ZA200602494B - Water dispersible film - Google Patents

Description

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TITLE OF INVENTION

[0001] In Vitro Generation of GABAergic Neurons from Embryonic Stem Cells and

Their Use in the Treatment of Neurological Disorders.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0002] Not applicable.

STATEMENIT REGARDING FEDERALLY SPONSORED

RESEARCH OR DEVELOPMENT

[0003] Not applicable.

REFERENCE TO A “Microfiche Appendix”

[0004] Not applicable.

BACKGROUND OF THE INVENTION L. FIELD OF THE INVENTION

[0005] The present disclosure relates to an improved method of producing terminally differentiated neuronal cells such as GABAergic neurons from pluripotent stem cells such as murine embryonic stem cells or human embryonic stem cells. The GABAergic neurons generated according to the present disclosure may serve as an excellent source for cell replacement therapy~ in neurodegenerative disorders and neuronal diseases such as, for example, stroke, ischemia, Parkinson’s disease, Alzheimer’s disease, epilepsy, and

Huntington's disease. 2. DESCRIPTION OF RELATED ART

[0006] Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the cemtral nervous system (CNS), and is widely distributed throughout the brain and expressed in interneurons modulating local circuits.

GABAergic neurons, which produce GABA, are the predominant inhibitory neurons in the mammalian CNS, and approximately 60-75% of all synapses in the CNS are

GABAergic (Schwartz, R.D ., 1988, Biochem. Pharmacol. 37:3369-75). GABAergic neurons are localized in the hippocampus, cerebellum, cerebral cortex, and hypothalamus, and GABA binds to at least three receptors, including GABA-A and

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GABA-B. GABA-A receptors medi ate fast inhibitory synaptic transmissions, neuronal excitability, and rapid changes in mood, such as seizure threshhold, anxiety, panic, and

Tesponse to stress (i.e, the “fight or flight” response). GABA-A receptors are also binding sites for benzodiazepines, ethanol, barbiturates, and neurosteroids. GABA-B receptors mediate slow inhibitory tran_smissions, and may be important in memory, mood, and pain.

[0007] The pathogenesis of several neurological disorders appears to involve a decrease in GABAergic neurotransm-ission, including some forms of epilepsy, chronic pain, anxiety, and other mood disorders. For example, a positron emission tomography (PET) study showed that patients with panic disorder have decreased GABA-A receptor binding (Malizia et al., 1998, Arch. Gen. Psychiatry 55:715-20). In addition, low plasma

GABA may be characteristic of a subgroup of patients with mood disorders (Brambilla ef al., 2003, Mol. Psychiatry 8:721-37). Certain drugs that enhance GABA activity have been shown effective in the treatmeent of these disorders, such as benodiazepines, valproate, and phenobarbital.

[0008] Many diseases of the cen-tral nervous system (CNS) such as Parkinson’s discase, Alzheimer’s disease, Multipele sclerosis, Huntington’s disease, amyotrophic lateral sclerosis, cerebral ischemia, and stroke are characterized by degeneration of neurons in the brain and spinal cord regions. Cells or neurons that degenerate or are otherwise damaged are not intrinsically replaced or repaired by the body, which can lead to permanent and irreversible damage (During ef al, 2001, Human Gene Ther. 12:1589-1591). Stroke or cerebral ischmemia can occur when a blood clot blocks a blood vessel or artery, interrupting blood flow= to an area of the brain, which causes the death of brain cells in the immediate area of thes block. The brain cells in the infarct usually die within minutes to a few hours after thee stroke or ischemia occurs. The death of these cells can lead to a release of chemicals that set off a chain reaction called the “ischemic cascade,” which endangers brain cells in the larger, surrounding area of brain tissue for which the blood supply is compromiseci. Without prompt medical treatment, this larger area of brain cells may also die, which ¢ an cause even more severe and long-term damage to the brain. Given the rapid pace of thee ischemic cascade, the “window of opportunity” for interventional treatment is only about six hours. Beyond this window of time,

4 PV reestablishment of blood flow and administration of neuroprotective agents may fail to help and can potentially cause further damage to brain functions (Padosch et al., 2001,

Anaesthesist 50:905-920; Nishino ancl Borlongan, 2000, Prog. Brain Res. 127:461-476).

[0009] When stroke occurs, the disruption of blood flow to the brain has a detrimental and potentially fatal effect on individual or groups of neurons. Starving large numbers of neurons of oxygen and wital nutrients in a specific area of the brain due to cerebral ischemia can lead to severe loss of functional capabilities in patients. For example, stroke patients may exper&ence loss of speech, memory, cognition, reduced mobility, or even paralysis. Withowat an adequate blood supply, brain cells lose their ability to produce energy, particularly adenosine triphosphate (ATP). If critical thresholds of this energy failure owccur, brain cells are damaged and die. Many researchers believe that an immense riumber of mechanisms cause brain cell damage and death following energy failure, with each of these mechanisms representing a potential route for therapeutic intervention.

[0010] One way brain cells resporad to energy failure is by elevating the concentration of intracellular calcium. These concentrations can be driven to dangerous levels by a process called excitotoxicity, in which brain cells release excessive amounts of glutamate, a neurotransmitter, which. leads to the degradation and destruction of vital cells located in the hippocampus, cortex, and thalamus region of the brain (Nishino and

Borlongan, 2000, Prog. Brain Res. 12.7:461-476). In addition, GABA-producing cells in the hippocampus region of the brain often degenerate after a stroke (Nishino and

Borlongan, 2000, Prog. Brain Res. 127:461-476). Based on neurohistopathological and neuropsychological investigations, several neuroprotective drug therapies have been developed to treat neurological disorders associated with cerebral ischemia or stroke, such as GABAergic agonists, calcium antagonists, glutamate antagonists, and antioxidants (Stutzmann ef al., 2002, CNS Drug Rev. 8:1-30; Rochelle et al., 2001, J.

Neurochem. 77:353-371; Blezer et aé., 2002 Eur. J. Pharmacol. 444:75-81). Currently there are hundreds of drugs and compounds in various stages of development for the prevention and acute interventional treatment of stroke (Rochelle et al, 2001, J.

Neurochem. 77:353-371). It is anticip-ated that several of these drugs will be submitted to the FDA for approval, and many are already engaged in the last phase of clinical trials.

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Among these, GABAergic drugs are found to be exceptionally effective in treating neurological disorders associated with cerebral ischemia or stroke.

[0011] Given the multi-dimensional nature of ischemic brain cell injury, however, stroke experts predict that no single drug-based timerapy will be able to completely protect the brain during and afier a stroke. Since current therapeutic alternatives do not adequately treat damage associated with cerebral ischemia or stroke, there is great interest in developing alternative therapies for v-arious neurodegenerative disorders and neuronal diseases. A cell-based therapy may be the only means available for comprehensively treating the damage caused bey such an event. Many neurological diseases and conditions are caused by the loss of neuronal cells in the brain and spinal cord regions. A wide spectrum of these neurolo gical diseases and conditions, including but not limited to Parkinson’s disease, Alzheimer's disease, Huntington's discase, and spinal cord injury, may be treatable with cell based therapies. For example, patients with

Parkinson’s disease have been successfully treated by transplanting dopaminergic neurons into the brain of affected individu als (Grisolia, 2002, Brain Res Bull 57:823-826). Therefore, when GABA-producsing cells are affected or damaged in cerebral ischemia or stroke patients, the replacement of these damaged GABA-producing cells with new and healthy GABA-producing cells would be an ideal therapy for the treatment of cerebral ischemia or stroke.

[0012] One major problem with cell transplantation as a therapeutic option for neurodegenerative disorders and neuronal diseases is the need for large quantities of neuronal cells, which are difficult to isolate from fetal or adult sources. One solution to this dilemma is the availability of pluripotent stem cells, which can be used to generate unlimited numbers of terminally differentiated cell types. Pluripotent embryonic stem (ES) cells are a viable alternative source of neuronal cells that may be used to treat various neurodegenerative disorders and neuronal diseases, ES cells can proliferate indefinitely in an undifferentiated state and are pluripotent, which means they are capable of differentiating into nearly all cell types present in the body. Because ES cells are capable of becoming almost all of the specialized cells of the body, they have the potential to generate replacement cells for a bxoad array of tissues and organs such as heart, pancreas, nervous tissue, muscle, cartilagze, and the like. ES cells can be derived

LS (he from the inner cell mass (ICM) of a blastocyst, which is a stage of embryo development that occurs prior to implantation. Humman ES cells may be derived from a human blastocyst at an early stage of the developing embryo lasting from the 4" to 7% day after fertilization. ES cells derived from the ICM can be cultured in vitro and under the appropriate conditions proliferate indefinitely.

[0013] ES cell lines have been successfully established for a number of species, including mouse (Evans et al, 1981, Nature 292:154-156), rat (Iannaccone et al., 1994,

Dev. Biol, 163:288-292), porcine (Evans et al., 1990, Theriogenology 33:125-128;

Notarianni ez al, 1990, J. Reprod. Fertil. Suppl. 41:51-6), sheep and goat (Meinecke-Tillmann and Meinecke, 1996, J. Animal Breeding and Genetics 113:413-426; Notarianni et al., 1991, J. R_eprod. Fertil. Suppl. 43:255-60), rabbit (Giles et al., 1993, Mol. Reprod. Dev. 36:130-138; Graves ef al, 1993, Mol. Reprod. Dev. 36:424-433), mink (Sukoyan ef al, Mol. Reprod. Dev. 1992, 33:418-431), hamster (Doetschman ez al., 1988, Dev. Biol. 127:224-227), domestic fowl (Pain ef al., 1996,

Development 122(8):2339-48), primate (U.S. Patent No. 5,843,780), and human (Thomson et al., 1998, Science 282:1145-1147; Reubinoff ef al., 2000, Nature Biotech. 18:399-403). Like other mammalian ES cells, human ES cells differentiate and form tissues of all three germ layers when injected into immunodeficient mice, proving their pluripotency. Published reports show that human ES cells have been maintained in culture for more than a year during whiich time they retained their pluripotency, self- renewing capacity, and normal karyotype (Thomson et.al., 1995, PNAS 92:7844-7848).

[0014] ES cells have been shown to differentiate into neurons and glial cells in both in vitro models (Bain et al., 1995, Dev. Biol. 168:342-357), as well as in vivo models (Brustle, et al, 1999, Science 285:754-56). Similarly, blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation (Deacon et al., 1998, Exp. Neurol. 149:28-41). Human or rodent stems cells are able to differentiate into specific neuronal types when grafted into either a developing central nervous system (Flax et al, 1998, Nat. Biotechnol. 16:1 033-39; Brustle et al, 1998, Nat. Biotechnol. 16:1040-44; Reubinoff et al., 2001, Nat. Biotechnol. 19:1034-40) or neurogenic areas of the adult CNS (Fricker ef al., 1999, J. Neurosci. 19:5990-6005; Shihabuddin ez al., 2000,

J. Neurosci. 20:8727-35).

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(0015) One method for generating GABA-producsing cells from immature neuronal cells has been reported (Rubenstein ez al, U.S. Patent No. 6,602,680, incorporated herein by reference). Rubenstein ef al. reported the production of GABAergic cells by increasing the activity of a DLX gene, for example DLX1, DLX2, or DLXS, in an immature neuronal cells. The increase in DLX activity causes differentiation of the immature neuronal cells into cells with the GABAergi«c phenotype. Methods for deriving

GABA-producing cells from mouse embryonic stem cells have also been reported (Hancock et al, 2000, Biochem. Biophys. Res. Comrmun. 271(2):418-21, Westmoreland et al, 2001, Biochem. Biophys. Res. Commun. 28<4(3):674-80; U.S. Publication No. 2003/0036195 Al, each specifically incorporated herein by reference), but these methods do not generate high percentages of GABAergic raeurons. Since large numbers of

GABAergic neurons are required for cell replacennent therapy, there is a need for : additional in vitro methods for generating large numbers of GABAergic neurons from pluripotent stem cells.

[0016] Methods that can generate high yields owf GABAergic neurons have great clinical significance for cell transplantation therapy, particularly for patients suffering from cerebral ischemia or stroke. To date, available therapies are extremely limited for treating the neuropathology associated with cerebral ischemia and stroke, so there is great interest in developing alternative therapies. Cell-boased therapies will require large numbers of cells or neurons for treatment, which is neot possible if fetal or adult tissue is the only source available for the cells and neurons. For example, about 1 million dopaminergic producing cells must be transplanted into a single Parkinson’s disease animal model to study the functional recovery of motor function (Grisolia, 2002, Brain

Res. Bull. 57:823-826). Obtaining such a large number of cells using fetal material raises many ethical problems. Generation of GABAergic reurons from pluripotent stem cells offers a potentially unlimited supply of GABAergic neurons to use in cell based- therapies. But methods that yield high percentages of GABAergic neurons are necessary to make this source practical, particularly since it is likely that large numbers of

GABAergic neurons will be needed for therapeutic methods utilizing these cells.

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BRIEF SUMMARY OF THE INVENTION

[0017] The present disclosure relates to improved methods of producing neuroprogenitor cells, as well as terminally differentiated neuronal cells or glial cells, from pluripotent stem cells such as embryonic stem (ES) cells. In a preferred . embodiment, the pluripotent stem ‘cells or ES cells are murine cells. The cells generated herein include but are not limited to cells with the phenotypic characteristics of neuroprogenitor cells, neuronal cells such as GABA. ergic, dopaminergic, serotonergic, and glutamatergic neurons, as well as glial cells such as oligodendrocytes and astrocytes.

