WO2015006474A1 - Methods of treating a subject with a cognitive dysfunction using spinal cord-derived neural stem cells - Google Patents

Abstract

The disclosure relates generally to methods of treating a subject with a cognitive dysfunction by administering to the brain of the subject an effective amount of spinal cord neural stem cells. The present disclosure provides methods for treating a subject with a cognitive dysfunction including, for example, a subject that has received radiotherapy for the treatment of a primary or secondary brain tumor. Such methods may be useful for treating symptoms resulting from exposure to radiation or symptoms of neurological diseases. In one aspect, the present disclosure provides methods for treating a subject with a cognitive dysfunction, for example, a human, by introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of the subject's brain (e.g., hippocampus).

Description

METHODS OF TREATING A SUBJECT WITH A COGNITIVE DYSFUNCTION USING SPINAL CORD-DERIVED NEURAL STEM CELLS PRIORITY CLAIM

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/844, 165 filed on July 9, 2013. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This work was supported by grants from the United States Government funded through the National Institutes of Health. The U.S. Government has certain rights in this invention.

FIELD

[0003] The present invention relates generally to methods for treating a subject with a cognitive dysfunction by introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of the subject's brain.

BACKGROUND

[0004] Debilitating and progressive cognitive dysfunctions are commonly associated with neurological diseases and can also be an unintended side effect of various therapeutic regimens such as radiotherapy. Such cognitive dysfunctions encompass a broad range of cognitive domains, including memory, communication, perception and concentration, problem-solving and decision-making, among others.

[0005] Palliative and preventative treatments to alleviate the symptoms of cognitive dysfunctions could improve the quality of life for both the patient and the patient's caregiver as well as minimize hospitalization or institutional care. As cognitive dysfunction affects millions of Americans, with numbers only increasing as the population ages, such treatments are desirable from both an economic and quality of life perceptive.

SUMMARY

[0006] The present disclosure provides methods for treating a subject with a cognitive dysfunction including, for example, a subject that has received radiotherapy for the treatment of a primary or secondary brain tumor. Such methods may be useful for treating symptoms resulting from exposure to radiation or symptoms of neurological diseases. In one aspect, the present disclosure provides methods for treating a subject with a cognitive dysfunction, for example, a human, by introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of the subject's brain (e.g., hippocampus).

[0007] The present disclosure also provides methods of treating a subject (e.g., a human) with a cognitive dysfunction by obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain (e.g., hippocampus).

[0008] The present disclosure also provides methods of augmenting neural cell numbers, increasing synaptic connections and/or providing paracrine support in a subject's brain, the methods comprising: obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain (e.g., hippocampus).

[0009] The present disclosure also provides methods for treating a subject with a brain cancer by administering radiation therapy to the brain of the subject, and introducing a therapeutically effective amount of spinal cord-derived neural stem cells to the subject's brain, wherein the therapeutically effective amount of spinal cord-derived neural stem cells is effective to treat a cognitive dysfunction.

[0010] In an embodiment of each or any of the above or below-mentioned embodiments, the spinal cord-derived neural stem cells are embryonic spinal cord-derived neural stem cells.

[0011] In an embodiment of each or any of the above or below-mentioned embodiments, the spinal cord-derived neural stem cells are fetal spinal cord-derived neural stem cells, wherein the fetal spinal cord-derived neural stem cells are obtained from a fetus being a gestational age of about 5 to about 20 weeks.

[0012] In an embodiment of each or any of the above or below-mentioned embodiments, the spinal cord-derived neural stem cells are human spinal cord-derived neural stem cells.

[0013] In an embodiment of each or any of the above or below-mentioned embodiments, the spinal cord-derived neural stem cells are expanded to form an expanded spinal cord-derived neural stem cell population. [0014] In an embodiment of each or any of the above or below-mentioned embodiments, expanding the spinal cord-derived neural stem cells includes culturing the spinal cord-derived neural stem cells in the absence of serum.

[0015] In an embodiment of each or any of the above or below-mentioned embodiments, expanding the spinal cord-derived neural stem cells includes exposing the spinal cord-derived neural stem cells to at least one growth factor. In some embodiments, the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof.

[0016] In an embodiment of each or any of the above or below-mentioned embodiments, the spinal cord-derived neural stem cells differentiate into neurons that engraft in vivo into the brain tissue of the subject. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the spinal cord-derived neural stem cells are capable of generating neurons in brain tissue of the subject.

[0017] In an embodiment of each or any of the above or below-mentioned embodiments, the cognitive dysfunction is induced by prior exposure of the subject to radiation.

[0018] In an embodiment of each or any of the above or below-mentioned embodiments, the cognitive dysfunction is associated with traumatic brain injury, diabetes, dementia, depression, aging, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, Multiple Sclerosis, epilepsy, cerebrovascular disease or substance abuse.

[0019] In an embodiment of each or any of the above or below-mentioned embodiments, the cognitive dysfunction is dementia, delirium or amnesia. In some embodiments, the dementia is vascular dementia, dementia with Lewy bodies, mixed dementia, frontotemporal dementia.

[0020] In an embodiment of each or any of the above or below-mentioned embodiments, the cognitive dysfunction includes speech impairment, confusion, disorientation, loss of memory, learning, perception, judgment, initiative, attention, planning, multitasking, spatial or analytical skills, reasoning ability, or combinations thereof.

[0021] In an embodiment of each or any of the above or below-mentioned embodiments, introducing the therapeutically effective amount of spinal cord-derived neural stem cells includes injecting at least a portion of the therapeutically effective amount of spinal cord-derived neural stem cells into a plurality of areas of the brain tissue of the subject including, for example, the cerebral hemispheres, cerebral cortex, subcortex, motor cortex, basal ganglia, striatum, internal capsule, thalamus, hypothalamus, hippocampus, corpus callosum, midbrain, substantia nigra, brainstem and cerebellum. [0022] In an embodiment of each or any of the above or below-mentioned embodiments, the subject may be administered one or more immunosuppressive drugs.

[0023] In an embodiment of each or any of the above or below-mentioned embodiments, the spinal cord-derived neural stems are injected into the subject's hippocampus.

[0024] These and other embodiments of the invention are described in further detail herein below.

BRIEF DESCRIPTION OF THE FIGURES

[0025] The foregoing summary, as well as the following detailed description of the disclosure, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the disclosure, shown in the figures are embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements, examples and instrumentalities shown.

[0026] Figure 1A-B: Figure 1A shows a schematic of the research design. Two month old athymic nude rats receiving 10Gy head-only γ-irradiation were transplanted two days later with human fetal-derived neural stem cells (NSI566 NSCs). At 1 -month posttransplantation surgery, animals were administered a novel place recognition and fear conditioning tasks. Three weeks later, after cognitive testing, animals were euthanized for immunohistochemical analysis. Non-irradiated control and irradiated animals receiving sterile hibernation buffer served as sham surgery groups. Figure 1 B shows immunocytochemical analysis of NSI566 NSCs and following in vitro differentiation. Undifferentiated NSI566 NSCs (far left panel) stained with DAPI show strong expression and co-localization of the multipotent marker Nestin. Following in vitro differentiation, NSI566 NSCs express both neuronal (MAP2- and SMI312-positive) and astrocytic (GFAP) cells (right panels). (Scale bars: Nestin, 10 μηι; MAP2, SMI312 and GFAP, 50 μηι).

[0027] Figure 2A-D shows that transplantation of NSI566 NSCs improves radiation- induced cognitive impairments at 1-month post-transplantation. Figure 2A shows NSI566 NSC-transplanted animals (IRR+NSI) explore more than controls (CON) and irradiated- sham (IRR) animals during the initial familiarization phase of NPR task (p < 0.001 , post hoc). Exploration ratios were calculated as, timenovei/time_novei⁺timefamiiiar, for the first minute of 5min (Figure 2B) and 24h (Figure 2C) test sessions in the NPR task. Figure 2B shows that for the 5 minute NPR test, IRR animals spent a significantly lower proportion of time exploring the novel place (p<0.001 vs. CON and vs. IRR+NSI, post hoc), while CON and IRR+NSI animals did not differ. IRR animals did not spend more time exploring the novel place than expected by chance (dashed line at 50%). Figure 2C shows that for the 24h test, after the initial familiarization phase, when animals were presented the same two objects, with one moved to a new spatial location, none of the groups spent more time exploring the novel place than expected by chance. Figure 2D shows that the IRR+NSI and CON animals exhibit similar time spent freezing following administration of a context test, compared to IRR rats which spent significantly less time freezing (p=0.014, post hoc comparisons, indicated by arrow). Figure 2D shows that 48 hours after the initial training phase, the context was changed, which resulted in a substantial reduction in freezing behavior in all groups (pre-cue bars). Further, freezing levels were restored in all groups following the tone sound (cue test bars). Data are presented as means + SEM. p-values were derived from post hoc comparisons. ^*, p= 0.014 indicates significant difference versus CON and IRR+NSI, and ^**, p=0.001 indicates significant difference versus IRR animals.

[0028] Figure 3 shows survival and location of transplanted NSI566 NSCs. At about two months post-transplantation, NSI566 NSCs are located near the injection (cells displayed as white) (Nt, needle track; Tc, transplant core; Figure 3A-E, 5 to 60x magnification) and CA1 and corpus callosum (CC) areas. Transplanted NSI566 NSCs did not show extensive migration patterns in the host hippocampus (DG, dentate gyrus; DH, dentate hilus, CA3 subfields). Transplanted NSI566 NSCs were detected with human specific nuclear antigen (Ku80) and counterstained with nuclear dye (TOTO-3). The insert in Figure 3E represents orthogonal reconstruction of confocal Z-stacks. (Scale bars: A-B, 100 μηη; C, 50 μηη; D, 20 μηη; E, 10 μηη and E-insert, 5 μηη).

