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Title:  Cell expansion system for use in neural transplantation

United States Patent:  6,787,356

Issued:  September 7, 2004

Inventors:  Studer; Lorenz (New York, NY); McKay; Ron D. (Bethesda, MD)

Assignee:  The United States of America as represented by the Department of Health and (Washington, DC)

Appl. No.:  744384

Filed:  March 16, 2001

PCT Filed:  July 23, 1999

PCT NO:  PCT/US99/16825

PCT PUB.NO.:  WO00/05343

PCT PUB. Date:  February 3, 2000

Abstract

The invention provides a method of culturing cells which includes a proliferating step in which the number of precursor cells is expanded and a differentiating step in which the expanded precursor cells develop into neuronal cells. The proliferating step includes the step of incubating the precursor cells in proliferating medium which includes basic fibroblast growth factor (bFGF). The differentiating step includes incubating the precursor cells in differentiation media in a manner effective to form a cellular aggregate that is not adhered to any surface of the incubation vessel. In a preferred embodiment, the cells arc incubated in a roller tube. The differentiation media can also include at least one differentiating agent. The invention also provides a method for treating a neurological disorder, such as Parkinson's disease, a method of introducing a gene product into a brain of a patient, an assay for neurologically active substances, and a cell culture.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a method of culturing cells. The method includes a proliferating step in which the number of precursor cell is expanded. The proliferating step includes the step of incubating the precursor cells in proliferating medium which includes basic fibroblast growth factor (bFGF). Preferably the precursor cells are obtained from a human fetal tissue between embryonic week 5 to embryonic week 8. The method also includes a differentiating step in which the expanded precursor cells develop into neuronal cells. The differentiating step includes incubating the precursor cells in differentiation media, wherein the cells are agitated during the incubation in a manner effective to form a cellular aggregate that is not adhered to any surface of the incubation vessel. In a preferred embodiment, the cells are incubated in a roller tube.

According to the invention, the differentiation media can also include at least one differentiating agent. Examples of differentiating agents include cyclic adenosine monophosphae (cAMP), dopamine, cAMP modulating agents and ascorbic acid. Preferably cAMP is N6,2-O-Dibutyrladenosine 3':5' Cyclic Monophosphate (dbcAMP) and/or 8-Bromoadenosine 3':5' Cyclic Monophosphate. These derivatives of cAMP are preferred for their enhanced ability to enter the cell. Preferably the cAMP modulating agent is capable of stimulating the cAMP pathway in the cell. More preferably, the cAMP modulating agent is a cAMP agonist. An example of a cAMP modulating agent includes forskolin.

The method of the invention can be used to culture a variety of cells, a preferably neuronal cells, including, but not limited to, dopaminergic neuron cells, cholinergic neuronal cells and serotonergic cells. Examples of suitable precursor cells include mesencephalic cells, basal forebrain cells or spinal cord cells and nucleus raphe cells. The invention also provides a method for treating a neurological disorder, such as Parkinson's disease, a method of introducing a gene product into a brain of a patient, and an assay for neurologically active substances. Advantageously, the method of the invention allows direct transplantation of the cultured cells without the need for enzymatic digestion to remove the cultured cells from a culture dish matrix.

The invention also provides a cell culture which includes about 80% to about 95% neurons. The percentage of glial cells in the cell culture is about 1% to about 5%, more preferably about 2% to about 3%, most preferably about 2% to about 2.5% for astrocytes and about 0.5% to 2%, more preferably 1% to 1.5%, and most preferably about 1% to 1.1% for oligodendrocytes. In contrast, no published work as of yet records percentages of neurons higher than 50%. Furthermore, the percentage of astrocytes in our culture system is extremely low as compared to any other known system.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the invention provides a method for generating neuronal cells by proliferating and differentiating precursor cells. In the proliferating step, the number of precursor cells is increased. According to the invention, precursor cells are incubated in the presence of proliferating media which includes basic fibroblast growth factor (bFGF). More preferably the precursor cells are obtained from a human fetal tissue between embryonic week 5 to embryonic week 8. The proliferating step of the invention provides an expansion of precursor cells by more than 1000-fold and thus decreases the problem of obtaining sufficient cells for a reliable, effective clinical cell transplantation.