The present disclosure demonstrates that pluripotent stem cells, for example murine ES cells, can differentiate into a high proportion of GAB.Acrgic neurons (e.g., at least about 60%). The percentage of GABAergic neurons generated according to the methods of the present disclosure is higher than previously described amethods. GABAergic neurons can be utilized for multi-potential cell-based therapies, for example cell replacement therapy, or to treat ncurodegenerative disorders and neuronal diseases, including, for example, stroke, cerebral ischemia, epilepsy, Parkinson’s disease, Huntington’s disease,

Alzheimer’s disease, chronic pain, anxiety, and other mood disorders.

[0018] The present disclosure provides a differentiated cell population in an in vitro culture obtained by differentiating pluripotent stem cells, wherein at least 60% of the differentiated neural cells are GABAergic neurons, cells that exhibit GABAergic neuron phenotypes, or cells that produce gamma aminobutyric acid (GABA). Preferably the

GABAergic neurons express GAD65, GAD67, GABA -A receptor, or GABA-B receptor, or a combination thereof. In other embodiments, at I east about 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the differentiated cells are

GABAergic neurons or produce GABA. Preferably, th € GABAergic neurons or cells that } produce GABA are derived from ES cells, more preferably human or murine ES cells. In other embodiments, the differentiated cell population also comprises at least about 15% dopaminergic neurons, at least about 10% glutamatergic neurons, at least about 5% serotonergic neurons, at least about 5% oligodendrocytes, at least about 5% astrocytes, or a combination of these amounts.

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[0019] The present disclosure also provides methods for generating differentiated cell popuH ations from pluripotent stem cells comprising the following steps: (a) expanding a culture of pluripotent stem cells; (b) culturing the pluripotent stem cells to select for meuroprogenitor cells that are positive for nestin; (©) expanding the nestin-positive neuroprogenitor cells; and (d) differentiating the nestin-positive cells to generate a differentiated cell population by culturing the cells in a differentiation media which comprises cytosine B-d-Arabino furanoside (Ara-C). [0020n] In a preferred embodiment, the pluripotent s€em cells are ES cells, more preferably human or murine ES cells. [0021 } In other embodiments, the above methods further include the step of culturing the pluripotent stem cells of step (b) to form embryofd bodies. Preferably, these embryoid bodies are cultured under conditions which select for ncuroprogenitor cells that are positive for nestin, for example by culturing the pluripotent stem cells or embryoid bodies in serum-free medium, preferably for 6-10 days. Im preferred embodiments, the serum-free medium is ITSFn serum-free defined medium, which preferably includes one or mo-t¢ soluble factors selected from the group consisting of insulin, sodium selenite, basic fibroblast growth factor, transferrin, and fibronectin. Preferably, the ITSFn serum- free defined medium comprises insulin, sodium selenite, transferrin, and fibronectin. In prefermed embodiments, these methods will generate meuroprogenitor cells which preferably comprise at least about 60-75% nestin-positive cells, more preferably about 80-90% nestin-positive cells, and most preferably about 95-99% nestin-positive cells. [0022E In certain embodiments, the above methods further include the step of expancling the nestin-positive neuroprogenitor cells of step (c) in CNS expansion media, preferably for 6-10 days. Preferably, the CNS expansion rmedia comprises one or more soluble factors selected from the group consisting of N2 supplement, B27 supplement, and a mneural-inducing agent. In a preferred embodiment, the neural-inducing agent is basic fibroblast growth factor (bFGF). In another preferred embodiment, the nestin- positiv-e neuroprogenitor cells are plated on a culture dish pre-coated with poly-L-

") “ omithine, poly-L-laminin, or a combination of the two. The nestin-positive neuroprogenitor cells may be expanded in culture and serially passed for one or more population doublings. These cells may also be cryopreserved in liquid nitrogen.

[0023] The nestin-positive neuroprogenitor cells are preferably grown in differentiation media for 2 or more days as set forth in step (d) of the above methods.

Preferably, the differentiation media also comprises N2 supplement, B27 supplement, or both, but not basic fibroblast growth factor (bFGF). In preferred embodiments, after the neuroprogenitor cells are grown in the differentiation media which contains cytosine

B-d-Arabino furanoside (Ara-C), the cells are further cultured in differentiation media without Ara-C, preferably for 8-16 days, more preferably for 12 days.

[0024] The present disclosure also provides methods of generating GABAergic neurons from neuroprogenitor cells, comprising enriching the neuroprogenitor cells for cells that are positive for nestin, and differentiating the mestin-positive cells to generate

GABAergic neurons by culturing the cells in the presermce of Ara-C. After the nestin- positive cells are cultured in differentiation media with ATa-C, they are preferably further differentiated in differentiation media without Ara-C. Preferably, at least about 40-99% of the nestin-positive cells differentiate into GABAergic raeurons using these methods.

[0025] In preferred embodiments, the methods disclosed above are used to generate differentiated cell populations, which preferably comprise about 60-80% GABAergic neurons, more preferably about 75-90% GABAergic neurons, and most preferably about 95-99% GABAergic neurons. In other embodiments, the-se methods are used to generate differentiated cell populations, which preferably comprise about 15-30% dopaminergic + neurons, more preferably about 20-40% dopaminergic neurons, and most preferably about 25-50% dopaminergic neurons. In certain embodinnents, these methods are used to generate differentiated cell populations, which preferably comprise about 5-20% serotonergic or glutamatergic neurons, more preferably about 10-25% serotonergic or glutamatergic neurons, and most preferably about 15-30%4% serotonergic or glutamatergic neurons. In other embodiments, these methods are used to generate differentiated cell populations, which preferably comprise about 5-20% oligodendrocytes or astrocytes,

v i more preferably aabout 10-25% oligodendrocytes or astrocytes, and mo st preferably about 15-30% oligodenedrocytes or astrocytes.

[0026] The present disclosure further provides an in vitro transplantation model to study the efficacy, survivability, and functionality of differentiated cells, preferably neuronal or neural cells, in a host-like environment, such as a brain environment. For example, GABA ergic neurons described herein are cultured with adult brain cells, preferably neural or hippocampal cells. Preferably the GABAergic meurons are plated onto the adult hippocampal cells. The cells are cultured together for at least 3-20 days, more preferably 71 week, and the survival of the neuronal or neural cells is determined.

Preferably at least about 80% of the neuronal or neural cells survive, more preferably 90-99% of the cells survive. A high survival rate indicates that the cells are likely to function in an aciult brain environment, and may be used to treat neurodegenerative disorders or neuronal diseases.

[0027] The p resent disclosure also provides methods for treating subjects with neurodegeneratives disorders or neuronal diseases by administering to a subject neuroprogenitor c=ells or differentiated neuronal cells derived from pluripotent stem cells, for example muririe or human ES cells, as described herein. The cells clerived herein may also be used for «cell replacement therapy in the subject. For example, a differentiated neuronal cell population may be derived as follows: (a) expancling a culture of pluripotent stem cells; (b) culturing the pluripotent stem cells to select for neuroprogenitor cells that are positive for nestin; (c) expanding the nestin-positive neuroprogenitor cells; and (d) differemtiating the nestin-positive cells to generate a differeratiated cell popula_tion by culturing the cells in a differentiation media which comprises cytosirae B-d-Arabino furanoside (Ara-C),

[0028] In another preferred embodiment, the differentiated cells of step (d) are further differentiated in clifferentiation media without Ara-C. In preferred embodiments, the subject is a patiemt, more preferably a human patient. Preferably tlhe neuroprogenitor cells or differeratiated neuronal cells derived from pluripotent stem cells are

Vo histocompostible with the subject, for example if the neuroprogenitor cells or * differentiaated neuronal cells have essentially the same genome as the subject.

[0029] In certain embodiments, GABAergic, dopaminergic, serotonergic, and glutamatergic neurons, as well as glial cells such as oligodendrocytes and astrocyte=s, are isolated from the differentiated neuronal cell population and administered to the patient.

In a prefered embodiment, GABAergic neurons are administered to the subject. These cells, as well as neuroprogenitor or differentiated ncuronal cell populations, can be administered to the subject to treat a variety of neurodegenerative disorders or neuronal diseases, including but not limited to stroke, cerebral ischemia, epilepsy, Parkinsson’s disease, Fluntington’s disease, Alzheimer’s disease, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, and other CNS disorders, as well as chronic “pain, anxiety, amd other mood disorders. These subjects may also be treated by cell replacement therapy. Preferably the cells are administered by transplantation, for example by transplanting the desired cells into the brain of the subject.

[0030] Another embodiment of the present disclosure is a method of freatimg a subject with a neurodegenerative disorder or neuronal disease comprising the following steps: (2) «expanding a culture of pluripotent stem cells; (b) «culturing the pluripotent stem cells to select for neuroprogenitor cells that mare positive for nestin; (c) expanding the nestin-positive neuroprogenitor cells; (d) differentiating the nestin-positive cells to generate a differentiated neural ceell population by culturing the cells in a differentiation media which comprise Ss cytosine f-d-Arabino furanoside (Ara-C); and (e) txansplanting a therapeutically effective amount of the differentiated neural cell population into the central nervous system of the patient.

[0031] Im a preferred embodiment, the pluripotent stem cells are murine or human ES cells. In other preferred embodiments, subject is a patient, more preferably a hurman patient. Pre-ferably the differentiated neural cell population is histocompatible with the patient. In another embodiment, step (d) further comprises differentiating the cells in the

>, IE differentiation media for 2 or myore days, and subsequently differentiating the cells in a second differentiation media that does not contain cytosine f-d-Arabino furanoside (Ara-C). In preferred emboddiments, GABAergic neurons are isolated from the differentiated neural cell population and administered to the subject, for example to the brain of the subject, preferabsly by transplantation. In certain embodiments, the neurodegenerative disorder or meuronal disease is selected from the group consisting of

Parkinson’s disease, Alzheimers disease, Huntington’s disease, Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressive supranuclear palsy, spinal cord injury, amyotrophic lateral sclerosis (A 1S), epilepsy, stroke, and ischemia.

[0032] In other embodiments, the neuroprogenitor cell population or differentiated neuronal cells derived from pluripotent stem cells as described herein can be used to screen compounds, for example small molecules and drugs, for their effect on the cell population, particular differentiated neural or glial cells, or the activity of these cells.

The compounds can also be screzened for neural cell toxicity or modulation. For example, a compound can be evaluated by adding the compound to a population of differentiated. neural cells, such as GABAexgic neurons, and comparing the survival, morphology, phenotype, functional activity, or other characteristics of the cells with differentiated neural cells cultured under similar conditions but not exposed to the compound. The compounds can be screened, for example, to determine whether they effect changes ira neurotransmitter synthesis, rele ase, or uptake by the cells.

[0033] Another embodiment of the present disclosure is a method of generating an irz vitro transplantation model for neural cells, comprising the following steps: (a) isolating adult hippocampal cells; (b) dissociating and culturing the hippocampal cells to generate = hippocampal cell culture; and (¢) culturing neural cells on the hippocampal cell culture; wherein the survival of the neural cells on the hippocampal cell culture is evaluated. In other embodiments, synaptic formation between the neural cells and the hippocampal cel culture are evaluated. In one embodiment of the present disclosure, the adult hippocampal cells are isolate from a mouse. In certain embodiments, the neural cellls wv cultured on the hippocampal cell cul ture are, for example, GABAergic, dopaminergic, serotonergic, or glutamatergic neurons. In other embodiments, glial cells such as oligodendrocytes and astrocytes are cultured on the hippocampal cell culture. The neural cells or glial cells cultured on the hippocampal cell culture may be derived using the methods disclosed herein, or by derived or isolated by other methods well known to those of skill in the art. Preferably, at least about 50% of the neural cells cultured on the hippocampal cell culture survive for at least a week, more preferably about 60%, 70%, 80%, 90%, or 95%. In one preferred embodiment, greater than about 90% of GABAergic neurons survive after one week of cul ture.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0034] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by» reference to one or more of these drawings in corabination with the detailed descripstion of specific embodiments presented herein.

[0035] Figure 1 shows a diagrammatic representation of the derivation of

GABAergic neurons from murine ES cells, as well as in vitro transplantation of the

GABAergic neurons with hippocampeal neurons derived from adult murine brain.

[0036] Figure 2 shows a diagrammatic representation of the steps for murine ES cell differentiation into terminally differentiated neuronal cells, which includes: (1) expansion of undifferentiated cells; (2) formation of embryoid bodies; (3) selection of nestin- positive cells; (4) expansion of nesstin-positive cells; and (5) differentiation of neural progenitors into terminally differenti ated neuronal cells.

[0037] Figure 3 shows the locali zation of the following markers by immunoreactivity in the neuronal population derived firom murine ES cells: (2) GAD-65 and GAD-67; (b)

GAT-1 and GAT-2; (c) Glutamate and GABA; and (d) Nestin and MAP-2. The immunoreactivity was studied in 4 different stages: expansion of nestin-positive cells, and 8, 12, and 16 days after differentiation.

[0038] Figure 4 shows co-localization of GABA and GAD-65/GAD-67 immunoreactivity in GABAergic meurons derived from murine ES cells using the methods disclosed herein. The presence of GABA immunofluorescence in the GAD-65 and GAD-67 positive cells confirms that these GABAergic neurons produce GABA.

[0039] Figure 5 shows the gene expression profile of GABAergic-neuron-specific factors in murine ES cells, and at various stages during the in vitro differentiation of these cells into GABAergic neurons. These stages were undifferentiated murine ES cells (Un), embryoid bodies (EB), nestin-selection (N), nestin-expansion (NE), 2 days of differentiation (D-2); 4 days of differentiation (D-4); 8 days of differentiation (D-8); 12 days of differentiation (D-12); and 16 days of differentiation (D-16). The expression of the following GABAergic neuron specific genes were analyzed: GADI1, GAD2, VIAAT, and GAD | embryonic transcripts. GADI expression was observed in all stages, including in the undifferentiated ES cells. GAD2 expression was observed in the nestin- selection, nestin-expansion, and all differentiated stages, but no expression was present in the undifferentiated and embryoid bodies stages. VIAAT expression was observed only in the differential stages, particularly day 8 and day 12 of differentiation. Interestingly,

VIAAT was not expressed after 16 days of differentiation. The expression of the GAD1 embryonic gene was observed in all stages except in the undifferentiated stage. [3-actin, a housekeeping gene, was detected in all stages as a positive control.