[0029] Figure 4 shows differentiation of transplanted NSI566 NSCs in the irradiated hippocampus. At about two months post-transplantation, Ku80-positive (human specific nuclear antigen) NSI566 NSCs differentiated into immature (doublecoritin, DCX, Figure 4A and a) and mature (neuron specific nuclear antigen, NeuN, Figure 4B and b) neurons as visualized by dual labeling of neuron-specific markers with Ku80. A similar pattern of differentiation was observed for immature (glial fibrillary acidic protein, GFAP, Figure 4C and c) and mature (S100 protein, Figure 4D and d) astrocytes. Arrows indicate representative dual-labeled NSI566 NSC transplanted-derived cells (Figure 4A-D). Confocal z-stack orthogonal reconstructions of dual-labeled cells are shown for neuronal (NeuN, a; DCX, b) and astrocytic (GFAP, c; S100, d) phenotypes (Figure 4a-d). DG, dentate gyrus; CC, corpus callosum. Scale bars: A-D, 50 μηη and a-d, 10μηι).

DETAILED DESCRIPTION

[0030] The disclosed methods relate to the treatment of a subject with a cognitive dysfunction and may be used to ameliorate complex learning and memory deficits. Cognitive dysfunctions are commonly associated with neurological disorders and can also be unintended side effects of various therapeutic regimens such as radiotherapy {e.g., radiotherapy administered for the treatment of a brain tumor). The types of dysfunctions encompass a broad range of cognitive domains, including memory, communication, perception and concentration, problem-solving and decision-making, among others. Despite the prevalence of cognitive dysfunction there remains a lack of suitable and efficacious treatment options. The present application provides methods for the treatment of a subject with a cognitive dysfunction by introducing spinal cord-derived neural stems cells to the subject's brain.

[0031] Prior to this invention, it was uncertain whether spinal cord-derived neural stems cells could survive and differentiate at a site in the brain that has been subjected to ionizing radiation (e.g., radiotherapy). This disclosure surprisingly demonstrates that spinal cord-derived neural stems cells differentiate into neurons and astrocytes in the radiation- treated brain, increase synaptic connectivities, and ameliorate any cognitive dysfunctions that arise as a result of treatment of a subject with radiation.

[0032] Presently, there is a lack of treatment options to combat the devastating side effects of cranial radiotherapy which is often administered for the treatment of a brain tumor. Notably, studies have highlighted the importance of preserving a pool of multipotent cells in the hippocampus in order to reduce the likelihood of developing a cognitive dysfunction subsequent to radiotherapy. However, such studies have also revealed that these niches of multipotent cells often harbor glioma stem cells which may lead to a reoccurrence of a brain tumor if not eliminated. Therefore, while sparing the hippocampus and the neural stem cells that reside within may lead to improved cognitive outcomes, targeting the hippocampus may improve progression free survival at the expense of debilitating cognitive decrements. As such, the administration of spinal cord-derived neural stem cells to the brain of a subject treated with radiotherapy represents a potential solution to treat any cognitive dysfunctions that may arise in the treated subject without sacrificing progression free survival.

[0033] Thus, the present disclosure also provides methods for treating a subject with a brain cancer by: administering radiation therapy or chemotherapy to the subject, obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of the subject's brain {e.g., hippocampus). [0034] Early in development, neural cells delineate themselves from each other depending on their location within the central nervous system. Fundamental regional differences exist between these neural cell populations regarding their proliferative capabilities, gene expression, ability or likelihood to generate a specific terminally differentiated cell type and migration patterns. (See, Kelly et al., PLoS One. (2009) 4(1 ): e4123. The disclosed methods surprisingly show that, despite understood regional differences between neural stem cells (NSCs) of the spinal cord and brain, spinal cord- derived NSCs are not constrained in their capacity to differentiate into appropriate neuronal subtypes and stably engrafted into regions of the brain.

[0035] Without wishing to be bound by a theory of the invention, it is believed that the methods of the invention restore cognitive dysfunction by: augmenting neuronal cell numbers, increasing synaptic connectivities and/or providing paracrine support. As used herein, the term, "NSCs" can also refer to neural or neuronal progenitors, or neuroepithelial precursors. NSCs can be functionally defined according to their capacity to differentiate into each of the three major cell types of the CNS: neurons, astrocytes, and oligodendrocytes.

[0036] The present disclosure provides methods of treating a subject with a cognitive dysfunction (e.g., a human) by obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population (e.g., injecting 70,000 NSI566 NSCs in 1 μΙ_ of cell suspension) to one or more areas of the subject's brain (e.g., hippocampus).

[0037] The present disclosure also provides methods of augmenting neural cell numbers, increasing synaptic connections and/or providing paracrine support in a subject's brain, the methods comprising: obtaining at least one neural stem cell from spinal cord tissue of a human, expanding the at least one neural stem cell to form an expanded neural stem cell population, concentrating the expanded neural stem cell population, and introducing a therapeutically effective amount of the expanded neural stem cell population (e.g., injecting 70,000 NSI566 NSCs in 1 μΙ_ of cell suspension) to one or more areas of the subject's brain (e.g., hippocampus).

[0038] The present disclosure also provides methods for treating a subject with a brain cancer by administering radiation therapy to the brain of the subject, and introducing a therapeutically effective amount of spinal cord-derived neural stem cells (e.g., injecting 70,000 NSI566 NSCs in 1 μΙ_ of cell suspension) to the subject's brain, wherein the therapeutically effective amount of spinal cord-derived neural stem cells is effective to treat a cognitive dysfunction.

[0039] In some embodiments, "treating" or "treatment" of a disease, disorder, or condition includes at least partially: (1 ) preventing the disease, disorder, or condition, i.e. causing the clinical symptoms of the disease, disorder, or condition not to develop in a mammal that is exposed to or predisposed to the disease, disorder, or condition but does not yet experience or display symptoms of the disease, disorder, or condition; (2) inhibiting the disease, disorder, or condition, i.e., arresting or reducing the development of the disease, disorder, or condition or its clinical symptoms; or (3) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, or condition or its clinical symptoms.

[0040] In some embodiments, "effective amount," as used herein, refers to the amount of spinal cord-derived neural stem cells that is required to confer a therapeutic effect on the subject. A "therapeutically effective amount," as used herein, refers to a sufficient amount spinal cord-derived neural stem cells being administered which will relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, in some embodiments, an "effective amount" for therapeutic uses is the amount of the spinal cord-derived neural stem cells required to provide a clinically significant decrease in disease symptoms without undue adverse side effects. In some embodiments, an appropriate "effective amount" in any individual case is determined using techniques, such as a dose escalation study. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. In other embodiments, an "effective amount" of spinal cord-derived neural stem cells is an amount effective to achieve a desired pharmacologic effect or therapeutic improvement without undue adverse side effects. In other embodiments, it is understood that "an effect amount" or "a therapeutically effective amount" varies from subject to subject, due to variation in metabolism, age, weight, general condition of the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0041] Cognitive dysfunction may include amnesia, dementia, and delirium. As used herein, the term "amnesia" refers to a deficit in memory. As used herein, the term "dementia" refers to a loss of global cognitive ability in a previously unimpaired person, which may be mild to severe in degree. It is understood in the field that dementia is not a single disease, but a non-specific syndrome generally describing diseases associated with loss of memory and/or other mental abilities severe enough to interfere with daily life. The most common type of dementia is Alzheimer's disease, but other types of dementia include, but are not limited to, vascular dementia, dementia with Lewy bodies, mixed dementia, Creutzfeldt-Jakob disease, Huntington's disease, or diseases associated with cerebrovascular disease and substance abuse such as normal pressure hydrocephalus and Wernicke-Korsakoff syndrome, respectively. As used herein, the term "delirium" refers to sudden severe confusion and disorientation, which develops with a relative rapid onset and fluctuates in intensity.

[0042] As used herein, the term "associated with", when used in the context to a condition or disease associated with cognitive impairment, means that the condition or disease may lead to cognitive impairment, may result from cognitive impairment, may be characterized by cognitive impairment or may otherwise be associated with or involve cognitive impairment in any way. The cognitive impairment associated with the condition or disease may be of any degree, for example mild, moderate or severe. The association between the condition or disease and cognitive impairment may be direct or indirect and it should be understood that cognitive impairment need not be the predominant feature of the condition or disease, nor necessarily be a feature of the condition or disease in every individual.

[0043] As used herein, the term, "neural stem cell" or "NSC" refers to a multipotential stem cell that can be functionally defined according to their capacity to differentiate into each of the three major cell types of the central nervous system (CNS): neurons, astrocytes, and oligodendrocytes. As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of self-renewal, meaning that with each cell division at least one daughter cell will also be a stem cell. NSCs can also refer to neural or neuronal progenitors, or neuroepithelial precursors.

[0044] In one embodiment, the NSCs are multipotent such that each cell has the capacity to differentiate into a neuron, astrocyte or oligodendrocyte. In another embodiment, the NSCs are bipotent such that each cell has the capacity to differentiate into two of the three cell types of the CNS. In another embodiment, the NSCs include at least bipotent cells generating both neurons and astrocytes in vitro and include at least unipotent cells generating neurons in vivo.

[0045] Growth conditions can influence the differentiation direction of the cells toward one cell type or another, indicating that the cells are not committed toward a single lineage. In culture conditions that favor neuronal differentiation, cells, particularly from human CNS, are largely bipotent for neurons and astrocytes and differentiation into oligodendrocytes is minimal. Thus, the differentiated cell cultures of the disclosed methods may give rise to neurons and astrocytes. [0046] In an embodiment, the NSCs are isolated from the CNS. As used herein, the term "isolated" with reference to a cell, refers to a cell that is in an environment different from that which the cell naturally occurs (e.g. where the cell naturally occurs in an organism) and the cell is removed from its natural environment.

