antigens specific for astrocytes, oligodendrocytes, and neurons. To confirm that the cells are indeed stem cells, a single cell can be plated at low density and monitored to determine if a single cell can give rise to the three phenotypes (B. A. Reynolds and S. Weiss, Dev. Biol. 175: 1 (1996)). Stem cells were also tagged with a retrovirus in vitro, after which the clones of cells derived from the original tagged cell were shown by Southern analysis to be derived from a single cell (T. D. Palmer, J. Takahashi, F. H. Gage, Mol. Cell.

Neurosci. 8: 389 (1997)).

It is thought that progenitor cells differentiate into the neuronal phenotype and then differentiate into specific types of neuron (for example, dopaminergic, serotonergic, cholinergic, hippocampal pyramidal cells, striatal neurons) (S. Weiss, et al., Trends Neurosci. 19,387 (1996)). It is important to produce cell types of a specific phenotype. For example, dopamine neurons are needed in large numbers as a potential therapy for Parkinson's disease. Parkinson's disease is caused by the loss of dopamine neurons and a large body of work suggests that replacement of these neurons by transplantation into patients will reverse the symptoms of Parkinson's disease (C. W. Olanow, et al. Trends Neurosci. 19,102 (1996)). This work to date has been done using neurons obtained from aborted human fetuses. However, the supply of tissue is unreliable and insufficient. In general, only a small percentage of stem cells can be induced in vitro to assume a neuronal phenotype and of those cells assuming the neuronal phenotype only a small percentage will assume the dopaminergic neuronal phenotype. The success rate in generating neurons varies a great deal.

Thus there is a need for factors and methods to induce conversion of stem cells into the neuronal phenotype (S. Weiss, et al., Trends Neurosci. 19: 387 (1996)).

However, no factors or methods have yet been described which mediate the conversion of stem cells into neurons.

SUMMARY OF THE INVENTION The present inventors have discovered a simple technique for converting stem cells into a neuronal phenotype. Following this method, it is possible to convert most or all stem cells into neurons.

The invention provides a method for producing a neuron comprising: (a) incubating a stem cell in a growth medium; (b) separating the growth medium from the stem cell; (c) heat-treating the growth medium to produce a heat-treated medium; and (d) subsequently incubating a stem cell in a treated medium which includes the heat-treated medium to produce a neuron.

The invention also provides a method for producing neurons comprising: (a) isolating a stem cell; (b) incubating the stem cell in a growth medium containing a growth factor; (c) separating the growth medium from the stem cell; (d) heat-treating the growth medium to produce a heat-treated medium; and (e) subsequently incubating the stem cells in a treated medium which includes the heat-treated medium, to produce neurons.

The invention further provides a conditioned medium for growing neurons from stem cells comprising a medium prepared by the steps of : (a) isolating a stem cell; (b) incubating the stem cell in a growth medium containing a growth factor; (c) separating the growth medium from the stem cell; and (d) heat-treating the growth medium to produce a conditioned medium.

In an embodiment the treated medium includes the growth medium. In a further embodiment, the treated medium includes the growth medium of step (c).

In embodiments, the stem cell may be a brain stem cell, an adult stem cell, a striatal stem cell, a mammalian stem cell, a human stem cell, a mouse stem cell and/ or a stem cell from a subependymal zone.

In embodiments, the heat-treating is at a temperature of between 40°Cand 65C, between 45°Cand 60C, between 50°Cand 60C, or about 56°C. The heat-treating may be for a duration of between 1 minute and one week, between five minutes and one day, between 10 minutes and one hour, or about 30 minutes.

In an embodiment the treated medium comprises about 50% the growth medium and about 50% the heat-treated medium.

The growth factor may be EGF, bFGF, or EGF and bFGF. The growth factor may have a concentration of about 20 ng/ml. The growth medium may be DMEM/F12.

In embodiments, the incubating may be carried out to a density of appoximately 12,500 cells/ml, it may be carried out at about 37 °C and/or it may be carried out at about 5% CO2.

In one embodiment the incubating is carried out for about 2 weeks. The separating may be by centrifugation.

The subsequent incubation may be carried out by plating isolated stem cells.

The isolated stem cells may be isolated from neurospheres.

The method of the invention also embodies further inducing the neurons to differentiate.

In another embodiment, the invention comprises a neuron produced by any of the methods of the invention. In another embodiment, the invention is the use of the neuron to treat a neural disease. The neural disease may be Parkinson's disease,

Huntington's disease, brain or spinal cord injury, epilepsy, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, tumors, or stroke.