[0040] Figure 6 is a comparative analysis of neuronal populations derived from murine ES cells. The total numbers of cells at different stages of derivation were counted using morphometrical analysis. The stages of derivation analyzed were expansion of (a) nestin-positive cells (NE); (b) 8 days of differentiation; (c) 12 days of differentiation; and (d) 16 days of differentiation. The cells were quantified according to: (1) total neurons; (2) glutamate expression; (3) GABA expression; (4) tyrosine hydroxylase (TH) expression; and (5) HT expression.

[0041] Figure 7 shows extracellular GABA levels as determined by Reverse Phase

High Performance Liquid Chromatography (RP-HPLC) in the following stages: nestin-expansion (NE); 8 days of differentiation (D-8); 12 days of differentiation (D-12); and 16 days of differentiation (D-16). GABA was only detected in the differentiated stages, and confirms that the neurons derived from murine ES cells using the methods disclosed herein produce GABA,

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[0042] Figure 8 shows immunoreactivity of cells derived from murine ES cells using the methods disclosed herein after 12 days of differentiation with anti-GABA-A receptor antibodies. Approximately 80% of the cells were positive for immunofluorescence with anti-GABA-A receptor antibodies.

[0043] Figure 9 shows immunoreactivity of cells derived from murine ES cells using the methods disclosed herein after 12 days of differentiation with anti-GABA-B receptor antibodies. Approximately 25% of the cells were positive for immunofluorescence with anti-GABA-B receptor antibodies.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present disclosure provides methaods for the efficient generation of cells of neural lineage that are differentiated from pluripotent stem cells. The cells generated herein include but are not limited to cells wwith the phenotypic characteristics of neuroprogenitor cells, GABAergic, dopamine=rgic, serotonergic, and glutamatergic neurons, as well as glial cells such as oligodendrocytes and astrocytes. Cells generated herein are identified by phenotypic characteristics, morphological characteristics, and/or cell markers, which are readily appreciated by th ose of skill in the art of evaluating such cells. As used herein, the term “neuroprogenitor cells” is interchangeable with the terms neural or neuronal progenitor cells, as well as neural or neuronal precursor cells, and refers to a cell that can generate progeny that ares either neuronal cells, such as neuronal precursors, neural cells, or neurons, or glial cells, such as glial precursors, astrocytes, or oligodendrocytes. The methods disclosed herein involve culturing cells in a combination of soluble factors and environmental conditions which encourage the cells to differentiate into cells of neural lineage. The methods disclo sed herein are preferably used to derive

GABAergic neurons from pluripotent stem cells.

[0045] These precursor and differentiated nesural cells can be used for a number of applications, including therapeutic and experimental applications, as well as in vitro drug development and screening, such as screening a scompound for neural cell toxicity or the ability to modulate the function of neuronal cells. Generation of precursor and differentiated neural cells such as GABAergic neurons, as well as other specialized neuronal cell types, from pluripotent stem cells -offers a potentially unlimited supply of these neurons, with tremendous potential benefit to individuals suffering from debilitating neurodegenerative disorders and neuronal diseases, including but not limited to stroke, ischemia, Huntingtons disease, epilepsy, chronic pain, anxiety, and other mood disorders. The precursor and differentiated neural cells described herein are typically the progeny of the cell population from which they were derived, and therefore will have essentially the same genome as the parent population, including a parent population that has been genetically altered, transformed, or transfected.

[0046] A preferred embodiment of the present disclosure is directed to improved methods for generating GABAergic neurons from pluripotent stem cells, preferably mammalian embryonic stem (ES) cells or mammalian embryonic germ (EG) cells. In particularly preferred embodiments, the mammalian ES or EG cells are murine or human

ES cells or EG cells. These neurons are derived from pluripotent stem cells by culturing the cells in the presence of certain soluble factors and environmental conditions.

[0047] As used herein, the term “GABAergic neurons” refer to neuronal cells that express, produce, or secrete the neurotransmitter GABA. Preferably, the terminal differentiation of GABAergic neurons involves the activation and regulation of the genes required for GABA synthesis, as well as vesicular packaging and release. In other preferred embodiments, GABAergic neurons express both the GABA-A and GABA-B receptors. GABA belongs to the chemical family glutamic acid decarboxylase (GAD), and GAD is the key enzyme in the synthesis of GABA. Mammalian species express two isoforms of GAD designated GAD] and GAD2, which are expressed at various levels in different brain regions. GAD] and GAD2 are also known as GAD67 and GADG5 respectively, which is indicative of their relative molecular masses in kDa. Since GAD65 and GAD67 are the enzymes that synthesize GABA, they both can be used as markers for identifying GABAergic neurons. In addition, the vesicle inhibitory amino acid transporter (VIAAT) is required for the synaptic packaging of GABA, and is also a marker for identifying GABAergic neurons. The pathogenesis of several neurodegenerative disorders and neuronal discases appears to involve the loss of

GABAergic neurons or a decrease in GABAergic neurotransmission, including some forms of stroke, ischemia, Huntingtons disease, epilepsy, chronic pain, anxiety, and other mood disorders.

[0048] The present disclosure is directed to improved methods of clifferentiating pluripotent stermn cells into neuroprogenitor cells, as well as into a differentiated population of neeural cells having phenotypic, molecular, and/or cellular characteristics similar to cells of neural lineage. In a preferred embodiment, the pluripoteent stem cells are murine or Iauman ES cells, which are differentiated into neural cells, preferably

GABAergic neumrons, using specific culture conditions. The present disclosure also relates to cells and cell populations produced by the disclosed methodss. In certain embodiments, th ¢ disclosed methods comprise the following steps: 1. A population of pluripotent stem cells are isolated; the pluripotent sstem cells are preferably murine or human ES cells. 2. The plurlipotent stem cells are expanded to provide sufficient starting= material. 3. The pluripotent stem cells are cultured in suspension to generamte embryoid bodies. 4. The embryoid bodies are replated on a substrate and incubated in a serum-free medium wwhich selects for neuroprogenitor cells. 5. The neuroprogenitor cells are expanded in an expansion medium, which comprisess soluble factors related to the nervous system. 6. The neuroprogenitor cells are differentiated into mature neurons in dill fferentiation medium, preferably the medium comprises a combination of soBuble factors related tO the nervous system, as well as cytosine B-d-Arabinc> furanoside (Ara-C).

[0049] Sourcees of Pluripotent Stem Cells

[0050] The meethods disclosed herein for the differentiation of cells of ne=ural lineage from pluripotent sstem cells involve the use of specific culture conditions, which direct differentiation of a remarkably high proportion of pluripotent stem cells into specific neuronal cell type=s. Pluripotent stem cells are derived from pre-embryonic, embryonic, or fetal tissues an _y time after fertilization, which, under the appropriate cornditions, are able to differentiate into several different cell types that are derivatives of alll three germ layers (endoderm, mesoderm, and ectoderm). Cells of neural lineage can also be derived from stem cells isolated from fetal or adult tissue that have the capacity to dififerentiate or

. a. be reprogrammed into cells of neural lineage. Pluripotent stem cells include but are not limited to mammalian ES cell and JEG cells, preferably murine ES or EG cells, or primate or human ES cells and EG cells. Preferably, the undifferentiated pluripotent stem cells have the capacity to divide and proliferate indefinitely in culture. As used herein, the term “differentiation” refers to a process whereby undifferentiated pluripotent stem cells or precursors cells acquire a more specialized fate. For example, a differentiated cell has a phenotype which is characteristic of a particular cell type or tissue.

[0051] In a preferred embodiment, the ES cells and ES cell lines used herein are derived from the inner cell mass of a blastocyst. These blastocysts may be isolated from recovered in vivo fertilized preimplantation embryos, or from in vitro fertilization (IVF), for example embryos fertilized by~ conventional insemination, intracytoplasmic sperm injection, or ooplasm transfer. Hurman blastocysts are obtained from couples or donors who voluntarily donate their surplus embryos. These embryos are used for research purposes after acquiring written arad voluntary consent from these couples or donors.

Alternatively, blastocysts may be de rived by transfer of a somatic cell or cell nucleus into ‘an enucleated oocyte of human or non-human origin, which is then stimulated to develop to the blastocyst stage. The blastocysts used may also have been cryopreserved, or result from embryos which were cryopres erved at an earlier stage and allowed to continue to develop into a blastocyst stage embryo. The development of both the blastocyst and the inner cell mass will vary according to the species, and are well known to those of skill in the art.

[0052] Murine ES cells may be derived in vitro from preimplantation embryos such as blastocysts using techniques well known to those of skill in the art, such as standard immunosurgery techniques (Evans ez al., Nature 292:154-159, 1981; Martin, Proc. Natl.

Acad. Sci. USA 78:7634-7638, 1981 ,, each incorporated herein by reference). Mouse EG cells may be derived from fetal germ cells, again using methods well known to those of skill in the art (Matsui et al., Cell 7€0:841-847, 1992, incorporated herein by reference).

To maintain mouse ES cells in an undifferentiated state, the cells are preferably cultured in the presence of leukemia inhibitory factor (LIF) on fibroblast feeder layers (Williams et al., Nature 336:684-687, 1988, incorporated herein by reference).

[0053] Primate or human ES cells may be derived from a blastocyst using standard immunosurgery techniques as disclosed in U.S. Patent Nos. 5,843,780 and 6,200,806,

Thomson et al. (Science 282:1145-1147, 1.998) and Reubinoff et al. (Nature Biotech. 18:399-403, 2000), each specifically incorporated herein by reference. Although ES cells derived in any number of the ways known to one of skill in the art can be used in the disclosed methods, a preferred embodiment uses human ES cells derived by a unique method of laser ablation (U.S. Serial No. 1(3/226,711, specifically incorporated herein by reference). In brief, this method isolates ¢ ells from the inner cell mass of a blastocyst through laser ablation of part of the zona pellucida and trophectoderm of the blastocyst, which forms an aperture or hole in the blastocyst through which cells of the inner cells mass can be aspirated. These cells can them be further cultured to establish ES cell lines.

This technique is advantageous because it allows the isolation of cells of the inner cell mass without undergoing the conventional cumbersome procedure of immunosurgery. In addition, ES cell lines generated using this technique, in particular human ES cell lines, can be isolated in the absence of any amimal generated antibodies and sera, which minimizes the risk of any transmission of animal microbes to the ES cell lines. In another embodiment, human EG cells are used that are derived from primordial germ cells present in human fetal material (U.S. Patent No. 6,090,622, and Shamblott et al., 1998, Proc. Natl. Acad. Sci. USA. 95:13726-13731, each specifically incorporated herein by reference).

[0054] Preferably, ES cell lines can bes maintained in culture in an undifferentiated state for a prolonged period of time, for excample over one year, and maintain a normal euploid karyotype. Human ES cells may bse morphologically identified by high nucleus to cytoplasm ratios, prominent nucleoli, and compact colony formation, with often distinct cell borders and colonies that are often flatter than mouse ES cells. Human ES cells are also preferably immunoreactive wi-th markers for human pluripotent ES cells, for example SSEA-3, SSEA-4, GCTM-2 antigen, and TRA 1-60, as described by Thomson et al. (1998), Reubinoff et al. (2000), B-uehr and Mclaren (1993), each specifically incorporated herein by reference. Preferably the human ES cells also express alkaline phosphatase, as well as OCT-4. In other embodiments, ES cells are able to form embryoid bodies under non-adherent culture conditions (U.S. Patent Nos. 5,914,268 and

6,602,711, each incorporated herein by reference). These embryoid bodies can be used to derive differentiated derivatives of the endoderm, mesoderm, and ectoderm germ layers, as well as other desired cell lineages.

[0055] Pluripotent stem cells, particularly ES or EG cells, can be propagated continuously under culture condifions that maintain the cells in a substantially undifferentiated state. ES cells must be kept at an appropriate cell density and repeatedly dissociated and subcultured while fr equently exchanging the culture medium to prevent them from differentiating. For genesral techniques relating to cell culture and culturing

ES cells, the practitioner can refer to standard textbooks and reviews, for example: E. J.

Robertson, “Teratocarcinomas and ewnbryonic stem cells: A practical approach” ed., IRL

Press Ltd. 1987; Hu and Aunins, 1997, Curr. Opin. Biotechnol. 8(2):148-53; Kitano, 1991, Biotechnology 17:73-106; Spier, 1991, Curr. Opin. Biotechnol. 2:375-79; Birch and Arathoon, 1990, Bioprocess Technol. 10:251-70; Xu et al., 2001, Nat. Biotechnol. 19(10):971-4; and Lebkowski et al., 2001, Cancer J. 7 Suppl. 2:583-93; each specifically incorporated herein by reference.

[0056] Traditionally, ES cells are cultured in ES medium on a layer of feeder cells.

Feeder cell layers are cells of one tEssue type that are co-cultured with ES cells, and provide an environment in which the ES cells may grow without undergoing substantial differentiation. Methods for culturings ES cells on feeder layers are well known to those of skill in the art (U.S. Patent Nos. 5,843,780 and 6,200,806, WO 99/20741, U.S. Serial

Nos. 09/530,346 and 09/849,022, WO 01/51616, each specifically incorporated herein by reference). The feeder layer preferably reduces, inhibits, or prevents differentiation of ES cells. Feeder layers are typically an ermbryonic fibroblast feeder layer of either human or mouse origin, for example mouse embryonic fibroblasts, human embryonic fibroblasts, human fibroblast-like cells or mesenchymal cells derived from human embryonic stem cells, or STO cells.