[0047] NSCs may be isolated from an area which is naturally neurogenic for a desired population of neurons and from embryonic, fetal, post-natal, juvenile or adult tissue. The desired population of cells may include the cells of a specific neuronal phenotype which can replace or supplement such phenotype lost or inactive in the course of disease progression. In an embodiment, the NSCs are isolated from the subventricular zone (SVZ) or from the subgranular zone of the dentate gyrus (DG). In preferred embodiments, the NSCs are isolated from the spinal cord in which neurogenesis of ventral motor-neurons is substantial and obtained at a gestational age of human fetal development during which neurogenesis of ventral motor-neurons is substantial.

[0048] Accordingly, in an embodiment, NSCs are isolated from the spinal cord at a gestational age of about 6.5 to about 20 weeks. Preferably, NSCs are isolated from the spinal cord at a gestational age of about 7 to about 9 weeks. In another embodiment the NSCs are isolated from embryonic spinal cord tissue. In yet another embodiment, neural stem cells are isolated from a human. It should be appreciated that the proportion of the isolatable NSC population can vary with the age of the donor. Expansion capacity of the cell populations can also vary with the age of the donor.

[0049] The NSCs of the ventral midbrain, for example, are distinct from the NSCs obtained from the spinal cord at the same gestational stage. In particular, the NSCs from the ventral midbrain exclusively give rise to tyrosine-hydroxylase-expressing dopaminergic neurons, whereas NSCs from the spinal cord exclusively generate acetylcholine-producing cholinergic neurons. Both cell types, however, simultaneously generate the more ubiquitous glutamate- and GABA-producing neurons. Therefore, in an embodiment, the disclosed methods include obtaining NSCs from the spinal cord to treat conditions ameliorated or attenuated, at least in part, by the implantation of acetylcholine-producing cholinergic neurons.

[0050] NSCs can also be isolated from post-natal and adult tissues. NSCs derived from post-natal and adult tissues are quantitatively equivalent with respect to their capacity to differentiate into neurons and glia, as well as in their growth and differentiation characteristics. However, the efficiency of in vitro isolation of NSCs from various post-natal and adult CNS can be much lower than isolation of NSCs from fetal tissues which harbor a more abundant population of NSCs. Nevertheless, as with fetal-derived NSCs, the disclosed methods enable at least about 30% of NSCs derived from neonatal and adult sources to differentiate into neurons in vitro. Thus, post-natal and adult tissues can be used as described above in the case of fetal-derived NSCs.

[0051] In an embodiment, human fetal spinal tissue is dissected under a microscope. A region of tissue corresponding to the lower cervical/upper thoracic segments is isolated. The NSCs are isolated, pooled, and expanded on poly-D-lysine coated culture vessels in a media containing fibronectin and basic fibroblast growth factor (bFGF; FGF-2). Cells are expanded and then concentrated to the desired target cell density of about 10,000 cells per microliter in a medium free of preservative and antibiotics. Concentrated cells may be used fresh for implantation or frozen for later use.

[0052] In an embodiment, the NSCs are derived from embryonic stem cells or induced pluripotent stem cells. As used herein, the term "embryonic stem cell," refers to a stem cell isolated from the developing embryo which can give rise to all of the cells of the body {e.g., cells of the ecto-, meso-, and/or endo-dermal cell lineages). The term "induced pluripotent stem cell," as used herein, refers to a stem cell derived from a somatic cell (e.g., a differentiated somatic cell) that has a higher potency than the somatic cell. Embryonic stem cells and induced pluripotent stem cells are capable of differentiation into more mature cells (e.g., neural stem cells or neural progenitor cells). Methods employed for growing and differentiating embryonic or induced pluripotent stem cells into NSCs in vitro can, for example, be such as those described in Daadi et al., PLoS One. 3(2):e1644 (2008).

[0053] In an embodiment, the NSCs can be diluted with an acceptable pharmaceutical carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which the cells of the disclosure are administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the neural stem cells and pharmaceutically acceptable carriers can be sterile. Water is a useful carrier when the cells are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use. The selection of a suitable carrier is within the skill of the ordinary artisan.

[0054] Various neuronal subtypes can be obtained from manipulation of embryonic stem cells expanded in culture. Thus, specific neuronal subtypes, based on the disclosed methods, can be isolated and purified from other irrelevant or unwanted cells to improve the result, as needed, and can be used for treatment of cognitive dysfunction.

[0055] The NSCs in the disclosed methods can be derived from one site and transplanted to another site within the same subject as an autograft. Furthermore, the NSCs in the disclosed methods can be derived from a genetically identical donor and transplanted as an isograft. Still further, the NSCs in the disclosed methods can be derived from a genetically non-identical member of the same species and transplanted as an allograft. Alternatively, NSCs can be derived from non-human origin and transplanted as a xenograft. With the development of powerful immunosuppressants, allograft and xenograft of non-human neural precursors, such as neural precursors of porcine origin, can be grafted into human subjects.

[0056] A sample tissue can be dissociated by any standard method. In one embodiment, tissue is dissociated by gentle mechanical trituration using a pipette and a divalent cation-free buffer (e.g. saline) to form a suspension of dissociated cells. Sufficient dissociation to obtain largely single cells is desired to avoid excessive local cell density.

[0057] For successful commercial application of NSCs, maintaining robust and consistent cultures that have stable expansion and differentiation capacities through many successive passages is desirable. As described above, the culture methods can be optimized to achieve long-term, stable expansion of an individual cell line of NSCs from different areas and ages of CNS development while maintaining their distinct progenitor properties. In one embodiment, stem cells can be cultured according to the methods set forth in U.S. 8,460,651 , U.S. 8,236,299, U.S. 7,691 ,629, U.S. 5,753,506, U.S. 6,040, 180, or U.S. 7,544,51 1 , the entireties of which are incorporated by reference herein.

[0058] In an embodiment, the NSCs of the disclosed methods can include pre- differentiated cells for transplantation. For maximum yield of the cells and for simplicity of the procedure, a confluent culture is harvested for transplantation which comprises primarily a population of undifferentiated cells. It should be appreciated, however, that a minor population of cells just starting to differentiate spontaneously can also exist due to the increased cell density. [0059] In an embodiment, the NSCs are concentrated in a solution such as the clinically usable, hibernation or freezing solutions described above. In an embodiment, the NSCs are concentrated to an appropriate cell density which can be the same or different from the cell density for administration of the cells. In an embodiment, the cell density for administration can vary from about 1 ,000 cells per microliter to about 1 ,000,000 cells per microliter depending upon factors such as the site of the injection, the minimum dose necessary for a beneficial effect, and toxicity side-effect considerations.

[0060] Low cell survival of donor cells using known methods has necessitated the delivery of a large quantity of cells to a relatively small area in order to attempt effective treatment. Injection volume, however, is hydrostatic pressure exerted on the host tissue and the prolonged injection time associated with high injection volumes exacerbates surgical risk. Additionally, over-injection of donor cells leads to compression and subsequent injury of the host parenchymal tissue. In attempting to compensate for volume constraints, known methods have required preparation of high cell density suspensions for the injections. However, a high cell density promotes tight clustering of the transplanted cells and inhibits cell migration or spreading preventing effective treatment beyond a limited area and compromising seamless integration into the host tissue.

[0061] In contrast, as a result of improved survival in vivo of the cells prepared by the disclosed methods, fewer number of cells are needed per injection. In fact, up to three to four times the number of injected cells have been shown to exist after six months from the time of injection demonstrating significant quantitative survival using the disclosed methods. Also, because of the quantitative survival, reproducible administration of desired cell doses can be achieved. Accordingly, in one embodiment, the NSCs are concentrated to a density of about 1 ,000 to about 1 ,000,000 cells per microliter. In one embodiment, the NSCs are concentrated to a density of about 2,000 to about 80,000 NSCs per microliter. In another embodiment, about 5,000 to about 50,000 NSCs per microliter have been used for effective engraftment. In another embodiment, about 10,000 to 30,000 NSCs per microliter are used. In a preferred embodiment, the NSCs are concentrated to a density of about 70,000 NSCs per microliter.

[0062] In another embodiment, the NSCs are concentrated to a density of about

1 ,000 to about 10,000 cells per microliter, about 10,000 to about 20,000 cells per microliter, about 20,000 to about 30,000 cells per microliter, about 30,000 to about 40,000 cells per microliter, about 40,000 to about 50,000 cells per microliter, about 50,000 to about 60,000 cells per microliter, about 60,000 to about 70,000 cells per microliter, about 70,000 to about 80,000 cells per microliter, about 80,000 to about 90,000 cells per microliter, or about 90,000 to about 100,000 cells per microliter. [0063] In another embodiment, the NSCs are concentrated to a density of about 100,000 to about 200,000 cells per microliter, about 200,000 to about 300,000 cells per microliter, about 300,000 to about 400,000 cells per microliter, about 400,000 to about 500,000 cells per microliter, about 500,000 to about 600,000 cells per microliter, about 600,000 to about 700,000 cells per microliter, about 700,000 to about 800,000 cells per microliter, about 800,000 to about 900,000 cells per microliter, about 900,000 to about 1 ,000,000 cells per microliter.

[0064] In another embodiment, the NSCs can be delivered to a treatment area suspended in an injection volume of less than about 100 microliters per injection site. For example, in the treatment of cognitive dysfunction of a human subject where multiple injections may be made, an injection volume of 0.1 and about 100 microliters per injection site can be used. In preferred embodiments, the NSCs can be delivered to a treatment area suspended in an injection volume of about 1 microliter per injection site.

[0065] In an embodiment, the disclosed methods include injecting NSCs at a cell density of about 1 ,000 to about 10,000 cells per microliter, about 10,000 to about 20,000 cells per microliter, about 20,000 to about 30,000 cells per microliter, about 30,000 to about 40,000 cells per microliter, about 40,000 to about 50,000 cells per microliter, about 50,000 to about 60,000 cells per microliter, about 60,000 to about 70,000 cells per microliter, about 70,000 to about 80,000 cells per microliter, about 80,000 to about 90,000 cells per microliter, or about 90,000 to about 100,000 cells per microliter into to one or more areas of the brain of the subject.