In the differentiating step, the precursor cells are induced to differentiate into neuronal cells. The differentiating step of the invention includes the step of incubating the precursor cells in a differentiation vessel in the presence of differentiating media in a manner effective to form a free-floating reaggregation of differentiated cells (e.g., an aggregation that is not adhered to a surface of the differentiation vessel). Formation of a free-floating reaggregate of differentiated cells in the differentiation step of the invention obviates the need for mechanical or enzymatic dissociation of the differentiated cells which generally causes disruption of axodendritic trees and cell loss. Additionally, the reaggregates of differentiated cells can be directly loaded into a stereotactic needle and transplanted in toto.

The differentiation media can include differentiating agents such as cyclic adenosine 3',5'-phosphate (cAMP), cAMP modulators, dopamine and ascorbic acid. Preferably cAMP is N6,2-O-dibutyrladenosine 3':5' cyclic monophosphate (dbcAMP) and/or 8-bromoadenosine 3':5' cyclic monophosphate. These derivatives of cAMP are preferred for their enhanced ability to enter the cell. Preferably the cAMP modulating agent is capable of stimulating the cAMP pathway in the cell. More preferably, the cAMP modulating agent is a cAMP agonist. An example of a cAMP modulating agent includes forskolin. As an alternative to dopamine, L-Dopa can be added as a differentiating agent. L-Dopa is converted in the cell into dopamine and released. Preferably ascorbic acid is L-ascorbic acid, although D-ascorbic acid can be used.

In a preferred embodiment, the differentiated cells are dopaminergic neurons. Dopaminergic neurons generated by the method of the invention are the first known dopaminergic neurons derived from in vitro expanded precursors that function in vivo. Dopaminergic neurons cultured using the method of the invention are particularly suitable for use in the treatment of Parkinson's disease.

I. Method of Culturing Cells

A first aspect of the invention provides a method for generating a differentiated cell culture by proliferating and differentiating a population of precursor cells. The method of the invention is applicable to a wide variety of precursor and differentiated cells. In a preferred embodiment, the method of the invention is used to generate differentiated neural cells.

A. Proliferating Step

In the proliferating step of the invention, precursor cells are incubated in proliferating media to increase the number of precursor cells. As used herein, the term "precursor cell" refers to a cell that is capable of differentiating to form a specific cell type, but does not yet express proteins associated with a specific cell type.

Typically, the precursor cells used in connection with the invention are cells that are associated with the central nervous system (CNS), referred to as CNS stem cells. The term "central nervous system" refers to the part of the nervous system of an animal that is the main site of integration of nervous activity. The central nervous system generally includes a brain or cerebral ganglia and a nerve cord. More preferably, the precursor cells are mammalian CNS stem cells, most preferably human fetal cells. CNS stem cells include cells from ventral midbrain, dorsal midbrain, lateral ganglionic eminence, hippocampus, cerebral cortex, striatum, septum, diencephalon, hindbrain and spinal cord. Preferably, the precursor cells are from the midbrain.

Most preferably, the method of the invention employs precursor cells obtained during a "sensitive period" before the cells have differentiated. As used herein, the "sensitive period" is the period during which precursor cells can be obtained that produce a very large number of a specific differentiated cell subtype. Preferably, the method of the invention employs precursor cells obtained from the midbrain before the cells differentiate into dopaminergic neurons. The inventors have found that later precursors under identical conditions produce much lower amounts of dopaminergic neurons in vitro. The sensitive period is the rat embryonic day 10 to embryonic day 12. In human fetal tissue the sensitive age is between embryonic week 5 to embryonic week 8.