[0057] ES cells are preferably cultumred in the presence of ES medium, which reduces, inhibits, or prevents the differentiation of the ES cells. Preferably, ES medium used to culture ES cells is supplemented with a nutrient serum, for example a serum or serum- based solution that supplies nutrients ef fective for maintaining the growth and viability of

ES cells. The nutrient serum may be animal serurm such as fetal bovine serum (FBS) or fetal calf serum (FCS) (U.S. Patent Nos. 5,<453,357, 5,670,372, and 5,690,296, incorporated herein by reference). As used hereira, FBS may be used in place of FCS, and vice versa. The ES medium may also be seruzm-free (WO 98/30679, WO 01/66697,

U.S. Serial No. 09/522,030, each specifically incorporated herein by reference). An example of suitable ES medium with serum for cul turing ES cells is Dulbecco's modified

Eagle's medium (DMEM), without sodium pyruvate, with high glucose content (70-90%) (GIBCO), supplemented with FBS or FCS (10—30%), f-mercaptoethanol (0.1 mM), non-essential amino acids (1%), and L-Glutamine 22 mM, 4 ng/ml basic fibroblast growth factor (bFGF), 50 U/ml penicillin, and 50 pg/ml stmeptomycin. The ES medium may also include 1000 U/ml of Leukemia inhibitory factor LIF). An example of suitable serum- free ES medium for culturing ES cells is 80% “KmockOut” Dulbecco's modified Eagle's medium (DMEM) (GIBCO), 20% KnockOut SR (a serum-free replacement, GIBCO),

B-mercaptoethanol (0.1 mM), non-cssential amino acids (1%), and L-Glutamine 1 mM.

[0058] ES cells may also be cultured under fe eder-free culture conditions. Methods for culturing ES cells in a feeder-free culture are “well known to those of skill in the art (U.S. Publ. No. 2002/0022268, WO 03/020920, U.S. Serial No. 10/235,094, each specifically incorporated herein by reference). ES cells in a feeder-free culture are preferably grown on a suitable culture substrate, for example an extracellular matrix, such as Matrigel® (Becton Dickenson) or laminin. Feseder-free cultures also preferably use conditioned medium to support the growth of ES cells. Conditioned medium is prepared by culturing a first population of either murine embryonic fibroblasts or human embryonic fibroblast cells in a medium for a sufficient period of time to produce “conditioned” medium, which will support the cielturing of ES cells without substantial differentiation. Alternatively, the feeder-free cultumre can combine an extracellular matrix with an effective medium that is added fresh to thee culture without being conditioned by another cell type (U.S. Publ. No. 2003/0017589, specifically incorporated herein by reference).

[6059] Preparation of Neuroprogenitor Cells

[0060] Isolated pluripotent stem cells may be expande=d and then subjected to culture conditions that cause them to differentiate into neuroprogenitor cells, For pluripotent stem cells to advance along the neural differentiation pmathway, the cells are cultured according to differentiation protocols disclosed herein. The pluripotent stem cells are cultured om a suitable substrate in a differentiation nutrient medium that contains differentiat-ion agents such as soluble factors and growth factors. Suitable substrates include bu are not limited to solid surfaces coated with =a positive-charge, for example poly-L-lysi me or polyomithine, substrates coated with exstracellular matrix components, for example fibronectin, laminin, PDGF, EGF, collagen \o/, human amniotic membrane, or Matrigel ®, or a combination thereof. Preferred differentiation nutrient mediums are those that ssupport the proliferation, differentiation, and ssurvival of desired neural cell types, and mmay include one or more suitable differentiation agents. As used herein, the term “growth factor” refers to proteins that bind to receptomrs on the cell surface with the primary ressult of activating cellular proliferation and differentiation. Suitable soluble factors inclcade but are not limited to neurotrophins, mitog=ens, stem cell factors, growth factors, differentiation factors (e.g, TGF-B Superfamily), TGF-p Superfamily agonists, neurotrophic factors, antioxidants, neurotransmitters, and smarvival factors. Many soluble factors are cyuite versatile, stimulating cellular division in riumerous different cell types, while others: are specific to particular cell types.

[0061] Suitable differentiation agents that specifically encourage the differentiation of neuronal cell types include but are not limited to progesterone, putrescine, laminin, insulin, sodi=uim selenite, transferrin, neurturin, sonic hedgeh_og (SHH), noggin, follistatin, epidermal growth factor (EGF), any type of fibroblast growth factor, cytosine

B-d-Arabino furanoside (Ara-C), growth and differentiationm factor 5 (GDF-5), members of the neur-otrophin family (nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin. 4 (NT-4), brain derived neurotropic factor (B.DNF)), transforming growth factor a (TG-F- a), transforming growth factor beta-3 (TGF B3), platelet-derived growth factor (PDG*F-AA), insulin-like growth factor (IGF-1), “bone morphogenic proteins (BMP-2, BMYP-4), glial cell derived neurotrophic factor (GD®NF), midkine, ascorbic acid,

= 2006/C 2498 dibutyryl cAMP, dopamine, and ligamds to receptors that complex with gp130 (e.g., LIF,

CNTF, SCF, IL-11, and IL-6). As used herein, the term “fibroblast growth factor” or “FGF” refers to any suitable fibroblast growth factor, derived from any organism that expresses such factors, and functional fragments thereof. A variety of FGFs are known to those of skill in the art, and include but are not limited to, FGF-1 (acidic fibroblast growth factor), FGF-2 (basic fibroblast growth factor), FGF-3 (int-2), FGF-4 (hst/K-

FGF), FGF-5, FGF-6, FGF-7, FGF-8 , and FGF-9. Differentiation nutrient mediums may also contain additives that help sustain cultures of neural cells, for example N2 and B27 additives (Gibco). Preferably, the differentiation agents retinoic acid, 13-cis retinoic acid, and trans-retinoic acid are not used in any of the methods disclosed herein.

[0062] The first step of differentiating the pluripotent stem cells involves inducing the cells to form embryoid bodies. Embryoid bodies are plated directly onto a suitable substrate with or without an extracellular matrix component such as fibronectin or laminin, and cultured in a suitable Fifferentiation nutrient medium adapted to promote differentiation into neuroprogenitor «cells, such as nestin-positive neuroprogenitor cells.

Nestin is a cell marker characteristic of neural precursors cells. In another embodiment, the pluripotent stem cells are first aggregated into a heterogeneous cell population by forming embryoid bodies, for exaample by culturing the pluripotent stem cells in suspension. These cells can be cultwred in nutrient medium with or without serum, as well as with one or more of thes differentiation agents listed above, to promote differentiation of cells in the embryo id bodies. Preferably the pluripotent stem cells are cultured in ES cell medium without L_IF.

[0063] As used herein, the term “embryoid bodies” refer to an aggregation of differentiated cells generated when pluripotent stem cells are grown in suspension culture, or overgrow in monolayer cultures. Embryoid bodies may also have undifferentiated cells in the aggregation of cells. Preferably this aggregation of cells is surrounded by primitive endoderm. EEmbryoid bodies typically contain cells derived from all three germ layers, ectoderm, mes oderm and endoderm. In mature human embryoid bodies, it is possible to discern cells bearing markers of various cell types, such as neuronal cells, haematopoietic cells, diver cells, and cardiac muscle cells. Some cells in mature embryoid bodies can behave functionally like differentiated cells. For example,

active cardiac muscle cells can causse an embryoid body to pulsate. Preferably the differentiation of pluripotent stem cells is controlled so that specific cell types can be obtained for therapeutic purposes.

[0064] The embryoid bodies are eultured until they reach sufficient size or desired differentiation, for example after 3-1 0 days of culture, preferably 4-8 days, and then plated onto a substrate. Preferably the substrate is coated with extracellular matrix components, including but not limited to poly-L-lysine, poly-L-ornithine, laminin, collagen, fibronectin, Matrigel®, or combinations thereof, The embryoid bodies are preferably plated directly onto the substrate without dispersing the cells. The embryoid bodies are then cultured under condllitions to encourage further differentiation of the plated cells into neuronal or CNS precursor cells. For example, the embryoid bodies may be cultured in a serum-free defined medium that is selective for nestin-positive cells, such as ITSFn medium. Alternatively, the expanded pluripotent stem cells can be plated directly on a substrate and cultured im serum-free defined medium to select for nestin- positive cells. Nestin is an in termediate filament protein cxpressed in the neuroepithelium.

[0065] Preferably, the serum-free defined medium used for expansion of embryoid bodies is DMEM:F-12 supplemented -with one or more growth factors selected from the group consisting of progesterone, putrescine, laminin, insulin, sodium selenite, transferrin, fibronectin, FGF, SHH, E-GF, and BDNF. More preferably, the serum-free defined medium is ITSFn medium, wvhich is supplemented with the nutrients insulin, sodium selenite, transferring, and fibronectin. Generally, the cells are grown under these conditions for a period of 5-16 days, more preferably for 7 days. In preferred embodiments, selection with the a bove serum-free defined medium enriches the population of viable nestin-positive acells to about 40-70%, more preferably to about 80%-90%, and most preferably to aborat 95%-99%.

[0066] Next, the neuronal or CNS precursor cells generated are expanded in CNS expansion media. As used herein, thes terms “expand” or “expansion” refer to a process by which the number or amount of celds is increased due to cell growth and division. The term “proliferate” may be used imterchangeably with “expand” or “expansion.”

Preferably the CNIS expansion media comprises a minimal essential medium such as

DMEM/F12, and i s supplemented with additives that help sustain cultures of neural cells, for example N2 ard B27 additives. The CNS expansion media also preferably includes one or more neurall-inducing agents to encourage proliferation of CNS precursor cells and to increase the efficiency of the generation of GABAergic neurons, for example basic fibroblast growth #actor (bFGF), as well as other factors that control G.ABAergic neuron fate during embryeogenesis in vivo. Preferably, the neuronal or CNS precursor cells are grown in the CNS expansion media for 4-10 days. Additionally, the cells are preferably plated on a surfac=e that permits adhesion of neuronal or CNS precursor cells, such as surfaces coated wwith poly-l-lysine, poly-L-omnithine, laminin, collagen, fibronectin,

Matrigel®, or com _binations thereof.

[0067] In one eembodiment, the embryoid bodies are generated fromm murine ES cells by culturing the cells on a bacteriological plate in the absence of feeder cells in an appropriate media . Preferably, murine ES cells are first dissociated for example by exposure to trypsim, followed by scraping and breakdown of the cells irato small clusters.

These clusters are then plated at an appropriate density onto bacteriological dishes that preferably have a mon-adhesive surface, which prevents attachment of the cells, thereby stimulating differentiation and formation of embryoid bodies. The cells are cultured in an appropriate mediu-m, for example ES medium, which preferably contains DMEM with high glucose or kmnockout DMEM supplemented with 10-20% FCS, FBS, or knockout serum replacement, as well as other supplements such as -mercaptoethanol, L-glutamine (2 mM), and antibiotics. The medium is changed at least every other day, and the embryoid bodies awre allowed to grow, preferably for about 4-8 days.

[0068] In anotTher embodiment, the embryoid bodies are generated from human ES cells by culturing sthe cells on a bacteriological plate in the absence of feeder cells in an appropriate media. Preferably the human ES cells are dissociated into clusters and then plated in non-adheerent plates to facilitate the develcpment of embryoid bodies. The appropriate media preferably contains DMEM with high glucose and is supplemented with 10-20% FCS. Other supplements may also be added to the media, such as 0.1 mM

‘ ‘oy 2-mercaptoethanol, 2 mM L-glutamine, 50 U/ml of penicillin, and 50 ug/ml of streptomycin. .

[0069] After the embryoid bodies are isolated, the embryoid bodies are replate=d on a culture plate coated with 0.1% to 0.2% gelatin in serum-frec medium for selec=tion of neuroprogenitor and CNS precursor cells, preferably nestin-positive cells. Prefera bly the serum-free medium is am basal medium such as DMEM:F-12, which is supplemented with growth factors. ITSFn medium, which is a medium that selects for nestin-positiv-e cells, contains the basal medi~um DMEM:F-12 (1:1) or IMDM medium, supplemented vith the growth factors insulin, sodium selenite, transferrin, and fibronectin.

[0070] Preferably, these neuroprogenitor or CNS precursor cells, preferably mnestin- positive cells, are next cultured in CNS expansion media which contain neural-inaducing growth factors that se=lect for neuronal precursors, preferably GABAergic neuronal precursors. One exarmple of CNS expansion media contains DMEM/F12 nmedium supplemented with N2, B27, and bFGF. The neuroprogenitor or CNS precursor ceells are also re-plated on anothe=r culture dish pre-coated with extra-cellular matrices, for example poly-L-lysine, poly-L-osrnithine, laminin, collagen, or combinations thereof. Al though not wishing to be bourmd by any particular mechanism, it is believed that these wvarious factors present in the @NS expansion media contribute to the overall increase in the percentage of neuronal cells and further induce differentiation of the GABAergic —meuron phenotype. In preferre-d embodiments, the nestin-positive precursor cells grown. in the

CNS expansion media f=or 5-8 days.

[0071] Differentiation of GABAergic Neurons

[0072] The neuroprcogenitor cells prepared according to the methods disclosed herein can be further differeratiated into high proportions of mature neurons, for emcample

GABAergic neurons, ass well as dopaminergic, serotonergic, and glutamatergic nesurons.

The neuroprogenitor cells can also be further differentiated into glial cells smuch as oligodendrocytes or astarocytes. Terminal differentiation of the neuroprogenitor cer CNS precursor cells is achie—ved by culturing the cells according to differentiation preotocols disclosed herein.