[0066] In some embodiments, the disclosed methods include injecting NSCs at a cell density of about 100,000 to about 200,000 cells per microliter, about 200,000 to about 300,000 cells per microliter, about 300,000 to about 400,000 cells per microliter, about 400,000 to about 500,000 cells per microliter, about 500,000 to about 600,000 cells per microliter, about 600,000 to about 700,000 cells per microliter, about 700,000 to about 800,000 cells per microliter, about 800,000 to about 900,000 cells per microliter, or about 900,000 to about 1 ,000,000 cells per microliter into to one or more areas of the brain of the subject.

[0067] In an embodiment, the disclosed methods include injecting NSCs at a cell density of about 5,000 to about 50,000 cells per microliter. In preferred embodiments, the disclosed methods include injecting NSCs at a cell density of about 70,000 cells per microliter.

[0068] In an embodiment, the disclosed methods include multiple injections of NSCs at a total cell number of about 4,000 to about 40,000 cells, about 40,000 to about 80,000 cells, about 80,000 to about 120,000 cells, about 120,000 to about 160,000 cells, about 160,000 to about 200,000 cells, about 200,000 to about 240,000 cells, about 240,000 to about 280,000 cells, about 280,000 to about 320,000 cells, about 320,000 to about 360,000 cells, or about 360,000 to about 400,000 cells introduced into one or more areas of the brain of the subject.

[0069] In some embodiments, the disclosed methods include multiple injections of

NSCs with a total cell number of about 400,000 to about 800,000 cells, about 800,000 to about 1 ,200,000 cells, about 1 ,200,000 to about 1 ,600,000 cells, about 1 ,600,000 to about 2,000,000 cells, about 2,000,000 to about 2,400,000 cells, about 2,400,000 to about 2,800,000 cells, about 2,800,000 to about 3,200,000 cells, about 3,200,000 to about 3,600,000 cells, or about 3,600,000 to about 4,000,000 cells introduced into one or more areas of the brain of the subject.

[0070] The volume of media in which the expanded NSCs are suspended for delivery to a treatment area can be referred to herein as the injection volume. The injection volume depends upon the injection site and the degenerative state of the tissue. More specifically, the lower limit of the injection volume can be determined by practical liquid handling of viscous suspensions of high cell density as well as the tendency of the cells to cluster. The upper limit of the injection volume can be determined by limits of compression force exerted by the injection volume that are necessary to avoid injuring the host tissue, as well as the practical surgery time.

[0071] Any suitable device for injecting the cells into a desired area can be employed in the disclosed methods. In an embodiment, a syringe capable of delivering sub-microliter volumes over a time period at a substantially constant flow rate is used. The cells can be loaded into the device through a needle or flexible tubing or any other suitable transfer device.

[0072] In another embodiment, the cells are injected at between about 2 and about

5 sites in the brain. In an embodiment, the cells are injected at between about 5 and about 10 sites in the brain. In an embodiment, the cells are injected at between about 10 to about 30 sites in the brain. In an embodiment, the cells are injected at between about 10 to about 50 sites in the brain. At least two of the sites can be separated by a distance of approximately 100 microns to about 5,000 microns. In an embodiment, the distance between injection sites is about 400 to about 600 microns. In an embodiment, the distance between injections sites is about 100 to about 200 microns, about 200 to about 300 microns, about 300 to about 400 microns, about 400 to about 500 microns, about 500 to about 600 microns, about 600 to about 700 microns, about 700 to about 800 microns, about 800 to about 900 microns, or about 900 to about 1 ,000 microns. In an embodiment, the distance between injection sites is about 1 ,000 to about 2,000 microns, about 2,000 to about 3,000 microns, about 3,000 to about 4,000 microns, or about 4,000 to about 5,000 microns. The distance between injections sites can be determined based on generating substantially uninterrupted and contiguous donor cell presence throughout the spinal cord tissue and based on the average volume of injections demonstrated to achieve about 2-3 month survival in animal models such as rats or pigs. The actual number of injections and distance between injections in humans can be extrapolated from results in animal models.

[0073] The NSCs of the disclosed methods can generate large numbers of neurons in vivo. When the NSCs are not overtly pre-differentiated prior to transplant, the NSCs can proliferate up to two to four cell divisions in vivo before differentiating, thereby further increasing the number of effective donor cells. Upon differentiation, the neurons secrete specific neurotransmitters. In addition, the neurons secrete into the milieu surrounding the transplant in vivo growth factors, enzymes and other proteins or substances which are beneficial for different conditions. Accordingly, a variety of conditions can be treated by the disclosed methods because of the ability of the implanted cells to generate large numbers of neurons in vivo and because the cognitive dysfunction may be caused by or result in missing elements including neuron-derived elements. Therefore, subjects suffering from cognitive dysfunctions due to lack of such neuron-derived elements, such as growth factors, enzymes and other proteins, can be treated effectively by the disclosed methods.

[0074] In an embodiment, the composition comprising an amount of NSCs may be administered to a subject in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, intravenous, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. Intracerebrospinal, intrathecal, intravenous, intraperitoneal, or subcutaneous administration of the cells is preferred, with intracerebrospinal, intrathecal, or intravenous routes being particularly preferred; however, other cell administration paradigms well known in the art can be used.

[0075] In an embodiment, introducing the therapeutically effective amount of the NSCs includes injecting at least a portion of the therapeutically effective amount into a plurality of areas of the brain of a subject.

[0076] In one embodiment, compositions of the NSCs of the invention are formulated as an injectable formulation and comprise, for example, an aqueous solution or suspension of the active ingredient suitable for intracerebrospinal delivery. When preparing the composition for injection, particularly for intracerebral delivery, a continuous phase can be present that comprises an aqueous solution of tonicity modifiers, buffered to a pH below about 7, or below about 6, for example about 2 to about 7, about 3 to about 6 or about 3 to about 5. The tonicity modifiers can comprise, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that render osmotic pressure of the formulation isotonic with blood. Alternatively, when a larger quantity of the tonicity modifier is used in the formulation, it can be diluted prior to injection with a pharmaceutically acceptable diluent to render the mixture isotonic with blood.

[0077] In some embodiments of any of the aforementioned methods, the composition comprising NSCs is administered once. In some embodiments of any of the aforementioned methods, administration of an initial dose the composition comprising NSCs is followed by the administration of one or more subsequent doses. Examples of dosing regimens (e.g., an interval between the first dose and one or more subsequent doses) that can be used in the methods of the disclosure include an interval of about once every week to about once every 12 months, an interval of about once every two weeks to about once every 6 months, an interval of about once every month to about once every 6 months, an interval of about once every month to about once every 3 months, or an interval of about once every 3 months to about once every 6 months. In some embodiments, administration is monthly, every two months, every three months, every four months, every five months, every six months, or upon disease recurrence.

[0078] In an embodiment, the NSCs are injected at between about 5 and about 50 sites. In an embodiment, the NSCs are injected at between about 10 to about 30 sites. At least two of the sites can be separated by a distance of approximately 100 microns to about 5000 microns. In an embodiment, the distance between injection sites is about 400 to about 600 microns. The actual number of injections in humans can be extrapolated from results in animal models.

[0079] The methods of the present disclosure may include administration of one or more immunosuppressive drugs prior to, concurrent with, or after the injection of the NSCs.

[0080] In some embodiments, the NSCs and immunosuppressive drug may be coadministered. The NSCs and immunosuppressive drug which make up the therapy may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The NSCs and immunosuppressive drug may also be administered sequentially, with either the NSCs or immunosuppressive drug being administered by a regimen calling for multiple step administration. Thus, a regimen may call for sequential administration of the NSCs and immunosuppressive drug with spaced- apart administration of the separate, active agents. The time period between the multiple administration steps may range from, for example, a few minutes to several hours to days, depending upon the properties of the NSCs and immunosuppressive drug such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the therapeutic compound, as well as depending upon the effect of food ingestion and the age and condition of the subject. Circadian variation of the target molecule concentration may also determine the optimal dose interval. The NSCs and immunosuppressive drug whether administered simultaneously, substantially simultaneously, or sequentially, may involve a regimen calling for administration of the NSCs by intravenous route and the immunosuppressive drug by an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues, for example. Whether the neural stem cells and immunosuppressive drug are administered orally, by inhalation spray, rectally, topically, buccally (for example, sublingual), or parenterally (for example, subcutaneous, intramuscular, intravenous and intradermal injections, or infusion techniques), separately or together, each such therapeutic compound will be contained in a suitable pharmaceutical formulation of pharmaceutically-acceptable excipients, diluents or other formulations components.

[0081] Any immunosuppressive drug is contemplated for in the present disclosure. As used herein, the term "immunosuppressive drug" denotes any drug aimed at decreasing or preventing activity of the subject's immune response.

[0082] Immunosuppressive drugs act to inhibit the proliferation and activity of all or a substantial portion of the immune cells within the body. Many immunosuppressive drugs function by inhibiting a step in the interleukin 2 (IL-2) signaling pathway. IL-2 is a cytokine that regulates the growth, proliferation, and activation of lymphocytes. For example, the immunosuppressive drug tacrolimus, considered one of the most potent immune system suppressors, is a calcineurin-dependent inhibitor that blocks IL-2 production and reduces proliferation of T-cells. Another immunosuppressive drug sirolimus acts in a calcineurin- independent fashion to inhibit the response of T- and B-cells to IL-2. Still other immunosuppressive drugs, such as mycophenolate mofetil and prednisolone, function by inhibiting key enzymes required for T- and B-cell growth or by binding to glucocorticoid receptors, respectively. It is well-recognized in the field that many immunosuppressive drugs have high inter- and intra-patient variability and require routine dosage adjustments to maintain appropriate trough levels for therapeutic concentrations.

[0083] In an embodiment of any of the above-described methods, the immunosuppressive drug comprises methylprednisolone. For methylprednisolone, an effective amount can range from about 4 to 1 ,000 mg per dose. A preferred dosage of methylprednisolone may be about 125 mg administered intravenously immediately prior to surgery. Methylprednisolone also goes by the trade names Medrol® and Solu-Medrol®. Effective dosages will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether methylprednisolone is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient. Determining the appropriate dosage of an immunosuppressive drug are customary methods to physicians skilled in the art.