According to the invention, precursor cells are triturated as described by Studer, L. in Current Protocols in Neuroscience (eds McKay, R. D. & Gerfen, C. R.) John Wiley & Sons Inc., New York, 1997) and dispersed in calcium/magnesium free HBSS (Hank's balanced salt solution) to a quasi single cell suspension. As used herein, the term "quasi single cell suspension" means that at least 80% of the cells are present as single cells and the remaining cells form small clusters of less than 200 cells. Preferably the suspension has a concentration of about 50x103 cells/ml to about 500x103 cells/ml, more preferably about 100x103 cells/ml to about 250x103 cells/ml, most preferably about 150x103 cells/ml to about 200x103 cells/ml.

The suspension of precursor cells is then plated onto proliferating medium. As used herein, the term "proliferating medium" refers to a solid or liquid substrate that can support cell growth. Typically, proliferating medium provides water, a source of energy, carbon, nitrogen, mineral elements, and vitamins. The medium may consist of pure compounds (defined medium) or crude animal or plant extracts (complex medium). The term "defined medium" refers to the fact that all the components and their concentrations are known. Usually the term is used in contrast to "serum-containing" medium where not all the components and their concentrations are known. The medium is preferably a solution of nutrients, water, salts, sugars, amino acids etc. containing the source of energy for the cells and also containing some hormones (e.g. progesterone) and trace elements (e.g. selenite) that enhance their general growth and survival. More preferably, the proliferating media includes serum free medium. Examples of serum free media include Dulbecco's Modified Eagle's Medium (DMEM) and Neurobasal.RTM. medium. Preferably, the proliferating media includes supplements such as N2 supplement and/or B27 supplement. More preferably, the proliferating media is coated a substance that makes the surface of the petri dish sticky and facilitates attachment of the stem cells. Preferably, the surface is coated with polyornithine and fibronectin before the cells are plated. Polyomithine and fibronectin are preferred for use in the method of the invention because they facilitate adherence of the cells to the medium but still allow the cells to proliferate because they do not cause cell differentiation (in contrast to other substrates used in cell culture, such as laminin).

According to the invention, the proliferating medium is supplemented with basic fibroblast growth factor (bFGF). Preferably the medium is supplemented with bFGF on a daily basis, for example, by adding a solution containing bFGF dissolved in buffer. An example of a suitable buffer is phosphate buffered saline (PBS) and bovine serum albumin. A preferred bFGF solution contains about 5 .mu.g/ml to about 20 .mu.g/ml bFGF, more preferably about 7 .mu.g/ml to about 15 .mu.g/ml bFGF, most preferably about 10 .mu.g/ml to about 10.5 .mu.g/ml bFGF. Preferably the bFGF solution contains about 0.05% to about 0.5%, more preferably about 0.1% to about 0.2% bovine serum albumin.

About 1 .mu.l to about 20 .mu.l, more preferably about 5 .mu.l to about 10 .mu.l of the bFGF solution is added to a cell culture containing about 5 ml medium to produce a final concentration of about 5 ng/ml to about 100 ng/ml bFGF (final concentration in the medium), more preferably about 10 to about 20 ng/ml (final concentration in the medium).

The precursor cells are incubated for about 5 to about 10 days, more preferably about 5 to about 7 days. Although expansion beyond the first week in culture leads to an exponential increase in cell number, the percentage of dopamine neurons obtained from long term expanded precursors decreases rapidly, corresponding to only about 1-2% of the total population.

Additional factors that promote proliferation of precursor cells include fibroblast growth factor 4 (FGF4) and vasointestinal peptide (VIP).

B. Differentiating Step

In the differentiating step, the precursor cells are transformed into differentiated cells. As used herein, a "differentiated cell" is a cell that produces specialized proteins associated with a specific cell type. The method is applicable to a variety of cell types. However, the method is preferably used to culture differentiated neural cells. As used herein, the term "neuronal cells" refers to neurons. In contrast, the term "neural cells" refers more broadly to cells associated with the central nervous system (CNS) of an organism, for example, neurons, glial cells, precursor cells, etc. For example, the method of the invention can be used to generate differentiated neuronal cells such as dopaminergic, cholinergic or serotonergic neuronal cells from precursor cells from the midbrain/hindbrain junction, the basal forebrain and spinal cord or nucleus raphe, respectively. The invention is most preferably directed towards a method of proliferating and differentiating mesencephalon precursor cells to form functional dopaminergic neurons.