, vy

[0073] Preferably, the nestin-positive neuroprogenitor or CNS cells are expanded 2, 3,4,5,6,7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days in a differentiation media which facilitates the differentiation of the neuroprogenitor or CNS cells into terminally differemtiated neural cells or mature neurons. In addition, Rifferentiation may be facilitate by withdrawing some or all of the factors that promoted the differentiation, proliferation, or both of the neuroprogenitor or CNS cells, such as -FFGF. For example, the expanded neu roprogenitor or CNS cells may be differentiated by cwalturing the cells in a differentiation media containing DMEM:F-12 medium or Neurobasal A medium, supplemented with FCS, N2, B27, or a combination thereof, with or writhout B-FGF. The differentiation m-edia may also contain an array of additional factors £0 enhance terminal differentiation ox GABAergic neuron yield, for example Ara-C. In one preferred embodiment, the neuroprogenitor or CNS cells are cultured in di ferentiation media containing Ara-C for one or more days, and then cultured in differenti ation media without

Ara-C for 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In another preferred embodiment, the neuroprogenitor or CNS cells are cultured in differentiation media that contamins DMEM:F12 media supplemented with N2 and B27 for one day; next cultured in diffe-rentiation media that contains DMEM:F12 media sugoplemented with 5%

FBS, B27, and Ara-C for two days; and finally cultured in the same differentiation media without Ara-C for 2-16 days, preferably 12 days.

[0074] Preferably, a high percentage of the neuroprogenitor cells differentiate into

GABAergic neurons, for example at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, © 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the cells. In one preferred embodiment, thie pluripotent stem cells expanded and differentiated according to the methods disclosed herein give rise to a high percentage (at least about 60%) of

GABAergic neurons. In addition, the GABAergic neurons may be further purified from a population ofS differentiated neural cells by methods well known to those of skill in the art, such as immunolabeling and fluorescence sorting, for example solid phase adsorption, FA_CS, MACS, and the like. Other differentiated neural cells derived herein, for example dopaminergic, serotonergic, and glutamatergic meurons, as well as oligodendrocytes and astrocytes, may also be isolated using similar -methods.

[0075] Uses for Neuroprogenitor Cells and. Differentiated Neural Cells

[0076] The neuroprogenitor cells and diffesrentiated neural cells described herein (e.g., GABAergic, dopaminergic, serotonergic, and glutamatergic neurons, as well as oligodendrocytes and astrocytes) can be utilized for various applications, such as therapeutic applications, as well as for in vitro and in vivo assessment and screening of various compounds such as small molecule druges for their effects on these cells. These cells can also be used to prepare cDNA expression libraries to analyze the expression patterns of these cells, as well as to prepare moxoclonal or polyclonal antibodies that are specific to markers for the particular cells used, using techniques that are well known to those of skill in the art. These cells can also be use therapeutically to the benefit of individuals suffering from debilitating neumodegenerative disorders and neuronal diseases.

[0077] The present disclosure provides for the use of the neuroprogenitor cells and differentiated neural cells described herein to tareat or prevent various neurodegenerative disorders and neuronal diseases in which neurons or glial cells are injured or die in the central nervous system (CNS) or spinal cord. Subjects in need of such therapy will be treated by a therapeutically effective amount of such cells to restore functions in the CNS or peripheral nervous system (PNS). As uased herein, a “therapeutically effective amount” of cells is an amount sufficient to arr-est or ameliorate the physiological effects in a subject caused by the loss, damage, or de generation of neural cells, such as mature neurons (e.g., GABAergic, dopaminergic, amd serotonergic neurons), astrocytes, and oligodendrocytes. For example, these cells could be used therapeutically by transplanting them directly into parenchymal or intrathecal sites of the CNS, depending on the disease or condition being treated.

[0078] These cells may be used to treat acute or chronic damage to the nervous system, as well as debilitating neurodegeneratiive disorders and neuronal diseases, which include disorders or diseases of the nervoums system, including the CNS and PNS.

Neurodegenerative disorders and neuronal diseases include but are not limited to

Parkinson’s disease, Alzheimer’s disease, Huntington's disease, Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressive supranuclear palsy, spinal cord injury,

/

Cl ot amyotrophic lateral sclerosis (ALS), epilepsy, stroke, ischemia injury or trauma to the nervous system, neurotoxic injury, and the like. Certain neurological disorders can also be treated wwith differentiated neural cells derived from plurip otent cells, for example disorders associated with cognition and psychology including but not limited to anxiety disorders, mood disorders, addiction, obsessive-compulsive disorders (OCD), personality disorders, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), ancl schizophrenia.

[0079] Orme embodiment of the present disclosure relates to methods of treating or preventing raeurodegenerative disorders or neuronal diseases characterized by the degeneration or destruction of GABAergic neurons by administra tion of a therapeutically effective amount of GABAergic neurons derived from pluripoterat stem cells, preferably murine or human pluripotent stem cells. Preferably, a human peatient suffering from a neurodegener ative disorder or neuronal disease is treated by engr-afting a therapeutically effective amo=unt of neuroprogenitor cells and differentiated neural cells of the present disclosure inteo the patient. When the patient suffers from cerebral ischemia or stroke, preferably thes administration of a therapeutically effective armount of GABAergic neurons will produce a reduction in the amount or severity of thes symptoms associated with the cerebr ral ischemia or stroke such as memory loss, cogniti ve disorders, or motor disorders.

[0080] The therapeutically effective amount of cells used will lepend on the needs of the subject, the subject’s age, physiological condition and health, the desired therapeutic effect, the sizes of the area of tissue that is to be targeted for therapy, the site of implantation, the extent of pathology (e.g., the level of neurormal degeneration), the chosen route ofS delivery, and the treatment strategy. For example, treatment of a disorder affecting a larger region of the brain could require a larger numbewr of cells to achieve a therapeutic effect when compared to a smaller target region. Cells may also be administered to more than one site in a given target tissue, with mealtiple small grafts of low cell doses. The cells of the present disclosure may be completely dissociated before administration, such as to create a suspension of single cells, or nearly completely dissociated befowre administration, such as to create small aggregatess of cells. The cells may be administered in a manner that allows them to graft or migrate to the intended

: be tissue site and reconstit=ute or regenerate a functionally deficient area. Preferably th_e cells are used for autologoums therapy, thereby minimizing or eliminating immune rejection problems after transpMantation, e.g. histocompatibility with the intended rec=ipient.

Alternatively, the cells =are used for allogenic therapy.

[0081] A suitable range of cells that can be administered to achieve a therampeutic effect can be from abowut 100 to about 1,000,000 neurons, preferably from about _500 to about 500,000 neurons, or from about 1000 neurons to about 100,000 neurons—. The number of cells administered will depend heavily on the number that survives therampeutic administration. Therap=eutic concentrations of neural cells administered to a subject may range from about 10, 7100, 500, 1000, 5000, 10,000, 15,000, 20,000, 25,000, 3 0,000, 35,000, 40,000, 45,0000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 15 0,000, 200,000, 250,000, 3002,000, 350,000, 400,000, 450,000 to about 500,000 cells per microliter of a pharmaceutically acceptable carrier. Ranges of concentrations of celilsin a carrier include, for exzample, 100-50,000 cells/pl, 1000-10,000 cells/ul, 5000-225,000 cells/ul, 15,000-45,000 cells/ul, 20,000-50,000 cells/pl, 55,000-200,000 cells/pl, 100,000-40,000 cells/pul. , 150,000-50,000 cells/pl, etc. The number of cells grafted into a transplant site will also saffect therapeutic efficacy.

[0082] For therapeutic applications, it is often preferable that populatio-ns of precursors or differenti_ated neural cells are substantially pure of any undifferertiated pluripotent stem cells. One strategy for removing pluripotent stem cells from a therapeutic preparation is to transfect the cells with a vector that has a gene whmich is preferentially expressed . in undifferentiated cells, the expression of which selects a_gainst the pluripotent stem c-ells. Suitable promoters that are preferentially expresssed in undifferentiated cells are the telomerase reverse transcriptase (TERT) promoter aed the

OCT-4 promoter. The gene expressed in the vector may for example be lytic to thee cell, such as a toxin, or it ma=y be selected against by the application of an external agent.

[0083] The ability tto generate GABAergic neurons, as well as dopaminergi c¢ and serotonergic neurons, from pluripotent stem cells as disclosed herein is of great clinical relevance for therapeuttically preventing or treating a variety of neurodegenesrative disorders and neuronal cliseases. For example, GABAergic neurons can be used to treat or prevent neurodegenerative disorders and neuronal diseases which are characterized by abnormalities in fast inhibitory synaptic transmissions, neuronal excitability, and rapid changes in mood, such as seizure threshold, anxiety, panic, and response to stress (i.e. the “fight or flight” response), as well as abnormalities in memory, mood, or pain functions. For example, GABAergic neurons can be used to treat or prevent Parkinson’s disease, Alzheimer’s disease, epilepsy, Lewy body dementia, multiple sclerosis, cerebellar ataxia, progressive supranuclear palsy, spinal cord injury, amyotrophic lateral sclerosis (ALS), and Huntington's disease, as well as stroke, ischemia, cerebral ischemia, injury or trauma to the nervous system, neurotoxic injury, and the like. GABAergic neurons can also be used to treat or prevent certain neurological disorders including but not limited to disorders associated with cognition and psychology such as anxiety disorders, mood disorders, addiction, obsessive-compulsive disorders (OCD), personality disorders, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), and schizophrenia.

[0084] Parkinson’s disease is a motor disorder caused by progressive degeneration of dopaminergic-producing cells in the substantia niga of the midbrain. The cell-based therapy of transplanting dopaminergic neurons into the substantia nigra of a patient with

Parkinson’s disease has been found therapeutically effective, but symptomatic relief is incomplete (Lindvall, O., 1 997, Neuroreport. 8( 14):iii-x). Therefore, the transplantation of dopaminergic neurons may not be sufficient to cure Parkinson’s disease, and recent findings suggest that there is another brain region, namely the subthalamic nucleus (STN), involved in the neuropathology of Parkinson’s disease (Bergman et al, 1998,

Trends Neurosci. 21:32-38; Luo ef al, 2002, Science 298:425-29). The subthalamic region of the brain contains both glutamatergic and GABAergic neurons (Nishino ef al, 1988, Japn. J. Pharmacol. 48:331-339). In a patient with Parkinson's disease, neurons located in the STN degenerate along with neurons of the substantia nigra (During et al, 2001, Hum. Gene Ther. 12(12):1589-91). Therefore, glutamatergic and GABAergic neurons may also degenerate in patients with Parkinson’s disease (Luo et al, 2002,

Science 298(11):425-429).

[0085] Recent findings suggest that the introduction of GAD, an enzyme which is critical for the biosynthesis of GABA, into the STN of a Parkinson's model reduces the

. Lf motor abnormalities associated with Parkinson’s disease (Luo et al, 2002, Science 298.425-29). Therefore, GABAergic neurons may also be useful for treating Parkinson’s disease, for example by administering or transplanting GABAergic neurons into the STN.

The GABAergic neurons may be administerec or transplanted alone, or in association or in combination with the administration or tran splantation of dopaminergic neurons in the substantia nigra. The GABAergic neurons may also be used to treat Parkinson’s disease in combination with drugs derived from plant , plant-based extracts, or synthetic sources that have anti-Parkinson’s, anti-neurodegenerative, or neuroprotective activities.

[0086] Alzheimer’s disease involves a deficit of mainly cholinergic cells in the nucleus basalis of the brain. Although celJular administration or transplantation of cholinergic cells is an effective therapy for Alzheimer’s disease, it may not be enough to cure the disease because other neuronal cell t-ypes are also lost in the brain, particularly the hippocampus, of Alzheimer’s patients. For example, Alzheimer’s patient suffer a tremendous loss of memory function, which may be due in part to the loss of hippocampal neurons, the majority of which are GABAergic neurons (Seidl et al., 2001,

Arch. Pharmacol. 363:139-145). In addition, significant loss of GABA content was found in the temporal cortex, occipital cortex, and cerebellum of Alzheimer’s patients (Seidl ef al, 2001). In one embodiment, GABAergic neurons derived from pluripotent stem cells as described herein are administered or transplanted into the hippocampal cortex region of the brain alone or in combina€ion with other neurons such as cholinergic neurons or dopaminergic neurons to treat Alzheimer’s patients,

[0087] There is an absolute need to improve the ability of cells to survive various neurodegenerative disorders and neuronal diseases, including but not limited to

Parkinson’s disease and Alzheimer’s disease, as well as cerebral ischemia and stroke.

Various factors influence neuronal degeneration and death. In a preferred embodiment, factors that induce neuronal degeneration and death, for example extracellular calcium, excessive release of glutamate, or release of oxygen radicals, are blocked before peuroprogenitor cells or differentiated neural cells described herein, for example

GABAergic neurons, are administered or transplanted in the brain of a patient. By blocking or antagonizing these factors at the site of cell administration or transplantation, a higher percentage of cells may survive the procedure.

a

[0088] In other embodiments, the present disclosure wrelates to the co-administration of one oer more neuronal survival factors with neuroproggenitor cells and differentiated neural cells of the present disclosure to treat a neurodegmenerative disorder or neuronal disease. The neuronal survival factor(s) may be administered prior to, in conjunction with, in combination with, or after the administration of thee desired cells. As used herein, a “neuronal survival factor” is any substance which causes neurons (either ir vitro or in vivo) that are contacted with the factor to survive for a pemriod of time greater than would . occur without the presence of the factor. Neuronal survival factors that may be used in the prese=nt therapeutic embodiment include but are not licmited to GABA agonists (e.g., benodiaz-epines, valproate, and phenobarbital), calcium antagonists, glutamate antagonists, antioxidants, tissue plasminogen activator (t-P®A), Glial-derived neurotrophic factor (GSDNF), nerve growth factor (NGF), ciliary neursotrophic factor (CNTF), brain derived meurotrophic factor (BDNF), neurotrophin-3 (NIT-3), neurotrophin-4 (NT-4),

FGF, IL- 1}, TNF, insulin-like growth factor (IGF-1, IGF—2), transforming growth factor beta (TGEF-B, TGF-P1), drugs derived from plant, planat-based extracts, or synthetic sources that have anti-Parkinson’s, anti-stroke, a=nti-cerebral ischemic, anti- neurodeg-enerative, or neuroprotective activities.