[0084] In an embodiment of any of the above-described methods, the immunosuppressive drug comprises prednisone. For prednisone, an effective amount can range from about 5 to 70 mg per dose. A preferred dosage of prednisone may be 60 mg delivered orally and tapered to 0 mg over 1 month. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the prednisone is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.

[0085] In an embodiment of any of the above-described methods, the immunosuppressive drug comprises basiliximab. For basiliximab, an effective amount can range from about 10 to 20 mg per dose. A preferred dosage of basiliximab may be 20 mg delivered intravenously, one dose given during transplantation and one given on postoperative day 4. Basiliximab also goes by the trade name Simulect®. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the basiliximab is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.

[0086] In an embodiment of any of the above-described methods, the immunosuppressive drug comprises tacrolimus. For tacrolimus, an effective amount can range from about 0.03 to 0.3 milligrams per kilogram per dose. Tacrolimus also goes by FK-506, fujimycin or trade names Prograf®, LCP-Tacro™, Advagraf®, and Protopic®. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the tacrolimus is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient. In an embodiment, a toxic dose of tacrolimus is administered to a subject.

[0087] Trough concentrations of tacrolimus are assessed to establish the appropriate dosing regimen. Therapeutic doses of tacrolimus have been reported to be 10- 20 ng/mL while doses greater than 20 ng/mL are associated with neurotoxicity. A preferred dosage of tacrolimus may maintain trough concentrations of about 4 to 8 ng/mL delivered orally twice a day.

[0088] In an embodiment of any of the above-described methods, the immunosuppressive drug comprises mycophenolate mofetil. For mycophenolate mofetil, an effective amount can range from about 1000 to 2000 milligrams per dose. A preferred dosage of mycophenolate mofetil may be 1 ,000 mg given orally twice a day. Mycophenolate mofetil also goes by mycophenolic acid and the trade name CellCept®. The salt mycophenolate sodium may also be used. Mycophenolate sodium goes by the trade name Myfortic®. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the mycophenolate mofetil is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.

[0089] In an embodiment of any of the above-described methods, the immunosuppressive drug comprises sirolimus. For sirolimus, an effective amount can range from about 1 to 20 milligrams per dose. Sirolimus also goes by the name rapamycin and the trade name Rapamune®. Effective doses will also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, whether the sirolimus is given as a combination therapy with another immunosuppressive drug, and also depends heavily on the individual patient.

[0090] The dosage may be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. The compositions may be given as a bolus dose, to maximize the circulating levels for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.

[0091] In some embodiments, compositions used in the methods described herein further comprise a pharmaceutically acceptable excipient. As used herein, the term "excipient" refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions. The pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.

[0092] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known in the art. "Administration" is not limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (for example, in capsules (e.g., as, powder, granules, microtablet, micropellets, etc.), suspensions, or tablets).

[0093] Administration to an individual may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art. [0094] In one embodiment of the present disclosure, an immunosuppressive drug is administered daily (or 1 to 5 times daily), weekly, or monthly. Illustratively, the composition is administered three times a week for five weeks and then weekly for an additional five weeks.

[0095] A dosage and dosage regimen may be administered to provide the optimal desired response (e.g., therapeutic response). The dose of an immunosuppressive drug may be measured in units of mg/kg of patient body weight. Alternatively, the dose of an immunosuppressive drug is measured in units of mg/kg of patient lean body weight (e.g., body weight minus body fat content), in units of mg/m² of patient body surface area, or in units of mg per dose (e.g., a fixed dose) administered to a patient. Any measurement of dose can be used in conjunction with the compositions and methods of the invention and dosage units can be converted by means standard in the art.

[0096] The method comprises the administration of an immunosuppressive drug of the present invention to a subject in need thereof. In one embodiment, the dosage regimen of immunosuppressive drug corresponds to once-a-day or twice-a-day dosages, and can include, for example, about 0.0001 mg/kg, about 0.0005 mg/kg, about 0.001 mg/kg, about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80, mg/kg, about 90 mg/kg, about 100 mg/kg, about 1 10 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg, about 150 mg/kg, about 160 mg/kg, about 170 mg/kg, about 180 mg/kg, about 190 mg/kg, about 200 mg/kg, about 220 mg/kg, about 240 mg/kg, about 250 mg/kg, about 500 mg/kg, about 750 mg/kg, or about 1 ,000 mg/kg (by body weight of the subject) dose of an immunosuppressive drug of the present invention, and can be modified in accordance with a variety of factors. These specific mg/kg amounts can vary, for example, from about 0.01 % to about 20% or more, depending on the application and desired therapeutic result. Other factors include the type of subject, the age, weight, sex, diet, and medical condition of the subject and the severity of the disease. Thus, the dosage regimen actually employed can vary widely and therefore deviate from the dosage regimen set forth above.

[0097] An immunosuppressive drug for use in any of the aforementioned methods may be administered in one or more doses (e.g., an initial dose optionally followed by one or more subsequent doses). Those skilled in the art will appreciate that dosages are generally higher and/or frequency of administration greater for initial treatment as compared with maintenance regimens. In certain embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more or eleven or more subsequent doses of the antibody are administered. The aforementioned dosage amounts refer to mg (immunosuppressive drug)/kg (weight of the individual to be treated).

[0098] An immunosuppressive drug thereof for use in any of the aforementioned methods may also be administered as a fixed dose, independent of a dose per subject weight ratio.

[0099] In some embodiments, the immunosuppressive drug is administered in one or more fixed doses of about 1000 mg or less, 500 mg or less, or 250 mg or less, 100 mg or less, 90 mg or less, 80 mg or less, 70 mg or less, 60 mg or less, 50 mg or less, 40 mg or less, 30 mg or less, 20 mg or less, or 10 mg or less of immunosuppressive drug. In some embodiments, the immunosuppressive drug is administered in one or more doses of at least 0.01 mg, at least 0.5 mg of immunosuppressive drug, at least 1 mg of immunosuppressive drug, or at least 10 mg of immunosuppressive drug. In some embodiments, the immunosuppressive drug thereof is administered in one or more doses of 1 mg to 100 mg of immunosuppressive drug.

[00100] In certain embodiments, the fixed dose immunosuppressive drug is from about 1 mg to about 10 mg, about 1 mg to about 25 mg, about 10 mg to about 25 mg, about 10 mg to about 50 mg, about 10 mg to about 100 mg, about 25 mg to about 50 mg, about 25 mg to about 100 mg, about 50 mg to about 100 mg, about 50 mg to about 150 mg, about 100 mg to about 150 mg, about 100 mg to about 200 mg, about 150 mg to about 200 mg, about 150 mg to about 250 mg, about 200 mg to about 250 mg, about 200 mg to about 300 mg, about 250 mg to about 300 mg, about 250 mg to about 500 mg, about 300 mg to about 400 mg, about 400 mg to about 500 mg, about 400 mg to about 600 mg, about 500 mg to about 750 mg, about 600 mg to about 750 mg, about 700 mg to about 800 mg, or about 750 mg to about 1000 mg. In some embodiments, the fixed dose of immunosuppressive drug thereof is less than 100 mg.

[00101] In various embodiments, dosage units of the present invention contain, for example, about 1 ng to about 2000 mg, about 0.001 mg to about 750 mg, about 0.01 mg to about 500 mg, about 0.1 mg to about 300 mg or about 1 mg to about 100 mg of an immunosuppressive drug of the present invention. Illustratively, such unit dosage forms can contain about 0.001 mg, or about 0.01 mg, or about 0.1 mg, or about 1 mg, or about 2 mg, or about 5 mg, or about 10 mg, or about 15 mg, or about 20 mg, or about 30 mg, or about 40 mg, or about 50 mg, or about 60 mg, or about 70 mg, or about 80, mg, or about 90 mg, or about 100 mg, or about 1 10 mg, or about 120 mg, or about 130 mg, or about 140 mg, or about 150 mg, or about 160 mg, or about 170 mg, or about 180 mg, or about 190 mg, or about 200 mg, or about 300 mg, or about 400 mg, or about 500 mg, or about 750 mg, or about 1 ,000 mg of an immunosuppressive drug of the present invention.

[00102] Illustratively, dosage units each contain about 0.01 mg, about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 40 mg, about 80 mg, about 100 mg, about 250 mg, about 500 mg, or about 1000 mg of an immunosuppressive drug of the present invention. The dosage unit form can be selected to accommodate the desired frequency of administration used to achieve the specified daily dosage. In one embodiment, a composition of the invention will be administered to a subject in an amount sufficient to about 0.1 to about 15 mg, about 0.5 to about 10 mg, and or about 1 to about 5 mg of the active agent, for example methylprednisolone, prednisone, sirolimus, etc.

[00103] In some embodiments of any of the aforementioned methods, administration of an initial dose of immunosuppressive drug is followed by the administration of one or more subsequent doses. Examples of dosing regimens (e.g., an interval between the first dose and one or more subsequent doses) that can be used in the methods of the disclosure include an interval of about once every week to about once every 12 months, an interval of about once every two weeks to about once every 6 months, an interval of about once every month to about once every 6 months, an interval of about once every month to about once every 3 months, or an interval of about once every 3 months to about once every 6 months. In some embodiments, administration is monthly, every two months, every three months, every four months, every five months, or every six months.