DETAILED DESCRIPTION OF AN EMBODIMENT The present invention discloses a novel method that induces all or almost all stem cells in vitro to assume a neuronal-like phenotype.

During culturing of stem cells, the present inventors noticed that cell proliferation was significantly higher at high cell densities compared to lower cell densities. These findings suggested that the cells were releasing a factor or factors that were"conditioning"the mediums. Accordingly, the present inventors heat- treated the medium at 56°C for 30 minutes and incubated stem cells in this heat- inactivated conditioned medium. Instead of reducing the proliferation rate as expected, all of the stem cells incubated in this medium assumed a neuronal-like phenotype.

The method used to create the neurons is as follows. Stem cells were isolated from an adult brain. The isolated stem cells were placed in a conditioned medium and were incubated. The conditioned medium was then separated from the neurospheres.

The conditioned medium was heat-treated. The stem cells were then plated in a medium which includes the heat-treated conditioned medium. By this methodology, almost all stem cells formed neurons.

It will be appreciated that the process of the invention may be better regulated by the most efficient selection of parameters for both methods and materials.

However, it will also be appreciated that the invention encompasses various parameters of methods and materials which are effective.

Isolating neural stem cells in vitro may be accomplished by dissecting a region of the fetal or adult brain or fetal or adult skin cells known to contain dividing cells in

vivo. This can be successfully done, for example, with the subventricular zone (SVZ) or the hippocampus from an adult brain or many structures in the developing brain.

The in vitro culture of region-specific differentiated neurons may be derived from any mammalian multipotential CNS stem cell, including mouse and human. Other specific regions from which the stem cells are derived are selected from the group consisting of cortex, olfactory tubercle, retina, septum, lateral ganglionic eminence, medial ganglionic eminence, amygdala, hippocampus, thalamus, hypothalamus, ventral and dorsal mesencephalon, brain stem, cerebellum, and spinal cord.

Stem cells as defined herein include cells able to divide to regenerate themselves and, in addition, to produce progeny which can follow other developmental patterns.

Cells can be obtained from donor tissue by dissociation of individual cells from the connecting extracellular matrix of the tissue. 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. Dissociation of fetal cells can be carried out in tissue culture medium, while a preferable medium for dissociation of juvenile and adult cells is low Ca 2+ artificial cerebral spinal fluid (aCSF). Regular aCSF contains 124 mM NaCI, 5 mM KC1, 1.3 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM D- glucose. Low Ca 2+ aCSF contains the same ingredients except for MgCl2 at a concentration of 3.2 mM and CaCl2 at a concentration of 0.1 mM. Dissociated cells are centrifuged at low speed, between 200 and 2000 rpm, usually between 400 and 800 rpm, and then resuspended in culture medium. The neural cells can be cultured in suspension or on a fixed substrate. However, substrates tend to induce differentiation of the neural stem cell progeny. Thus, suspension cultures are preferred if large numbers of undifferentiated neural stem cell progeny are desired. 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 cm2 culture flasks. Cells cultured in suspension are resuspended at approximately 5 X

104 to 2 X 10'cells/ml, preferably 2.85 X 10 4 cells/ml. Cells plated on a fixed substrate are plated at approximately 2-3 X 10 3 cells/cm2, preferably 8.0 X 10 3 cells/cm 2.

Division of neural stem cells can be accomplished in numerous ways. They may undergo asymmetric division where cells are produced with different fates.

Neural stem cells may also undergo proliferative divisions, which expands the stem cell population, or differentiative divisions, which reduces the stem cell population, (Doe, C. Q., Fuerstenberg, S., and Peng, C. Y, JNeurobiology 36 : 111 (1998)). Neural stem cells are also likely to produce intermediate precursors termed progenitor cells that can generate specific subpopulations.

Many different mechanisms can be used for regulating the differentiation of neural stem cells and their progeny. These factors include cell division history, increased cell density, exposure to different levels or types of growth factors, either addition or withdrawal of or, alteration of substratum-linked factors (Fisher, L, Neurobiol. Dis. 4: 1 (1997); Morrison, S., Shah, N., and Anderson, D, Cell, 88: 287 (1997); Shen, Q., Qian, X., Capela, A., and Temple, S, JNeurobiology 36: 162 (1998)).

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 glutamin and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like. Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like. In some cases, the medium may contain serum derived from bovine, equine, chicken and the like. One embodiment for proliferation of neural stem cells is to use a defined, serum- free culture medium, as serum tends to induce differentiation and contains unknown components (i. e. is undefined). A defined culture medium is also desired if the cells are to be used for transplantation purposes.