As used herein, the term dopaminergic neuronal cells refers to those cells generally found in the region of the ventral midbrain (VM) known as the substantia nigra pars compacta that project to the striatum. The precursor cells are typically found near the midbrain/hindbrain junction of an intact brain. Dopaminergic neurons can be characterized by their secretion of dopamine as a neurotransmitter and high levels of expression of tyrosine hydroxylase (TH), an enzyme that catalyzes the rate limiting step in the biosynthesis of dopamine.

In the differentating step of the invention, precursor cells are combined with differentiation medium in an incubation vessel and incubated for an amount of time sufficient to allow for the differentiation of the precursor cells into neuronal cells. Neurobasal/B27 medium (Gibco, Life Technologies) with or without serum supplement (1-10% fetal bovine serum) is a preferred differentiation medium. Like proliferation medium, differentiation medium contains a variety of nutrients necessary for cell survival. However, in contrast to proliferation medium, differentiation medium does not include mitogens which enhance cellular proliferation, for example bFGF. Additionally, differentiation medium may contain supplements to enhance differentiation, such as B27. Those of skill in the art are familiar with a variety of media suitable for use as differentiation media. The yield of differentiated cells after expansion and differentiation is typically about 15% to about 20%.

1. Reaggregation System

According to the invention, a reaggregation system is used in the differentiating step. In contrast to conventional cell cultures in which cultured cells are adhered to a surface of incubation vessel, the reaggregation system of the invention is a three-dimensional system that allows conversion of precursor cells into differentiated cells which exist in the differentiating media as free-floating reaggregates. As used herein, the phrase "adhered to" refers to cellular attachment via receptors on the cell surface, such as the interaction between cells and a cell culture matrix containing laminin, fibronectin, polylysine or other attachment factors. Advantageously, the cells cultured according to the reaggregation system of the invention can be transplanted without enzymatic digestion to remove the cells from the culture dish matrix. Enzymatic digestion tends to kill most cultured cells.

In the reaggregation system of the invention, the incubation vessel is rotated during incubation in a manner effective to form a differentiated cell cluster that is not adhered to any of the surfaces of the incubation vessel. Examples of suitable incubation vessels include enclosed spheres, tubes or drums. Generally, suitable incubation vessels are polystyrene based plastic tubes, for example, Falcon #2095. The quality of the tubes is important as cells might adhere to other plastics despite the rotational movement. Additionally, other plastics might negatively influence cell survival during differentiation. Most preferably, the incubation vessel is a roller tube or drum such as the roller drum available from Bellco Glass, Inc. (product number 7736-10164). According to the invention, the incubation vessel is slowly rotated around a horizontal axis during incubation to prevent attachment of single cells to the incubation vessel and to further interaction among cells to enhance aggregation. Preferably, the incubation vessel revolves at a speed of about 1 to about 4 rotations per minute. Most preferably, the incubation vessel is placed in a tissue culture incubator and tilted 5 degrees.

According to the invention, the precursor cells are incubated in the differentiation medium for about 5 to about 10 days, more preferably about 5 to about 7 days.

2. Differentiation Factors

According to another aspect of the invention, the differentiation medium includes at least one differentiation factor which improves differentiation of precursor cells into differentiated cells. Differentiation factors of the invention include cyclic adenosine 3',5'-phosphate (cAMP), cAMP modulators, dopamine and ascorbic acid. As used herein, cAMP includes N6,2-O-dibutyrladenosine 3':5' cyclic monophosphate (dbcAMP) and/or 8-bromoadenosine 3':5' cyclic monophosphate. These derivatives of cAMP are preferred for their enhanced ability to enter the cell.