[00104] The disclosure also provides dosing regimens for use in any of the aforementioned methods, wherein the dosing regimens comprise more than one dosing interval for administration of the immunosuppressive drug. In some embodiments, the dosage regimen comprises at least two (e.g., two, three, four, five, six) different dosing intervals for administration of the immunosuppressive drug. In some embodiments, the dosage regimen comprises two different dosing intervals for administration of the immunosuppressive drug. In some embodiments, the dosing regimen comprises two different dosing intervals for administration of the immunosuppressive drug, wherein a first dosing interval comprises administration of one or more doses of immunosuppressive drug thereof and a second dosing interval comprises administration of one or more doses of the immunosuppressive drug thereof, and wherein the first dosing interval is shorter in time than the second dosing interval. For example, the first dosing interval may be days or weeks, and the second dosing interval may be months. In some embodiments, the first dosing interval is about 5 days to about 28 days, about 7 days to about 21 days, about 12 days to about 16 days, or about 14 days. In some embodiments, the second dosing interval is about 1 month to about 3 months, about 1 month to about 2 months, or about 1 month.

[00105] In some embodiments of any of the aforementioned methods, the dose can be escalated or reduced to maintain a constant dose in the blood or in a tissue. In related embodiments, the dose is escalated or reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% in order to maintain a desired level of the immunosuppressive drug.

[00106] In some embodiments of any of the aforementioned methods, the immunosuppressive drug is administered to a subject such that the interval between doses is a time sufficient to maintain a plasma concentration of said immunosuppressive drug in the subject at a level of at least about 0.1 μg/mL, at least about 0.3 μg/mL, at least about 1 μg/mL or at least about 2 μg/mL. In some embodiments, these plasma concentration values refer to values obtained for an individual that is treated with the immunosuppressive drug in accordance with the disclosure herein.

[00107] In some embodiments of any of the aforementioned methods, administration of an initial dose of the immunosuppressive drug is followed by the administration of one or more subsequent doses, and wherein said one or more subsequent doses are in an amount that is approximately the same or less than the initial dose.

[00108] In some embodiments of any of the aforementioned methods, administration of an initial dose of the immunosuppressive drug is followed by the administration of one or more subsequent doses, and wherein at least one of the subsequent doses is in an amount that is more than the initial dose.

[00109] In some embodiments of any of the aforementioned methods, an immunosuppressive drug is administered, wherein administration of an initial dose of the immunosuppressive drug is followed by the administration of one or more subsequent doses, and wherein the plasma concentration of said immunosuppressive drug in the human is permitted to decrease below a level of about 0.1 μg/mL, about 0.07 μg/mL, about 0.05 μg/mL, about 0.03 μg/mL or about 0.01 μg/mL for a period of time greater than about 1 week and less than about 6 months between administrations during a course of treatment with said initial dose and one or more subsequent doses. In some embodiments, the plasma concentration values refer to values obtained for an individual that is treated with immunosuppressive drug in accordance with the disclosure herein.

[00110] The amount of immunosuppressive drug necessary to elicit a therapeutic effect can be experimentally determined based on, for example, the absorption rate of the immunosuppressive drug into the blood serum or the bioavailability of the immunosuppressive drug. It is understood, however, that specific dose levels of the immunosuppressive drug of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject (including, for example, whether the subject is in a fasting or fed state), the time of administration, the rate of excretion, the drug combination, the severity of the diabetes mellitus and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for subject administration. Studies in animal models generally may be used for guidance regarding effective dosages for treatment of diabetic disorders or diseases in accordance with the present invention. In terms of treatment protocols, it should be appreciated that the dosage to be administered will depend on several factors, including the particular immunosuppressive drug that is administered, the route administered, the condition of the particular subject, etc. Generally speaking, one will desire to administer an amount of the immunosuppressive drug for a period of time that elicits a desired therapeutic effect, for example, lowering blood glucose level to acceptable levels, or improvement or elimination of symptoms, and other indicators as are selected as appropriate measures by those skilled in the art. Determination of these parameters is well within the skill of the art.

[00111] In some embodiments, the composition comprising the immunosuppressive drug may be administered to a subject in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous, intraperitoneal, or oral administration of the immunosuppressive drug is preferred, with intravenous or oral routes being particularly preferred.

[00112] In one embodiment, an immunosuppressive drug of the invention is formulated as an injectable formulation and comprises, for example, an aqueous solution or suspension of the active ingredient suitable for intravenous delivery. When preparing the immunosuppressive drug for injection, particularly for intravenous delivery, a continuous phase can be present that comprises an aqueous solution of tonicity modifiers, buffered to a pH below about 7, or below about 6, for example about 2 to about 7, about 3 to about 6 or about 3 to about 5. The tonicity modifiers can comprise, for example, sodium chloride, glucose, mannitol, trehalose, glycerol, or other pharmaceutical agents that render osmotic pressure of the formulation isotonic with blood. Alternatively, when a larger quantity of the tonicity modifier is used in the formulation, it can be diluted prior to injection with a pharmaceutically acceptable diluent to render the mixture isotonic with blood.

[00113] In another embodiment, the immunosuppressive drug of the present invention is administered by intravenous (IV) infusion or intra-arterial administration over a desired period (for example, bolus injection, 5 min, 15 min, 30 min, 1 hr, 2 hr, 3 hr, 6 hr, 24 hr, 48 hr, 72 hr or 96 hour infusions). In one embodiment of the present invention the period of administration is no greater than about 3 hours.

[00114] Without further description, it is believed that one of ordinary skill in the art may, using the preceding description and the following illustrative examples, make and utilize the agents of the present disclosure and practice the claimed methods. The following working examples are provided to facilitate the practice of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES

[00115] The present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The materials and methods as used in the following experimental examples are described below.

Example 1 : Expansion of Human Neural Stem Cells

[00116] Spinal cord from at least one donor of gestational age of approximately 7-

8.5 weeks was obtained. A single contiguous tissue of the spinal cord was dissociated in Ca ⁺⁺ and Mg ⁺⁺ -free phosphate buffered saline using mechanical trituration. The resulting cell suspension was then seeded into tissue culture plates pre-coated with both poly-L- ornithine or poly-D-lysine and human fibronectin or other extracellular matrix proteins. Tissue culture-treated plates or flasks were then incubated with 100 μg/ml poly-D-lysine for 1 hour at room temperature. They were then washed three times with water and dried. They were then incubated with 25 mg/ml for 5 minutes at room temperature. Sometimes, 10 mg/ml fibronectin for 1 hour at room temperature was used. Sometimes, 1 mg/ml fibronectin for 18 hours at 37°C. was used. Culture media consisting of N2 (DMEM/F12 plus insulin, transferrin, selenium, putrescine, and progesterone) was supplemented with 1 human recombinant basic fibroblast growth factor (bFGF). In an embodiment, a range of 0.1 ng/ml-100 ng/ml can be used. In an embodiment, 10 ng/ml of bFGF was used.

[00117] The resulting initial culture consists of post-mitotic neurons and proliferative NSCs in a monolayer. Subsequently, after approximately five to about twenty days in culture, the dividing, nestin-positive, NSCs dominate the culture over the non-dividing neurons or the slowly-dividing glia. Under these culture conditions, NSCs are selectively favored for expansion. The expanding NSC population was passaged by mild enzymatic treatment, such as using trypsin. The cells were then cultured in media free of serum or substantially free of serum. Although low concentration of serum may be tolerated by the cells, it is best to avoid exposing the cells to serum since serum contains many cytokines such as LIF and CNTF which promote glial differentiation of the NSCs. Thus, during passage, the enzyme used was stopped by adding specific enzyme inhibitor, such as trypsin inhibitor, rather than serum. At each passage, the number of harvested cells were counted, and a fraction was re-seeded for further expansion. Using this method, human NSCs can be expanded beyond 10¹⁸ -fold increase in population while maintaining their growth and differentiation properties. During the expansion, almost all cells express nestin, the in vivo marker of mitotic neuroepithelial cells, and are absent of antigens of differentiated neurons and glia such as type 3-beta tubulin and GFAP. The cells were also negative by immunostaining for PSA-NCAM, a possible marker of committed neuronal progenitors, 04 and GalC, markers of oligodendrocytes, and RC2, a marker of radial glia. Thus, determined by immunostaining, the NSCs stably maintain their expression of antigen profile throughout the prolonged expansion period.

Example 2: Differentiation of Human Spinal Cord Neural Stem/Progenitor Cells

[00118] At any point during expansion of the NSCs, the cultures can be differentiated by withdrawal of the mitogen in the culture such as bFGF. Differentiation of NSCs ensues within about 1-3 days after the removal of mitogen, and distinct heterogeneous cell morphologies are apparent. By approximately day 4-7 of differentiation, neuron-specific antigens, such as MAP2c, tau, and type III beta-tubulin, can be visualized by immunostaining. By approximately day 12-14, elongated, fasciculated axonal processes are evident throughout the culture along with clear polarization of subcellular protein trafficking. By approximately day 28, synaptic proteins, such as synapsin and synaptophysin, localize into axon terminals, appearing as punctate staining. Additional feeder layer of astrocytes can be provided to further promote long-term maturation of the neurons. Differentiation of human spinal NSCs generates mixed cultures of neurons and glia wherein the neurons robustly express neuron-specific antigens such as tau, MAP2ab and type3 beta tubulin and comprises approximately 50% of the culture. Additionally, the culture spontaneously generates long, bundled, axon cables that stretch for several centimeters. A significant proportion of the neurons are GABAergic with cholinergic motor neurons also being present in the culture. Presence of significant GABA neurons in culture predicts usefulness of the human spinal NSCs for treating various neurological conditions caused by decreased GABA production in certain circuitry. Likewise, presence of cholinergic neurons demonstrates that the human spinal NSCs are capable of motor neuron differentiation and predicts their usefulness for treating various motor neuron diseases caused by gradual degeneration of motor neurons. For treatment, the NSCs may be expanded with or without further phenotype-enhancing conditions, harvested, and injected into a neural area of deficiency.

[00119] In an exemplary method, prior to transplantation, spinal cord-derived NSCs (hereon referred to as, "NSI566 NSCs"), were tested in vitro for the expression of multipotent and neural markers. Robust expression of the neural stem cell marker nestin validated the undifferentiated state of the NSI566 NSCs (Figure 1 B). Additional in vitro differentiation analysis demonstrated the capability of NSI566 NSCs to generate cell types positive for the neuronal neurofilament proteins MAP2 and SMI312 and astocytic GFAP following growth factor deprivation for 7 days (Figure 1 B).