Conditions for culturing should be close to physiological conditions. The pH of the culture medium should be close to physiological pH, preferably between pH 6- 8, more preferably between about pH 7 to 7.8, with pH 7.4 being most preferred.

Physiological temperatures range between about 30 °Cto 40 °C. Cells are preferably cultured at temperatures between about 32 °Cto about 38 C, and more preferably between about 35 °Cto about 37C.

The culture medium is supplemented with at least one proliferation-inducing growth factor. As used herein, the term"growth factor"refers to a protein, peptide or other molecule having a growth, proliferative, differentiative, or trophic effect on neural stem cells and/or neural stem cell progeny. Growth factors which may be used for inducing proliferation include any trophic factor that allows neural stem cells and precursor cells to proliferate, including any molecule which binds to a receptor on the surface of the cell to exert a trophic, or growth-inducing effect on the cell. Preferred 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 a (TGF-a), and combinations thereof.

Numerous specific factors have been identified and among them are Nurrl, a transcription factor of the thyroid hormone/retinoic acid nuclear receptor superfamily (Wagner, J., et al., Nature Biotechnology 17: 653 (1999)), or a combination of FGF2 and epidermal growth factor (EGF) (B. A. Reynolds and S. Weiss, Science 255: 1707 (1992); Morshead, C. M. et al., Neuron 13: 1071 (1994)). EGF is particularly useful as a primary mitogen to expand the most primitive cells. Also useful for primitive cells is FGF-2 with or without EGF. Transforming growth factor a (TGF-a), another ligand for the EGF receptor, has also been shown to have a proliferative effect (Tropepe, V., Craig, C. G., Morshead, C. M., and Van der Kooy, D: JNeuroscience, 17: 785 (1997)). In addition to proliferation-inducing growth factors, other growth factors may be added to the culture medium that influence proliferation and differentiation of the cells including NGF, platelet-derived growth factor (PDGF), thyrotropin releasing hormone (TRH), transforming growth factor ps (TGFps), insulin-like growth factor (IGFJ and the like.

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. Titration experiments can be performed to determine the optimal concentration of a particular growth factor.

Within 3-4 days in the presence of a proliferation-inducing growth factor, a multipotent neural stem cell begins to divide giving rise to a cluster of undifferentiated cells referred to herein as a"neurosphere".

Following the methods of the invention, the cells all extend projections (neurites) and these neurites form synapses with one another. The extending terminals of these neurites form growth cones, the specialization that guides the extending neurites.

A multiplicity of mechanisms may trigger differentiation (see, for example, U. S. patent nos. 5,851,832 to Weiss et al and U. S. patent nos. 6,040,180 to Johe).

Cell-cell interactions can alter the response of stem cells to growth factors suggesting that environmental signals can also play a role in regulating the differentiative outcome of the stem cell population.

It is believed that conversion into the neuronal phenotype is the first step prior to conversion into specific neuronal phenotypes (S. Weiss, et al., Trends Neurosci.

19: 387 (1996)). Following the methods of the present invention, it is possible to convert most or all stem cells into neurons. This allows one to then proceed to convert all the neurons into a desired specific phenotype. For example, one could convert all the neurons into dopaminergic neurons, and thus provide a pure population of dopaminergic neurons for the treatment of Parkinson's disease. Parkinson's disease is caused by the loss of dopamine neurons and a large body of work suggests that replacement of these neurons by transplantation into patients will reverse the symptoms of Parkinson's disease (C. W. Olanow, et al. Trends Neurosci. 19: 102 (1996)).

The present invention will be further illustrated by referring to the following examples which are given by way of illustration.

EXAMPLES Stem cells were isolated from the subependymal zone of the striatum of adult mouse brain according to Reynolds and Weiss (1992). Isolated stem cells were placed in a DMEM/F 12-containing 20 ng/ml of both EGF and bFGF at a density of approximately 12,500 cells/ml and were incubated at 37°C with 5% CO2 according to B. A. Reynolds and S. Weiss, Science 255: 1707 (1992) or B. A. Reynolds and S.

Weiss, Dev. Biol. 175: 1 (1996). Cells were allowed to proliferate for up to 2 weeks.

The conditioned medium was then separated from the neurospheres by centrifugation.

The conditioned medium was heat-treated for 30 minutes at 56°C. Single stem cells dissociated from neurospheres were then plated in a medium consisting of 50% heat- treated conditioned medium and 50% stem cell medium as described in step 2. The resulting cells were found to be neurons.