As used herein, the term cAMP modulator refers to a molecule that is capable of up-regulating or stimulating cAMP production within a cell. Preferably, the cAMP modulating agent is a cAMP agonist. The cAMP modulator can be exogenous to the patient's system or may be an endogenous compound. For example, the cAMP modulator can be a naturally occurring peptide, protein or enzyme, or a peptide or protein fragment which catalyzes the production of cAMP. An examples of a suitable cAMP modulator is forskolin.

As an alternative to dopamine, the differentiating agent can includes substances which stimulate dopamine production or are converted into dopamine in vivo ("dopamine simulator"). For example, L-Dopa can be added as a differentiating agent. L-Dopa is converted in the cell into dopamine and released. Ascorbic acid includes L-ascorbic acid and D-ascorbic acid, although L-ascorbic acid is preferred.

Preferably, the differentiated media includes at least one of cAMP, forskolin, dopamine and ascorbic acid. Preferably, cAMP is present in a concentration from about 1 .mu.M to about 5 mM, more preferably about 10 .mu.M to about 1 Mm. Typically, differentiation media containing cAMP results in about a 50% to about 300% increase in the total number of differentiated neural cells from precursor cells. Most typically, an increase in tyrosine hydroxylase immunoreactive (TH-ir) cells, also called dopaminergic cells is observed. Preferably forskolin is present in the differentiation medium at a concentration from about 1 .mu.M to about 100 .mu.M, more preferably about 2 .mu.M to about 10 .mu.M. Typically, differentiation media containing forskolin results in about a 40% to about 150% increase in the total number of differentiated neural cells, particularly TH-ir cells. Preferably dopamine is present at a concentration from about 0.1 .mu.M to about 1 mM, more preferably about 1 .mu.M to about 1 mM to provide about a 300% to about 700% increase in the total number of TH-ir cells. The effects of cAMP, forskolin and dopamine appear to be additive.

Addition of ascorbic acid to a cell culture during differentiation dramatically increases the percentage of resulting functional neuronal cells, particularly TH-ir cells. Preferably ascorbic acid is included in connection with dopamine to prevent auto-oxidation of dopamine in the medium (the effects of dopamine and ascorbic acid are additive). Although, ascorbic acid by itself has a stronger effect than dopamine on induction of dopaminergic neurons. Typically an average about 5 to about 20 fold (500 to 2000%) increase in TH-ir neurons is observed. This surprising effect of ascorbic acid is not observed with other antioxydative agent. Without intending to be bound by theory, it is believed that ascorbic acid acts via a mechanism distinct from its antioxydative properties. Because ascorbic acid is also an essential nutritional supplement (Vitamin C), it is unlikely to have unwanted side effects.

II. Differentiated Cell Culture

The invention also provides a differentiated cell culture which includes neural cells that function in vivo. Preferably, the differentiated cell culture includes dopaminergic neurons that function in vivo. As used herein, the phrase "function in vivo" means that the cells can survive in a relevant animal model, retain their identity after transplantation, integrate into the host brain, and improve animal behavior.

The invention also provides a cell culture which includes about 80% to about 95% neurons. The percentage of glial cells in the cell culture is about 1% to about 5%, more preferably about 2% to about 3%, most preferably about 2% to about 2.5% for astrocytes and about 0.5% to 2%, more preferably 1% to 1.5%, and most preferably about 1% to 1.1% for oligodendrocytes. In contrast, no published work as of yet records percentages of neurons higher than 50%. Furthermore, the percentage of astrocytes in our culture system is extremely low as compared to any other known system.

III. Methods of Use

A. Treatment of Neurological Disorders

In one embodiment, the present invention provides a method of treating a patient suffering from a neurological disorder, such as a central nervous system disorder, or alleviating the symptoms of such a disorder, by administering cells cultured according to the method of the invention to the patient's brain. As used herein, the terms "treating" and "treatment" refer to curative therapy, prophylactic therapy, and preventative therapy. The term "therapy" as used herein, refers to therapeutic methods for reducing or eliminating the symptoms of the particular disorder for which treatment is sought. The term "patient" 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. Examples of neurological disorders include Parkinson's disease, Huntington's disease, Alzheimer's disease, severe seizure disorders including epilepsy, familial dysautonomia as well as injury or trauma to the nervous system, such as neurotoxic injury or disorders of mood and behavior such as addiction and schizophrenia.