Example 3: Transplantation of Spinal-Cord Derived Neural Stem Cells to the Brain

[00120] A neuronal stem cell may be isolated, expanded in vitro and then introduced (e.g., transplanted) to one or more areas in a subject (e.g., a subject's brain) afflicted with cognitive dysfunction.

[00121] In an exemplary method for treatment of cognitive dysfunction, constitutively immunodeficient athymic nude (ATN) rats (stain 0N01 , Cr:NIH-rnu) were injected with NSI566 NSCs. All rats were maintained in an animal facility for the duration of the experiments and all animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Male ATN rats (2 months old, purchased from Frederick National Laboratory, NCI, MD, USA) were maintained in sterile housing conditions (20°C ± 1 °C; 70% humidity; 12 hour each light and dark cycle) and had free access to sterilized diet and water. Animals were divided into three groups: 0 Gy (no irradiation), sham surgery controls (CON, n=12), 10 Gy irradiated sham surgery (IRR, n=9), and 10 Gy irradiated with transplanted NSI566 NSCs (IRR+NSI, n=8). Rats were anesthetized, eyes and body were shielded and the head was exposed to cranial γ-irradiation (10 Gy) using a ¹³⁷Cs irradiator (J.L. Shepard, Mark I, CA, USA) at a dose rate of 2.07 Gy/min, as described in Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45) :19150-19155.

[00122] The NSI566 NSCs were transplanted in irradiated rats as represented in the schematic in Figure 1A. At two days post-irradiation, each rat received bilateral, intra- hippocampal transplantation of 70,000 NSI566 NSCs (IRR+NSI) in 1 μΙ_ of cell suspension using a 33-gauge microsyringe at an injection rate 0.25 μΙ_/Γηίη . Both hippocampi received 4 distinct injections (total 2.8 x 10^λ5 live NSI566 NSCs per hemisphere) using precise stereotaxic coordinates, as described in Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45): 19150-19155. Sham-operated control (CON) and irradiated (IRR) rats received sterile vehicle (hibernation buffer) at the same stereotaxic coordinates.

[00123] To evaluate the outcome of NSI566 NSC-transplantation on function, at 1- month post-transplantation, rats from CON, IRR and IRR+NSI groups were tested on novel place recognition(NPR), contextual and cued fear conditions (FC) tasks as described in Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45):19150-19155; Acharya et al., Cancer Res. (201 1 ) 71 (14)4834-4845; Christie et al., J. Vis. Exp. (201 1 ) 56:3108. FC was administered in three phases over three days: a training phase, a context test and a cue test. Cued FC memory has shown to rely on intact amygdala function, while contextual FC memory additionally engages the hippocampus. For all phases, rats were place in a PhenoTyper 3000 (Noldus Information Technology, Leesburg, VA) comprised of a video camera for observation and live tracking during FC trials. The animals were tested on NPR followed by FC. For both tasks, video recording and automated tracking of the animals was carried out using a Noldus Ethovision XT (version 7.0; Noldus Information Technology, Leesburg, VA), as described in Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45): 19150- 19155; Acharya et al., Cancer Res. (201 1 ) 71 (14)4834-4845; Christie et al., J. Vis. Exp. (201 1 ) 56:3108.; Christie et al., Clin. Cancer. Res. (2012) 18(7):1954-1965.

[00124] Statistical analyses were carried out using PASW Statistics 18 (SPSS, IBM Corporation, Somers, NY). All analyses were 2-tailed and a value of p < 0.05 was considered statistically significant. In all cases, normal distribution of the data (Kolmogorov-Smirnov test) and homogeneity of variance (Levene's test of equality of error variances) were confirmed. When a statistically significant overall group effect was found, multiple comparisons were made using Fisher's protected least significant different (FPLSD) post hoc tests to compare the individual groups.

[00125] For the NPR tasks, exploration ratio, or the proportion of total time spent exploring the novel spatial location (t_novei/tnovei⁺ t_famiiiar) was used as the main dependent measure. The behavior of the animals during minute 1 of the 5-minute and 24-hour test phases was analyzed. For details see, Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45):19150-19155 and Christie et al., Clin. Cancer Res.(2012) 18(7):1954-1965. The number of animals included for NPR task analyses were: CON (n=12), IRR (n=9) and IRR+NSI (n=8).

[00126] For the FC task, percentage of time spent freezing was used as the main dependent measure. Freezing was assessed during the final minute of the baseline (i.e. before tone-shock pairings were administered) and post-training (after tone-shock pairings) phases. For the context test, freezing was assessed over the entire 5-minute trial. For the pre-cue test, freezing was assessed during the first minute, in which no tone was sounded, and for the cue test, freezing was assessed across the three minute interval that the tone was sounded and for the final minute of the trial in which no tone was sounded. Repeated measures ANOVA were used to assess group (between subjects factor) and phase (within subjects factor) effects on freezing behavior. For details see Christie et al., Clin. Cancer Res.(2012) 18(7): 1954-1965. A separate cohort of animals for each group (CON, IRR, and IRR+NSI), n=8 animals were analyzed for the FC task analyses.

[00127] As shown in Figure 2, animals treated with NSI566 NSCs exhibited improvements in cognition 1 month post-transplantation in rats receiving head-only irradiation, as assessed by two well characterized an widely used cognitive task as compared to controls. For the novel place recognition (NPR) task, animals were first familiarized with two identical objects in specific spatial locations in an open field arena and total time spent exploring both identical objects was assessed. Following a 5 minute retention interval, animals were placed in the same arena with one object moved to a novel spatial location. In contrast to irradiated animals that underwent sham transplantation surgery, the performance of NSI566 NSC-engrafted animals was indistinguishable from unirradiated controls on the 5min NPR task (Figure 2B), with both controls and transplanted animals showing significant preference for exploring the novel place. Differences in exploration ratios between cohorts re-tested 24h later on the NPR task were not found to be significant, as animals only showed a trend for exploring the novel location at this time (Figure 2C).

[00128] Using the FC task, a specific impairment was detected (Figure 2D). Animals subjected to cranial irradiation spent significantly less time engaged in freezing behavior than controls during the context phase of the task. Interestingly, animals engrafted with NSI566 NSCs demonstrated intact freezing behavior and were statistically indistinguishable from controls in their contextual fear memory. The amount of post- training freezing observed was comparable between all experimental cohorts, indicating that irradiation, sham surgery and/or transplantation of NSI566 NSCs did not affect initial acquisition of the conditioned freezing response. Similarly, the experimental condition imposed on animal cohorts did not affect freezing behavior during the cue test phase, indicating intact acquisition and memory for the conditioned tone stimulus. Importantly, these studies demonstrate that intrahippocampal transplantation of spinal cord-derived neural stem cells can rescue cognition following exposure to cranial irradiation.

[00129] Approximately two months after the rats were subjected to the transplantation, and following cognition testing, 8 animals in each group were euthanized for immunohistochemical analysis to determine the pattern of integration of transplanted NSI566 NSCs (Figure 3). Animals were deeply anesthetized with isoflurane and euthanized by intracardiac perfusion with 4% paraformaldehyde (Acros Organics, NJ, USC). Tissues were processed in a sucrose gradient (10-30%) and 30 μΓη-thick sections cut coronally through the hippocampus using a cryostat (Leica Microsystems, Wetzlar, Germany) were then stored in phosphate buffered saline (PBS) with 0.02% sodium azide (Sigma-Aldrich, MO, USA).

[00130] Immunohistochemical studies employed both monoclonal and polyclonal antibodies. Primary antibodies were as follows: anti-Ku80 (human specific DNA telomere- binding nuclear protein, mouse, 1 :100, STEM101™, StemCell Inc., Cambridge, UK), anti- DCX (doublecortin, goat, Santa Cruz Biotechnology Inc., CA, USA), anti-NeuN (neuron specific nuclear antigen, rabbit, 1 :250, Millipore, MA, USA), anti-GFAP (glial fibrillary acidic protein, rabbit, 1 :500, Millipore, MA, USA), anti-MAP2 (neuronal filament protein, monoclonal AP20, mouse, 1 :200, Sigma-Aldrich, MO, USA), anti-SMI312 (pan-axonal neurofilament, mouse, 1 :200, EMD Millipore, MA, USA), anti-nestin (rabbit, 1 :200, EMD Millipore, MA, USA). The secondary antibodies and detection reagents included biotinylated horse anti-goat IgG (1 :200, Vector Labs, CA, USA), donkey anti-mouse and anti-rabbit conjugated with Alexa Fluor 488 or 594 (1 :200, Invitrogen, CA, USA) and TOTO- 3 iodide (infrared nuclear counterstain, Invitrogen, CA, USA).

[00131] To identify and track differentiated fates of transplanted human cells, representative sections were processed using dual immunofluorescence staining for human-specific nuclear antigen (Ku80) and various mature and immature neuronal (DCX, NeuN) and astro-glial (GFAP, S100) markers. Serial sections taken through the middle of the hippocampus were selected for staining and stores in tris-buffered saline (TBS, 100mM, pH 7.4, Sigma Aldrich, MO, USA) overnight. Free floating sections were first rinsed in TBS followed by Tris-A (TBS with 0.1 % Triton-X-100, Sigma-Aldrich, MO, USA), blocked with 10% normal donkey serum (NDS with Tris-A, Sigma-Aldrich, MO, USA) and incubated overnight in a mouse anti-Ku80 solution (1 :100) prepared in 3% NDS and Tris-A. The next day, the sections were treated with donkey anti-mouse IgG conjugated with Alexa Fluor 594 (1 :200) made with Tris-A and 3% NDS for 1 h. The sections were light-protected, washed with Tris-A and blocked in serum and primary antibodies for neuronal and glial markers. Color development was facilitated by Alexa Fluor conjugated secondary antibodies, as described above, and counter stained with TOTO-3 nuclear dye for visualization of hippocampal morphology. Immunostained sections were rinsed in TBS and mounted on clean Vectabond (Vector Labs, CA, USA) coated slides using SlowFade anti- fade mounting medium (Invitrogen, CA, USA).