[0089] In one preferred embodiment, neuronal surviv-al factors are co-administered with neuronal cells, preferably GABAergic neurons, to treat subjects suffering from cerebral i schemia or stroke. Currently, GABA agonists (e .g., benodiazepines, valproate, and phen-obarbital), calcium antagonists, glutamate antagonists, antioxidants, and other neuroprotective agents or drugs are the chemical agents used to treat cerebral ischemia or stroke. VWhile these drugs may help relieve symptoms asssociated with the neurological disorders of stroke or ischemia patients, they are unawble to cure these disorders.

Considering their critical role in stroke or ischemia patientss, these agents or drugs can be used to tre=at the neurological disorders and diseases associated with stroke and ischemia, in combiraation with GABAergic neurons derived from plLaripotent cells, before, during, or after th- erapeutic administration of the GABAergic neuromns.

[0090] In another preferred embodiment, tissue plassminogen activator (t-PA) is co-admini_stered before, during, or after the therapeutic administration of neuronal cells, preferably~ GABAergic neurons, to trcat subjects suffering from cerebral ischemia or

‘ a; stroke. Recombinant forms of t-PA have been used to remove blood clots or blockage associated with cerebral ischemia or streoke in patients, and is the only FDA approved therapy for cerebral ischemia or stroke. The prognosis of a patient suffering from a stroke or cerebral ischemia is improved by supplying blood as soon as possible to the damaged site(s), preferably the hippocampus, cortex, and thalamus regions of the brain, particularly before administering neuronal cells of any type in the damaged site(s) of the brain. This may be achieved by administering t-PA to the patient, which will improve the survival of the administered neurons im the host environment by unblocking blood vessels, thereby providing sufficient oxygzen and nutrients to the neurons,

[0091] As used herein, the terms “to treat”, “treatment”, or “therapy” refer to both therapeutic treatment and prophylactic or preventative measures. Therapeutic treatment includes but is not limited to reducing or Eliminating the symptoms of a particular disease or disorder, or slowing or attenuating the progression of, or curing an existing disease or disorder. Therefore, those in need of trezatment include those already diagnosed with a neurodegenerative disorder or neuronal disease, as well as those in which a neurodegenerative disorder or neuronal d_isease is to be prevented. The methods of the present disclosure can be used to treat amy mammal in need of treatment, including but not limited to humans, primates, and domestic, farm, pet, or sports animals, such as dogs, horses, cats, sheep, pigs, cattle, rats, mice, etc. A “disorder” is any condition that would benefit from treatment with neuroprogenitor cells, differentiated neural cells, or any type of cell derived according to the methods of the present disclosure. Examples of disorders and diseases that would benefit from treatment with cells of the present disclosure, in particular GABAergic neurons, are Parkinson’s disease, Alzheimer’s disease,

Huntington’s disease, Lewy body dementia, multiple sclerosis, cercbellar ataxia, progressive supranuclear palsy, spinal cord injury, amyotrophic lateral sclerosis (ALS), epilepsy, stroke, ischemia, and the like, as well as disorders associated with cognition and psychology including but not limited to anxiety disorders, obsessive-compulsive disorders (OCD), personality disorders, att ention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), and schizophrenia.

[0092] The methods of present disclosure may be advantageously carried out by direct administration of neuroprogenitor cells or differentiated neural cells of the present

V0 2005/021704 PCT/IB2004/002847 disclosure to the lesioned area. Methods of neurormal transplantation and cell culture are we=1l known to those of skill in the art, e.g., U.S. P at. No. 5,514,552; Yurek and Sladek, 19290, Annu. Rev. Neurosci. 13:415-440; Rosentha 1, 1998, Neuron 20:169-172; Vescovi et al, 1999, J. Neurotrauma 16(8):689-93; Vescovi et al., 1999, Exp. Neuro. 156(1):71- 83 3 Brustle ef al, 1999, Science 285:754-56; eac’h specifically incorporated herein by reference. The cells may be delivered alone or im combination with other factors, for ex=ample a neuronal survival factor, and may be delivered along with a pharmaceutically aceceptable vehicle. Ideally, such a vehicle woulld enhance the stability and delivery properties of the cells. [0€D93]) The present disclosure also provides for pharmaceutical compositions comtaining the cells which can be administerel using a suitable vehicle such as lipmosomes, microparticles, or microcapsules. Cells of the present disclosure may also be supplied in the form of a pharmaceutical composi-tion comprising an isotonic excipient, an_d prepared under conditions that are sufficiently sterile for human administration.

General principles of medicinal formulations of cell compositions is found in Cell

Tlzerapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, G.

M orstyn & W. Sheridan eds, Cambrigge Universitsy Press, 1996, and Hematopoietic Stem

Cell Therapy, E.. Ball, J. Lister & P. Law, Chuarchill Livingstone, 2000, specifically in-corporated herein by reference. Additionally, it may be desirable to administer a plaarmaceutical composition containing a neuronal survival factor locally to the area in need of treatment, which may be achieved by, for example, local infusion during surgery, injection, a catheter means, or implant means, wheerein such implant can be of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes or fibers.

[0094] The neuroprogenitor cells and differ-entiated neural cells of the present disclosure may be administered to a subject as eitlier a substantially homogenous, nearly homogeneous, or heterogeneous cell populatiom. A substantially homogenous cell population comprises greater than 75% of a simgle cell type, such as a GABAergic ne=uron, more preferably greater than 90%, and mosst preferably greater than 95%-99%. A hesterogeneous cell population will consist of two car more cell types mixed in a single cell population, for example GABAergic newons, dopaminergic neurons, serotonergic neurons, Schwann cells, oligodendrocytes, astrocytes, and glial cells. The cells may alsos be genetically altered by methods well Emnown to those of skill in the art to express or release trophic factors, growth factors, neuronal survival factors, or other therapeutic compounds in the damaged area of the brain, central nervous system, peripheral nervous system, or other tissues. The use of promoter and cell type combinations for proteim expression is generally known to those of skill in the art of molecular biology, fom . example, see Sambrook, et al. 1989, Molecular Cloning: A Laboratory Manual 2% Ed .

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, specifically incorporatecl herein by reference.

[0095] To achieve expression of trophic factors, growth factors, neuronal survival factors, or other therapeutic compounds in the neuroprogenitor cells and differentiatecd neural cells of the present disclosure, suztable regulatory elements can be derived from =a variety of sources, and may be readil=y selected by one of ordinary skill in the arf.

Examples of regulatory elements include a transcriptional promoter, enhancer, and RNA polymerase binding sequence, as well as a ribosomal binding sequence, including a translation initiation signal. Other a«dditional genetic elements, such as selectable markers, may also be incorporated inte the recombinant molecule. The recombinarmt molecule may be introduced into the pluripotent stem cells, or the neuroprogenitor cells or differentiated neural cells derived from the pluripotent stem cells, using in vitro delivery vehicles or in vivo techniques. Examples of delivery techniques include retroviral vectors, adenoviral vectors, D"NA virus vectors, liposomes, physical techniques such as microinjection, and transfection via electroporation or calcium phosphate precipitation, or other methods known ir the art for transfer of creating recombinant cells.

The genetically altered cells may be encapsulated in microspheres and implanted into cor in proximity to the diseased or damage=d tissue. Protocols employed are well-known to those skilled in the art, and may be found, for example, in Ausubel et al, Curremnt

Protocols in Molecular Biology, Joshn Wiley & Sons, New York, N.Y., 199 7, incorporated herein by reference,

[0096] Preferably, the cell transplant therapy of the present disclosure al=so incorporates some means of storing and preserving the neuroprogenitor cells amd differentiated cells for use in transpl ant surgery, for example long-term storage Wy cryopreservation, or short term storage in preservation medium. Cryopreserved embryonic messencephalic tissue has been successfully stored for up to 70 days and transplanted ass homografts in rodent (Collier ef al, Progress in Brain R-esearch, Vol. 78,

New York, Eksevier (1988), pp. 631-36, specifically incorporated herein by reference) and primate (Collier et al., 1987, Brain Res. 436:363-66, specifically incorporated herein by reference). It has also been demonstrated that embryonic mesencephalic cells can be successfully cultured after cryopreservation. Mesencephalic tissue can also be stored short-term (2-_S days) in preservation medium at 4°C and subsequently transplanted with surviving gra#ft volumes similar to those for fresh tissue (Sauer er al, 1989, Restor.

Neurol. Neumosci. (Suppl.:3.suprd Int. Symp. Neural Tranplan.):56, specifically incorporated Bherein by reference). Similar techniques may be emplceyed to store and preserve the meuroprogenitor cells and differentiated cells of the presemt disclosure, and such techniqu es are well known to those of skill in the art.

[0097] Araother use for the neuroprogenitor cells and differenti ated neural cells described herein is to screen for factors such as pharmaceutical commpounds, solvents, small molecu les, peptides, or polynucleotides, as well as for environm_ental factors such as culture co nditions or manipulations, that affect the phenotype or characteristics of these cells. For example, biologically active molecules present in —plant, plant-based extracts, or ira animal, human, or synthetic sources, may be screened ard evaluated using these cells. In addition, these cells can be used to assess candidate growth factors or differentiatiomn factors. For example, a candidate pharmaceutical compound can be added to neuroprogzenitor cells or mature neurons, either alone or in combination with other drugs, and ary changes in the morphology, phenotype, or functional activity in the cells can be assessed and evaluated. In another embodiment, GABAergic n.eurons are used to screen for factors that affect receptors (e.g., agonists or antagonis€s) of GABAergic neuron in thes CNS, PNS, or specific tissues or organs. GABAergic n:eurons can also be used to screzen for agonists and/or antagonists of neuropeptides, neurotransmitters, neurohormoraes, or GABA. GABAergic neurons can also used to teszt the neurotoxicity of biological 1y active molecules.

[0098] I addition, the neuroprogenitor cells and differentiated neural cells described herein may bye further modified at any stage of differentiation. For example, these cells may be genetically modified to have single or multiple genetic modifications, either transient or stable. Genetic alterations of these cells may be desirable for many reasons, such as to provide modified cells for g=ene therapy or replacement tissues for grafting or implantation. The cells of the present clisclosure can be genetically modified through the introduction of vectors expressing a Selectable marker under the control of a neural : specific promoter, which are well knowen to those of skill in the art. These cells may also be modified at any stage to express cemtain markers or genes that can be used to fisrther purify differentiated cells derived fromm pluripotent stem cells, or alternatively to imduce differentiation into particular cell lineages. These cells can be modified to reduce or prevent immune rejection after transplantation, i.e. histocompatibility with the intended recipient.

[0099] To increase the replicative capacity of cells generated using the present disclosure, these cells may be telomer@ized by genetically altering them with a suitable vector so that they express the telomesrase catalytic component (TERT). The T ERT sequence used may be derived from hurman or mouse (WO 98/14592 and WO 99/27 113, specifically incorporated herein by reference), as well as other mammalian spe=cies.

Alternatively, transcription of the endo=genous TERT gene can be increased. Methods used to genetically modify cells are vvell known to those of skill in the art. T hese methods utilize various molecular bio logy techniques, many of which are genemrally described in Sambrook, ef al. 1989, Molecular Cloning: A Laboratory Manual 2"® Ed.

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, specifically incorpor—ated herein by reference. [00100}) In Vitro Transplantation With Adult Brain Cells

[00101] The human brain has little capacity for self-repair, and developing therampies for brain damage caused by disease or Mnjury remains a great clinical challenge. While : there has been much interest in the p ossibility of treating brain damage by cellular transplantation, this approach is still at a_n early experimental stage. One major hurd3e in the transplantation of neural cells is thes survivability of these cells, since experiments show that most grafted neurons degenerate after the transplantation. For example, the typical survival rate of dopaminergic cells grafted into experimental animals or subj ects with Parkinson’s disease has been limit-ed to about 10% (Brundin et al, 1987, Ann. N.Y.

Acad. Sci. 495:473-96; Nakao ef al... 1995, Nat. Med. 1(3):226-31). Although the cause(s) of this low survival rate is unlsnown, it may be due to neurotoxic effects by the recipient’s brain cells, or insufficient ssupply of nutrient and oxygen to the transplanted cells. Clearly, factors that cause neureonal death should be further investigated so that neuronal death after transplantation may be minimized.

[00102] One way to identify and study these factors is to create a brain-like environment and then test the efficacy or survivability of neuronal or neural cells, whether derived from pluripotent stesm cells or isolated from other sources. One embodiment of the present disclosure creates this brain-like environment by using a technique of in vitro transplantation of disolated neuronal or neural cells with adult neural cells, preferably cells isolated from the adult hippocampus. As used herein, the term “in vitro transplantation” refers to culturing two different types of cells together in the same culture environment. In vitro transplan tation can be used to determine the compatibility of two types of cells under similar environments and to study and predict factors that affect the survivability and functionality of transplanted cells. This system can be used to assess the survivability and functionality of the isolated neuronal or neural cells, particularly those derived from plurip otent cells. This assessment will in turn help determine the ability of these cells to treat neurodegenerative disorders or neuronal diseases, including but not limited to stroke, ischemia, Parkinson’s disease, Alzhcimer’s disease, epilepsy, and Huntington's dise=ase. In a preferred embodiment, this technique is used to assess the survivability or efficacy of GABAergic neurons derived from pluripotent cells as disclosed hereira in a host-like environment. In preferred embodiments, the survivability of neuronal or neural cells, preferably GABAergic neurons, derived from pluripotent cellss is at least 90% in the adult hippocampal cell environment after in vitro transplantati on. In other embodiments, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%%, 70%, 75%, 80%, 85%, 95%, or 99% of the

GABAergic neurons survive in this envi ronment.