In this method of the invention, precursor cells are cultured in vitro as described above to form differentiated neuronal cells which are then transplanted into the brain of a patient in need thereof.

1. Formulations

After the cell reaggregate is formed according to the cell culturing method previously described, the reaggregate is suspended in a physiologically compatible carrier. As used herein, the term "physiologically compatible carrier" refers to a carrier that is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Those of skill in the art are familiar with physiologically compatible carriers. Examples of suitable carriers include cell culture medium (e.g., Eagle's minimal essential media), phosphate buffered saline, and Hank's balanced salt solution+/-glucose (HBSS).

The volume of cell suspension administered to a patient will vary depending on the site of implantation, treatment goal and amount of cells in solution. Typically the amount of cells administered to a patient will be a "therapeutically effective amount." As used herein, a therapeutically effective amount refers to the number of transplanted cells which are required to effect treatment of the particular disorder. For example, where the treatment is for Parkinson's disease, transplantation of a therapeutically effective amount of cells will typically produce a reduction in the amount and/or severity of the symptoms associated with that disorder, e.g., rigidity, akinesia and gait disorder.

It is estimated that a severe Parkinson's patient will need at least about 100,000 surviving dopamine cells per grafted side to have a substantial beneficial effect from the transplantation. As cell survival is low in brain tissue transplantation in general (5-10%) an estimated 1-4 million dopaminergic neurons should be transplanted. The invention delivers aggregates with about 15% TH cells and containing about 50,000-100,000 cells in total (7500-15000 TH cells/aggregate). This means that for a successful therapy about 100-500 aggregates should be transplanted per patient side. The aggregates are collected together and then loaded directly into a stereotactic needle. Aggregates do not need any special formulation if total duration of the implantation procedure is less than 1 hour. Preferentially, the prepared spheres are maintained in medium and will be loaded into the needle immediately before introducing the needle into the patients brain.

2. Methods of Administration

According to the invention, the cell reaggregate is administered to the patient's brain. The reaggregate may be implanted within the parenchyma of the brain, in the space containing cerebrospinal fluids, such as the sub-arachnoid space or ventricles, or extaneurally. As used herein, the term "extraneurally" is intended to indicate regions of the patient which are not within the central nervous system or peripheral nervous system, such as the celiac ganglion or sciatic nerve. "Central nervous system" is meant to include all structures within the dura mater.

Typically, the reaggregations are administered by injection into the brain of the patient. Injections can generally be made with a sterilized syringe having an 18-21 gauge needle. Although the exact size needle will depend on the species being treated, the needle should not be bigger than 1 mm diameter in any species. Those of skill in the art are familiar with techniques for administering cells to the brain of a patient.

3. Diseases

a. Parkinson's disease

Parkinson's disease (PD) is characterized by the progressive loss in function of dopaminergic neurons. The progressive loss of dopaminergic function interferes with the normal working of the neuronal circuitry necessary for motor control so that patients with PD show characteristic motor disturbances such as akinesia, rigidity and rest tremor. Other symptoms include pain, impaired olfaction, alterations of personality and depression. Quinn et al., (1997) Baillieres Clin. Neurol. 6:1-13.

According to the invention, dopaminergic neuronal cells are generated using the cell culturing method described above. The dopaminergic cells are then administered to the brain of the patient in need thereof to produce dopamine and restore behavioral deficits in the patient. Preferably, the cells are administered to the basal ganglia of the patient.

b. Alzheimer's disease

Alzheimer's disease involves a deficit in cholinergic cells in the nucleus basalis. Thus, a subject having Alzheimer's disease may be treated by administering cells cultured according to the method of the invention that are capable of producing acetylcholine.

c. Huntington's disease

Huntington's disease involves a gross wasting of the head of the caudate nucleus and putamen, usually accompanied by moderate disease of the gyrus. A subject suffering from Huntington's disease can be treated by implanting cells cultured according to the method of the invention that are capable of producing the neurotransmitters gamma amino butyric acid (GABA), acetylcholine, or a mixture thereof.

4. Gene Therapy

In an additional embodiment of the invention, the cultured cells may be transfected with a nucleic acid which encodes a neurologically relevant polypeptide. The term "neurologically relevant peptide" generally refers to a peptide or protein which catalyzes a reaction within the tissues of the central nervous system. Such peptides may be naturally occurring neural peptides, proteins or enzymes, or may be peptide or protein fragments which have therapeutic activity within the central nervous system.

According to this aspect of the invention, precursor cells are cultured in vitro as described above and an exogenous gene encoding a desired gene product is introduced into the cells, for example, by transfection. The transfected cultured cells can then be administered to a patient with a neurological disorder.

a. Genes of interest

Examples of neurologically relevant peptides include neural growth factors and enzymes used to catalyze the production of important neurochemicals or their intermediates. The peptide encoded by the nucleic acid may exogenous to the host or endogenous. For example, an endogenous gene that supplements or replaces deficient production of a peptide by the tissue of the host wherein such 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 may be an enzyme which catalyzes the production of a therapeutic or neurologically relevant compound. Again, such compounds may be exogenous to the patient's system or may be an endogenous compound whose synthetic pathway is otherwise impaired. Examples of neurologically relevant compounds include tyrosine hydroxylase, nerve growth factor (NGF), brain derived neurotrophic factor (BDGF), basic fibroblast growth factor (bFGF) and glial cell line derived growth factor (GDGF).

b. Gene constructs

Typically the gene of interest is cloned into an expression vector. As used herein, the term "expression vector" refers to a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to a nucleotide sequence that encodes the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression. Regulatory elements include elements such as a promoter, an initiation codon, a stop codon and a polyadenylation signal.

Expression of the cloned sequences occurs when the expression vector is introduced into an appropriate host cell. In this case, the preferred host cell is a neuronal cell. Procedures for preparing expression vectors are known to those of skill in the art and can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).

IV. Assay

Another aspect of the invention provides an assay for evaluating the effect of substances on differentiated cells, preferably differentiated neuronal cells. The assay can be used to discover drugs capable of regulating the survival, proliferation or genesis of neuronal cells. According to this aspect of the invention, a population of neuronal cells is produced by the cell culturing method described above. The population of cells is contacted with a substance of interest and the effect on the cell population is monitored. The impact on the cell population can be monitored, for example, by determining whether the substance causes an increase or decrease in the expression of a reporter gene by examining the level of its protein, RNA, biological activity or other methods. For example, in one immunocytochemical method, the dopaminergic cells are monitored to determine the impact of a substance on the expression of tyrosine hydroxylase.

Substances of interest include extracts from tissues or cells, conditioned media from primary cells or cell lines, polypeptides whether naturally occurring or recombinant, nucleotides (DNA or RNA) and non-protein molecules whether naturally occurring or chemically synthesized.

Claim 1 of 23 Claims

What is claimed is:

1. A method of generating a cell culture comprising dopaminergic neuron cells, said method comprising the sequential steps of:

a. providing precursor cells comprising human or rat fetal central nervous system cells;

b. proliferating precursor cells, said step of proliferating comprising:

i. incubating a suspension of said precursor cells in a proliferating medium which includes basic fibroblast growth factor (bFGF) to form proliferated precursor cells; and subsequently

c. differentiating said proliferated precursor cells, said step of differentiating comprising:

i. incubating said proliferated precursor cells in an incubation vessel which contains differentiation medium in a manner effective to form a reaggregation of differentiated dopaminergic neuron cells that is not adhered to any surface of the incubation vessel, wherein the differentiation medium includes ascorbic acid.



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