[00103] The novel in vitro transplantzation model disclosed herein can also be used to study the following parameters: (1) symaptic formation between and among transplanted cells and host cells; (2) factors invol ved in neuronal or neural death after cellular

Wa 2005/021704 PCT/IB2004/002847 transplantation; and (3) rate of neuronal or neural death. Not only will the in vitro transplantation model help to provide detailed infomation required for improving cell survivability and functionality after transplantation, it will also help neurobiologists or neuredsurgeons to decide on a strategy before transplanting neurons in a patient. * * *

[001404] The following examples are included to demonstrate preferred embodiments of th_e invention. It should be appreciated by those of skill in the art that the techniques discl-osed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute prefesrred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without depam-ting from the spirit and scope of the invention.

Example 1

[00105] The following example demonstrates the in vitro derivation of functional

GAB- Aergic neurons from murine embryonic stern cells. Figure 1 illustrates the deriv-ation of GABAergic neurons from murine ES cells, while Figure 2 illustrates the different steps for differentiating murine ES cells into terminally differentiated neurons.

[00106] 1) Culture and Expansion of Murine Embryonic Stem Cells:

[00107] The murine ES cells utilized in the present set of experiments were isolated from the inner cell mass of a mouse blastocyst usings techniques well known to those of skill An the art. The murine ES cells were of J-1 origsin (obtained from National Institute of Dezntal and Craniofacial Research, National Institute of Health, Bathesda, Maryland,

USA), and were at passage 14. The cells were maintained on mitomycin-C treated mouse embryonic fibroblast feeder cells, which are mitotically inactivated in ES cell medium.

The rnurine ES cells were cultured in the ES cell medium to expand the number of undifferentiated cells. Generally, ES cells can be <xpanded at least about 1000 fold witho=ut losing pluripotency. The ES medium used generally included a carbon source, a nitrogen source, and a buffer to maintain the desired pH. The ES cell medium consisted of D-ulbecco’s modified Eagles medium (DMEM) or knockout DMEM (Gibco),

supplemented with 10-20% ES cell qualified fetal bovine sermm (FBS) (Hyclone) or serum. replacement knockout serum (Gibco), 1% MEM non-essenttial amino acid solution, 2mM L-glutamine, and 0.1 mM P-mercaptoethanol. The “ES medium was also supplemented with leukemia inhibitory factor (LIF) at a concentration of 1000 units/ml (ESGIRO, Chemicon International Inc.) to inhibit differentiation o=f the murine ES cells. [0010:8] The murine ES cells were expanded by culturing and regularly passaging the

ES ce=1ls in ES cell medium to inhibit differentiation again using techniques well known to tho se of skill in the art. The ES cells were cultured on tissue cialture plates treated with 0.1% to 0.2% gelatin in phosphate buffered saline (PBS) at 37° &C for at least 1-2 hours.

The ESS cells were grown on mouse feeder cells inactivated -with mitomycin-C at a conce:miration of 1x10°/ml. The ES cells were incubated for 4 days at a temperature betwe=en about 35° C and 40° C, preferably about 37° C, between about 1% and 10% CO, atmossphere, more preferably at 5% CO, atmosphere. The ESS medium was changed every day or every other day depending on the growth of ES cells in culture. [0010 9] 2) Generation of Embryoid Bodies: [0011 O] After the undifferentiated murine ES cells were pro liferated and expanded, they wwvere cultured to form embryoid bodies. First, the ES cells vere dissociated by brief expos<ure to 0.05% trypsin-EDTA, followed by scraping and bre=akdown of the cells into small clusters. These clusters were then plated at a density of approximately 4 x 10° cells/ml onto 60 mm bacteriological dishes in the absence of feeder cells in an appro priate media. The bacteriological dishes used have a non-adhesive surface that prevents attachment, thereby stimulating differentiation of the ESS cells and formation of embrsy/oid bodies. These cells were cultured as a suspension culture in ES medium. The

ES medium used to culture these cells had DMEM with higgh glucose or knockout

DME_M which was supplemented with 10-20% FBS or knockout serum replacement, as well aas other supplements such as §-mercaptoethanol (0.1 mM)w , L-glutamine (2 mM), and amtibiotics. No bFGF or LIF was added to the ES medium_ On the following day, the ceell suspension was transferred to a new culture plate, leaving behind any cells attaclmed to the previous plate. The ES cell medjum was chan.ged every other day by centrifuging the cells out of the old medium, resuspending the ce=lls in fresh medium, and

‘ returning the cells tom a non-adhesive culture plate. The embryoid “bodies were allowed to grow for 4-8 days. At the end of the 4-8 days, the embryoid bocTies were collected and spun down at low speed (1000 rpm, 5 minutes) and resuspendesd in ES cell medium.

About 30-40 embryaoid bodies were then transferred to an uncoated tissue culture plate and incubated for 24 hours.

[00111] 3) Selecwtion and Expansion of Nestin-Positive Neuropmrogenitor Cells:

[00112] After 24 hours, nestin-positive cells (neuroprogenitor ecclls) were selected by replacing the ES cell medium with ITSFn (nestin selection) serum-free defined medium.

The ITSFn medium consisted of DMEM:F12 medium (Gibco) supplemented with the growth factors inscalin (5-25 pg/ml) (Sigma), sodium selenite (1-5 nM) (Sigma), transferrin (1-10 pg/ml) (Gibco), and fibronectin (1-5 pg/ml). Generally, the cells were incubated in the ITSSFn medium for about 6-10 days, more prefesably for about 7 days, with the ITSFn meclium being replenished every other day. Thee cells were generally grown at about 35° CC and 40° C, preferably about 37° C, and betw een about 1% and 10%

CO; atmosphere, meore preferably between about 2% and 6% C O, atmosphere. After complete selection, gpreferably for 7 days, the neuroprogenitor cells were characterized for mestin expression using an immunofluorescence techniques, which showed that approximately 90% eof the cells were positive for nestin expressiosn. The nestin-positive cells were subsequermtly expanded as described below.

[00113] The nestin-positive cells were dissociated using 0.05% trypsin-EDTA and plated onto poly-L-eorinithin/laminin coated plated containing (CNS expansion media.

The CNS expansion. media contained DMEM:F12 supplement vith N2 (10ug/ml) and

B27 (20ug/ml). A mnerve growth factor was also added to the CCNS expansion media,

B-FGF (10-20 ng/mml), which is known to enhance the neuroral productivity, The neuronal precursor cells were expanded for 6 days, to genera.te a large number of neuronal cells. The cells were grown in the CNS expansion media for 6 days, with the : media being replenished every two days.

[00114] 4) Differentiation of Neuronal Progenitor Cells: [0011S] The expanded neuronal progenitor cells were differentiated by culturing the cells in a differentiation media containing Neurobasal A medium ( Gibco), FCS (10-20%)

(HYCLONE), and B27 supplement (2-10%) (Gibco), but no B-FGF. Additionally, the differentiation media contained factors to enhance GABAergic neuron yield, preferably

Ara-C (Sigma Chemical Co. USA). Culturing the newmronal progenitor cells in differentiation media containing Ara-C (20 ug/ml) incr-eased the percentage of

GABAergic neurons in the neuronal population derived fron murine ES cells. After 3 days of culture in the differentiation media containing Ara-C, the cells were grown in the same media without Ara-C for 8, 12, or 16 days. When thee neuronal progenitor cells were cultured in differentiation media containing Ara-C for 3 days, and the cells were then grown in differentiation media without Ara-C for 16 dayws, at least about 60% of the neuronal progenitor cells differentiated into GABAergic neurons (Figure 6). {00116] 5) Characterization of Differentiated Neurons

[00117] An analysis of the total population of neuronal cel 1s generated using the above methods demonstrated that more than 90% of the differentiatzed cells were neuronal cells.

The differentiated neuronal cell types generated according to the present disclosure were evaluated both by the overall morphology of the cells, as well as the phenotypes identified by immunoflourescence. Immunofiourescence armalysis was carried out at the neuroprogenitor expansion stage, as well as after 4, 8, 12, aznd 16 days of differentiation as disclosed in the protocol above. First, the isolated cells w~ere grown in 2-well chamber slides, rinsed with PBS, and fixed for 10 minutes with 49~ paraformaldehyde at room temperature. Next, the cells were permeabilized with €0.2% Triton X-100 in PBS containing 1% normal goat serum, and then blocked witha 1% bovine serum albumin (BSA)/PBS for 1 hour at room temperature.

[00118] The cells were then incubated overnight at 4° C with a primary antibody (antibody dilution was made in 1% BSA). The following primary antibodies were employed in the immunoflourescence investigation: Monoclonal GABA, 1:200 (Chemicon Inc. USA); Polyclonal GABA, 1:500 (Chemicon Inc. USA); GAD65, 1:500 (Chemicon Inc. USA); GAD67, 1:500 (Chemicon Inc. US_A); GAT-1, 1:500 (Chemicon

Inc. USA), GAT-2, 1:500 (Chemicon Inc. USA); Glutammate, 1:500 (Chemicon Inc.

USA); Nestin, 1:50 (Chemicon Inc. USA); Oligodendroscytes, 1:500 (Chemicon Inc.

USA); Serotonin, 1:500 (Chemicon Inc. USA); Tyrosine baydroxylase, 1:800 (Chemicon

Inc. USA); MAP-2, 1:500 (Chemicon Inc, USA); and GFAP, 1:500 (Chemicon Inc.

USA). After overnight incubation with the primary antibody, the cells were washed with

PBS and incubated with an FITC 1abeled secondary antibody for one hour in a dark environment. The cells were then washed three times with PBS and covered with mounting media. The chamber slides were observed under fluorescence microscope to evaluate the immunopositive cells in the different stages of differentiation.

[00119] I[mmunofluorescence anakysis of the differentiated cultures revealed that about 90% of the differentiated neuronal cells were immunoreactive to neuron-specific markers, such as Gad-65, Gad-67_,, GAT-1, GAT-2, Glutamate, GABA, Nestin and

MAP-2 (Figure 3). Of the total neuronal cells in the culture, about 60% of the neuronal cells stained for GABA, the marker for GABAergic neurons, while about 15% of the neuronal cells stained for tyrosine hydroxylase (TH), the marker for dopaminergic neurons. In addition, about 10% of” the neuronal cells stained for glutamate, while about 5% of the neuronal cells stained for serotonin, the marker for serotonergic neurons.

Finally, about 5-10% of the cells stained as oligodendrocytes. The cell culture also included glial cells, typically between about 5% to 10% of the cell population. Analysis demonstrated that at least some off the differentiated neurons in the cell culture were synaptically active. Typically, mature neurons are identified by the presence of long axonal projections with lots of fine spine-like structures. The presence of myelin- associated protein-2 (MAP-2) in dhe axonal projections also indicates maturation of neurons.

[00120] The differentiated cells were also analyzed by double-labeling the cells with primary antibodies to determine whether the expression of GAD65 and GAD67 was colocalized (Figure 4). GAD65 and GAD67 are two genes expressed in GABAergic neurons that are required for the synthesis of GABA. The differentiated neuronal cell population was studied by employing a double-immunolabeling technique using

GADG65/GAD67 and GABA antibodies, as described above. The double-immunolabeling results suggested that neuronal cells that express GAD65 and GADG7 proteins also express GABA neurotransmitters (Figure 4). These results confirm that GABAergic neurons derived using the disclosed methods are GABA-producing cells. The neuronal cells that expressed GAD65 and GADG67 protein were also shown to express GABA

Va 0 2005/021704 PCT/[B2004/002847 dur-ing early development because glutamate was converted in GABAergic neurons in the pre=sence of GAD6S5 and GAD67 enzymes. [00 3121] The gene expression profiles of cells collectesd at different stages of diffferentiation were also analyzed. Cells were collected for analysis by Reverse

Transcriptase Polymerase Chain Reaction (RT-PCR) from each of the following stages of the disclosed method: (1) Undifferentiated; (2) Embryoid Bodies (EBS); (3) Nestin

Expansion; (4) 2 days of differentiation; (5) 4 days of differentiation; (6) 8 days of differentiation; (7) 12 days of differentiation; and (8) 16 days of differentiation. After the cells were collected they were pelleted, and total cellular RMA was extracted from the cell pellets using the RNeasy Qiagen kit. The isolated RNA ~was stored at —20° C. The totaal cellular RNA was treated with RNase-free RQ DNase (Promega Corp., Madison,

WL) to remove all traces of DNA. cDNA was synthesized ficom the isolated total RNA usimg Moloney Leukemia virus superscript II reverse tr-anscriptase following the ma-mufacturer’s instructions Random hexamer primers (G~-IBCO/BRL) were used to prime the reverse transcriptase (RT) reactions.

[00122] The cDNA synthesized by this reverse transcripstase reaction was used for

PC-R amplification with different sets of specific primers to determine which genes were expressed in the collected cells. The primers were designed to identify mRNAs exporessed in GABAergic neurons, specifically the glutamate d2 ecarboxylase genes (GAD1 ancl GAD?2), the alternatively spliced GAD1 embryonic RNA, and the vesical inhibitory am ino acid transporter (VIAAT) transcript. GADI is alternatively spliced during dewelopment, and the embryonic transcript is predominzntly expressed in neural stern/progenitor cells during fetal development. The exporession of a ubiquitously exporessed gene, B-Actin, was also monitored as a positive comtrol. These PCR reactions wesre carried out using only 10% of the total first strand reaction (cDNA synthesized by

RT") as the template and platinum Taq polymerase under stanclard PCR conditions, which are= well known to those of skill in the art. The general ecycling parameters used to ama plify DNA products were as follows; 1. denaturation of the template cDNA at 94° C foxr 30 seconds; 45 _—

2. annealing the primers at 55-65° C for 1 minute, depending on the primers used; and 3. incubating the reaction at 72° C feor 1 minute, and repeating steps 1-3 (cycles) between 25 and 40 times.

[00123] After the PCR reaction, the products w=ere run through an agarose gel using . electrophoresis along with a DNA size ladder. The expression of GAD1, GAD2, GAD! embryonic, VIAAT, and B-Actin were all analyze by RT-PCR using the primers as set

Forth in Table 1:

Table 1 : Primer sets used to amplify GAB= Aergic neuron specific genes

GAD1 (302bp) | Sense: CCT TCG CCT GCA _ACC TCC TCG AAC (SEQ ID NO:1)

Anti-sense: GCG CAG TTT GCT CCCCGTTCT T (SEQID NO:2)

GAD2 (583 bp) | Sense: ACT CTG GCA TTT CTA CAA GAT GIT AGT A (SEQ ID NO:3)

Anti-sense: GAA TCA CAC IGT CTG TTC CAA TCC CTA A (SEQ ID NO:4)

GAD1 embryonic | Sense: TGG TTG ACT GTA €GAG ACA CCC TGA ADT A (234 bp GADI; (SEQ ID NO:5) 320 bp GAD1 Anti-sense: TCC CAT CAC CTT TATTTG ACCATCC embryonic) (SEQ ID NO:6) “VIATT (572bp) | Sense: TCC TGT CCT TTT CTC CCG CCC CGC CGC C (SEQ ID NO:7)

Anti-sense: GCA CCA CCT CCC CGT CTT CGT TCT CCT C (SEQ ID NO:8)

IB-Actin (220 bp) | Sense: GGG TCA GAA GGA. CTC CTA TG (SEQ ID NO:9)

Anti-sense: GTA ACA ATG CCA CCA TGT TCA AT (SEQID NO:10)

[00124] The above analysis by RT-PCR demonstrated that many ES cell-derived neuronal cells spontaneously differentiated into neurons that expressed markers of the

GABAergic phenotype. A difference was found in the expression of GAD] and GAD2 throughout the different stages of cell culture analyzed. The GAD1 gene was expressed in almost all the stages (stage 1 to stage 5), including in the undifferentiated stage (Figure 5). This is consistent with a previous report of GAD] expression in ES cells (Bain et al., 1993, Brain Res. Mol. Brain Res. 17:23-30). GAD?2, on the other hand, was only expressed in the differentiated sta ge as shown in Figure 5. This result suggests that the GABAergic neurons generated using the disclosed methods have unequal phenotypic expression of both the GAD1 and GAID2 genes. The GAD1 embryonic spliced variant is expressed in all stages except the undifferentiated stage, while the neurotransmitters gene (VIAAT), which is known to transpoxt GABA, is only expressed in the differentiated stages (Figure 5). Expression of the housekeeping gene B-actin was used as a positive control (Figure 5).

[00125] Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was also used to analyze the expression of GABA by GABAergic neurons derived using the disclosed methods. Since a definitive characteristic of GABAergic neurons is the production of GABA, the functional capacity of ES cell-derived GABAergic neurons to produce GABA was evaluated by directly measuring the intracellular GABA levels using

RP-HPLC. The concentration of GABA detected in each sample was determined by comparison with a standard solution ef GABA injected into the column immediately before and after each experiment.

[00126] To begin, cells were collected at different stages of the disclosed method:

Nestin-expansion stage and differentizated cells isolated after 8, 12, and 16 days of differentiation. The culture supernatants were collected from the different stages, immediately treated with 7.5% orthoph_osphoric acid and metabisulphite (0.22 mg/ml) to stabilize the neurotransmitter for measurement, and stored at —80° C until analysis by

RP-HPLC. Intracellular levels of GABA were detected using isocratic HPLC method based on the electrochemical detection of GABA derivation with OPA/t-butylthiol (Kehr and Ungerstedt, 1988, J. Neurochem, 51(4):1308-10; Osborne et al., 1991, J. Neurosci.

Method 3-4: 99-106, both specifically incorporated herein by refe:rence). The HPLC data were normalized against GABA standards at varying flow rates and sensitivities (Figure 7p».

[00127] While no GABA was detected in the nestin or nestin expansion stages, the measured levels of GABA for day-8 of differentiation was 20.95 —pg/ml, while day-12 and day-16 of differentiation measured 26.18 pg/m) and 18.60 pg/ml respectively. This analysis demonstrated that the highest levels of GABA were produced by the GABAergic neurons derived above on day-12 of differentiation. The overall release of GABA from the cultured cells was 21.51 pg/ml.

[00128] Since the highest levels of GABA were produced “by GABAergic neurons derived On day-12 of differentiation, cells at this same stage of differentiation were evaluated. for the presence of GABA-A and GABA-B receptors. The distribution of functional neurotransmitter receptors, for example GABA-A ancl GABA-B receptors, on the surfacce of neurons, for example GABAergic neurons, is higzhly relevant to synaptic transmisszion and signal processing (Eder ef al., 2001, Eur. J. Nevarosci. 13:1065-69). It is known thaat both GABA-A and GABA-B receptors on mature GABAergic neurons are critical for normal functioning of these neurons. To determine whether the GABAergic neurons clerived from murine ES cells as described herein also e=xpress these GABAergic receptors , the cells were immunostained using anti-GABA-A receptor antibody, 1:250 (Chemicon, USA) and anti-GABA-B receptor antibody, 1:250 (Chemicon, USA) using the immuanofluorescence protocol previously described.

[00129] Immunofluorescence analysis of day-12 differentiateed cultures demonstrated that bottm GABA-A and GABA-B receptors are localized to differentiated GABAergic neurons. Interestingly, approximately 80% of the differenti ated cells expressed the

GABA-A\ receptor (Figure 8), while only approximately 25% eof the differentiated cells expressead the GABA-B receptor (Figure 9). Since expressiosn of both GABA-A and

GABA-E3 receptors are highly relevant for synaptic transmissicon and signal processing, isolating GABAergic neurons that express both receptors could be critical for improsing cell func tionality after cellular transplantation.

CL

Example 2

[00130] The following excample, applicants of the present disclosure use an in vitro transplantation model to study the efficacy, survivability, and functionality of

GABAergic neurons derived from murine embryonic stem cells. Figure 1 illustrates the in vitro transplantation mode=l.

[00131] The survivability and functionality of the GABAergic neurons derived from murine ES cells was studied by in vitro transplantation of the GABAergic neur-ons with hippocampal cells of the adult mouse brain. The hippocampal cells were isolated by first isolating cells from the hippocampus of the adult mouse brain, disassociating these cells with 0.05% trypsin-EDTA, and culturing the cells for one week in Neurobasal—A media supplemented with B27 (20 pg/ml) and N2 (10 pg/ml). The adult hippocampal brain cells were grown on 100 rmm tissue culture plates coated with poly-L-ornitThine and laminin or gelatin. After ome week, GABAergic neurons derived from murine ES cells and collected after 12 days of differentiation were plated on the adult hippocampal brain cells, and both cell types were cultured together for one week. Approximately 90% of the GABAergic neurons suxvived after one week of in vitro transplantation vwith adult hippocampal brain cells. This result suggests that neurons derived from pluripotent stem cells are functional in the adult brain environment and make synaptic connections with adult brain cells, which also» suggests that these cells may be used therapeutically to treat a variety of neurodegenerati ve disorders or neuronal diseases.

[00132] All of the compositions and methods disclosed and claimed hereXxn can be made and executed without undue experimentation in light of the present disclosure.

While the compositions ancl methods of this invention have been described ira terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit ana scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents descrit>ed herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deesmed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims (43)

CLAIMS WHAT IS CLAIMED IS:

1. A differentiated cell population in an in vro culture obtained by differentiating murine pluripotent stem cells, wherein at least 60% of the differentiated cells are GABAergic neurons.

2. The cell population of claim 1, wherein the murine pluripotent stem cells are murine embryonic stem cells.

3. The cell population of claim 1, wherein tthe GABAergic neurons express GAD65.

4, The cell population of claim 1, wherein the GABAergic neurons express GAD67.

5. The cell population of claim 1, wherein -the GABAergic neurons express GABA-A receptor.

6. The cell population of claim 1, wherein. the GABAergic neurons express GABA-B receptor.

7. The cell population of claim 1, whereira the GABAergic neurons express GABA-A and GABA-B receptors.

8. A differentiated cell population in an izz vitro culture obtained by differentiating murine pluripotent stem cells, whereim at least 60% of the differentiated cells produce gamma aminobutyric acid (G _ABA).

9. The cell population of claim 8, wherein the murine pluripotent stem cells are murine embryonic stem cells.

10. A method of generating a differentiated neural cell population from mammalian pluripotent stem cells comprising the following steps: (a) expanding a culture of pluripotent stem cells; (b) culturing the pluripotent stem cel Is to select for neuroprogenitor cells that are positive for nestin; (c) expanding the nestin-positive newroprogenitor cells; and

-~' pt (d) differentiating the nestin-positive cells to generate a differentiated cell population by culturing the cells in a differentiation media which comprises cytosine p-d-Arabino furanoside (Ara-C).

11. The cell population of claim 10, wherein the mammalian plurigootent stem cells are murine embryonic stem cells.

12. The cell population of claim 10, wherein the mammalian plurigootent stem cells are human embryonic stem cells.

13. The method of claim 10, wherein the differentiated cell popula_tion comprises at least about 60% GABAergic neurons.

14. The method of claim 10, wherein the differentiated cell popula-tion comprises at least about 15% dopaminergic neurons.

15. The method of claim 10, wherein the differentiated cell popula-tion comprises at least about 10% glutamatergic neurons.

16. The method of claim 10, wherein the differentiated cell populamtion comprises at least about 5% serotonergic neurons.

17. The method of claim 10, wherein the differentiated cell populastion comprises at least about 5% oligodendrocytes.

18. The method of claim 10, wherein the differentiated cell population comprises at least about 5% astrocytes.

19. The method of claim 10, wherein the neuroprogenitor cells that- are positive for nestin are selected by culturing the stem cells in serum-free mecdium.

20. The method of claim 19, wherein the serum-free medium is ITSFn serum-free defined medium.

21. The method of claim 20, wherein the cells are grown in the ITS Fn serum-free defined medium for 6-10 days.

22. The method of claim 19, wherein the serum-free medium comprises one or more soluble factors selected from the,group consisting of insulin, soclium selenite, transferrin, and fibronectin.

23. The method of claim 10, further comprising culturing the mammalian pluripotent stem cells of step (b) to form embryoid bodies.

24. The method of claim 23, wherein the embrsyoid bodies are cultured to select for peuroprogenitor cells that are positive for n_estin.

25. The method of claim 24, wherein the neuro progenitor cells comprise at least about 90% nestin-positive cells.

26. The method of claim 23, wherein the neuro progenitor cells that are positive for nestin are selected by culturing the embryoid bodies in serum-free medium.

27. The method of claim 26, wherein the serum-free medium is ITSFn serum-free defined medium.

28. The method of claim 26, wherein the serum-free medium comprises one or more soluble factors selected from the group con sisting of insulin, sodium selenite, basic fibroblast growth factor, transferrin, zand fibronectin.

29. The method of claim 10, further comprising expanding the nestin-positive neuroprogenitor cells of step (¢) in CNS expansion media.

30. The method of claim 29, wherein the nestin-positive neuroprogenitor cells are plated on a culture dish pre-coated with poky-L-ornithine or laminin.

31. The method of claim 29, wherein the CNS expansion media comprises one or more soluble factors selected from the group consisting of N2 supplement, B27 supplement, and a neural-inducing agent.

32. The method of claim 31, wherein the neura I-inducing agent is basic fibroblast growth factor (bFGF).

33. The method of claim 29, wherein the cells are grown in the CNS expansion media for 6-10 days. 34, The method of claim 10, wherein the differentiation media comprises N2 supplement, B27 supplement, or both, but rot basic fibroblast growth factor (bFGF).

WE) 2005/021704 PCT/1B2004/002847

35. The method of claim 10, wherein the cells are grown in the differentiation media for 2 or more days.

36. The method of claim 10, further comprising step (¢), where in the differentiated cell population of step (d) are further differentiated by culttaring the cells in a second differentiation media that does not contain cytosine {-d-Arabino furanoside (Ara-C).

37. The method of claim 36, wherein the differentiated cell population is grown in the second differentiation media for 8-16 days.

38. A method of generating GABAergic neurons from mamma lian neuroprogenitor cells, comprising enriching the neuroprogenitor cells for ce ls that are positive for nestin, and differentiating the nestin-positive cells to generate GABAergic neurons by culturing the cells in the presence of cytosine f—d-Arabino furanoside (Ara-C).

39. A method of generating an in vitro transplantation model for neural cells, comprising the following steps: (a) isolating adult hippocampal cells; (b) dissociating and culturing the hippocampal cells to generate a hippocampal cell culture; and (¢) culturing neural cells on the hippocampal cell cultumre; wherein the survival of the neural cells on the hippocampal_ cell culture is evaluated.

40. A method of claim 39, wherein the adult hippocampal cells are isolated from a mouse.

41. A method of claim 39, wherein the neural cells are GABAergic neurons.

42. A method of claim 41, wherein greater than 90% of the GAs BAergic neurons survive after one week of culture.

43. A method of claim 39, further comprising evaluating the synaptic formation between the neural cells and the hippocampal cell culture.