[00132] Laser scanning confocal analyses to identify phenotypic fate of graft- derived cells were performed using a Nikon Eclipse microscope (TE2000-U, EZ-C1 interface), as described previously in Acharya et al., Proc. Natl. Acad. Sci. (2009) 106(45):19150-19155; Acharya et al., Cancer Res. (201 1 ) 71 (14)4834-4845. Z-stack analysis (1 μηι intervals) and orthogonal image reconstruction were done using Nikon Elements AR software (v3.22, Nikon Instech Co. Ltd, Japan).

[00133] Examination of Ku80 (human-specific nuclear antigen) immunofluorescence stained sections from IRR+NSI group demonstrated the presence of grafts along the entire spot-temporal axis of the hippocampus (Figure 3). Visualization with Ku80 marker revealed that transplanted cells remained in clusters at the transplant site and very few Ku80⁺ cells were observed migrating away from the transplant core in the irradiated host hippocampus (Figure 3 C-D). With the exception of 2 animals (wherein transplant was found above DG), transplant-derived cells were located above the CA1 subfield with projections into the corpus callosum (CC), and they formed a transplant core (Tc, Figure 3 A-C). As shown in Figure 3, the location of transplant did not distort the host hippocampal cytoarchitecture (TOTO-3 nuclear counter stain, Figure 3).

[00134] To determine the phenotypic fate of transplant-derived cells in the irradiated animals following NSI transplantation, dual immunofluorescence and confocal Z- stack analyses were carried out in representative sections of IRR+NSI group. Examination of dual immunofluorescence-stained Ku80⁺ and various neuronal and astrocytic markers demonstrated the presence of both phenotypes in the irradiated hot hippocampus (Figure 4). Transplant-derived cells expressed immature (DCX⁺, Figure 4A) and mature (NeuN ⁺, Figure 4B) neuronal markers. Moreover, assessment of astrocytic differentiation using Ku80 revealed the presence of double-labeled immature (GFAP⁺, Figure 4C) and mature (S100⁺, Figure 4D) phenotypes. Orthogonal reconstructions of confocal Z-stacks are shown for each marker (Figure 4a-d). Qualitative examination of all animals indicated that the majority of transplanted cells differentiated into neurons that were still in the developmental stage (DCX⁺, Figure 4A) and located primarily in the transplant core, whereas a minority of transplant-derived cells expressed astrocytic markers (Figure 4C-D). These results indicate robust survival of grafted cells approximately 2 months following transplantation in the irradiated CNS. Further, while evidence suggests that NSI graft- derived cells undergo extensive proliferation within the transplant core, this does not appear to translate to teratomas formation over the duration of the study.

Example 5: Treatment of Subject with Cognitive Dysfunction

[00135] Studies are conducted to determine the effects treating cognitive dysfunction in a subject. For example, a multicenter, randomized, double-blind, placebo- controlled study is undertaken to evaluate treatment with a weight-based or fixed dose of spinal cord-derived neural stem cells (NSCs) in human subjects diagnosed with cognitive dysfunction. More specifically, a clinical study was performed to examine the efficacy and safety of introducing a therapeutically effective amount of spinal cord-derived NSCs to at least one area of the brain of the human subject. The composition is effective to treat cognitive dysfunction.

[00136] Surgery is performed using standard anesthetic and monitoring techniques. For each human subject 10 μΙ_ of the live NSI566 NSC cell suspension is microinjected into each site at a rate of 5 μΙ_/Γηίη over 2 minutes. Each injection contains approximately 100,000 cells (about 10,000 cells per microliter). The needle tip is left in place for 1 minute postinfusion to reduce suspension reflex. The process is completed over 5 to 10 distinct injections using precise stereotaxic coordinates. Control patients received sterile vehicle (hibernation buffer) at the same stereotaxic coordinates. All patients are monitored and cared for following injections by standard post operative care procedures.

[00137] Once the NSCs are present in the afflicted individual, they engraft and differentiate and thereby help treat the cognitive dysfunction. One advantage of this method is that it may be repeated, as needed, and thereby alleviate some or all of the cognitive dysfunction in the human subject. Optionally, cells may be differentiated into appropriate cell types in vitro before transplantation.

[00138] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, in accordance with one aspect of the subject matter herein, there is provided methods for treating a disease or disorder associated with cognitive dysfunction, the methods comprising: introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of brain tissue of the subject.

[00139] In accordance with another aspect of the subject matter herein, there is provided methods of treating cognitive dysfunction in a subject, said method comprising: obtaining at least one neural stem cell from spinal cord tissue of a human; expanding the at least one neural stem cell to form an expanded neural stem cell population; concentrating the expanded neural stem cell population; and introducing a therapeutically effective amount of the expanded neural stem cell population to one or more areas of brain tissue of the subject.

[00140] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the spinal cord-derived neural stem cells are embryonic spinal cord-derived neural stem cells. [00141] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the spinal cord-derived neural stem cells are fetal spinal cord-derived neural stem cells.

[00142] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the fetal spinal cord-derived neural stem cells are obtained from a fetus being a gestational age of about 5 to about 20 weeks.

[00143] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the spinal cord-derived neural stem cells are human spinal cord-derived neural stem cells.

[00144] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, expanding the spinal cord-derived neural stem cells to form an expanded spinal cord-derived neural stem cell population.

[00145] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, expanding the spinal cord-derived neural stem cells includes culturing the spinal cord-derived neural stem cells in the absence of serum.

[00146] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, expanding the spinal cord-derived neural stem cells includes exposing the spinal cord-derived neural stem cells to at least one growth factor.

[00147] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof.

[00148] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the spinal cord-derived neural stem cells differentiate into neurons that engraft in vivo into the brain tissue of the subject.

[00149] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,

90%, 95% or more of the spinal cord-derived neural stem cells are capable of generating neurons in brain tissue of the subject.

[00150] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the subject is human.

[00151] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, wherein the cognitive dysfunction is induced by prior exposure of the subject to radiation.

[00152] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the cognitive dysfunction is associated with Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, Multiple Sclerosis, cerebrovascular disease or substance abuse.

[00153] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the cognitive dysfunction is dementia, delirium or amnesia.

[00154] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the dementia is vascular dementia, dementia with Lewy bodies, mixed dementia, frontotemporal dementia.

[00155] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the cognitive dysfunction includes speech impairment, confusion, disorientation, loss of memory, learning, perception, judgment, initiative, attention, planning, multitasking, spatial or analytical skills, reasoning ability, or combinations thereof.

[00156] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, introducing the therapeutically effective amount of spinal cord-derived neural stem cells includes injecting at least a portion of the therapeutically effective amount of spinal cord-derived neural stem cells into a plurality of areas of the brain tissue of the subject

[00157] In accordance with another aspect which may be used or combined with any of the preceding or following aspects, the areas of the brain tissue include cerebral hemispheres, cerebral cortex, subcortex, motor cortex, striatum, internal capsule, thalamus, hypothalamus, hippocampus, midbrain, brainstem and cerebellum.

[00158] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[00159] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [00160] The terms "a," "an," "the" and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[00161] Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[00162] Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[00163] Specific embodiments disclosed herein can be further limited in the claims using consisting of or and consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the disclosure so claimed are inherently or expressly described and enabled herein.

[00164] It is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

[00165] While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.

Claims

A method for treating a subject with a cognitive dysfunction, the method comprising: introducing a therapeutically effective amount of spinal cord-derived neural stem cells to one or more areas of the subject's brain.

The method of claim 1 , wherein the spinal cord-derived neural stem cells are embryonic spinal cord-derived neural stem cells.

The method of claim 1 , wherein the spinal cord-derived neural stem cells are fetal spinal cord-derived neural stem cells.

The method of claim 3, wherein the fetal spinal cord-derived neural stem cells are obtained from a fetus being a gestational age of about 5 to about 20 weeks.

The method of claim 1 , wherein the spinal cord-derived neural stem cells are human spinal cord-derived neural stem cells.

The method of claim 1 further comprising expanding the spinal cord-derived neural stem cells to form an expanded spinal cord-derived neural stem cell population.

The method of claim 6, wherein expanding the spinal cord-derived neural stem cells includes culturing the spinal cord-derived neural stem cells in the absence of serum.

The method of claim 6, wherein expanding the spinal cord-derived neural stem cells includes exposing the spinal cord-derived neural stem cells to at least one growth factor.

The method of claim 8, wherein the growth factor is selected from the group consisting of bFGF, EGF, TGF-alpha, aFGF and combinations thereof.

The method of claim 1 , wherein the spinal cord-derived neural stem cells differentiate into neurons that engraft in vivo into the brain tissue of the subject.

1 1 . The method of claim 1 , wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the spinal cord-derived neural stem cells are capable of generating neurons in brain tissue of the subject.

12. The method of claim 1 , wherein the subject is human.

13. The method of claim 1 , wherein the cognitive dysfunction is induced by prior exposure of the subject to radiation.

14. The method of claim 1 , wherein the cognitive dysfunction is associated with traumatic brain injury, diabetes, dementia, depression, aging, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, Multiple Sclerosis, epilepsy, cerebrovascular disease or substance abuse.

15. The method of claim 1 , wherein the cognitive dysfunction is dementia, delirium or amnesia.

16. The method of claim 15, wherein the dementia is vascular dementia, dementia with Lewy bodies, mixed dementia, frontotemporal dementia.

17. The method of claim 1 , wherein the cognitive dysfunction includes speech impairment, confusion, disorientation, loss of memory, learning, perception, judgment, initiative, attention, planning, multitasking, spatial or analytical skills, reasoning ability, or combinations thereof.

18. The method of claim 1 , wherein introducing the therapeutically effective amount of spinal cord-derived neural stem cells includes injecting at least a portion of the therapeutically effective amount of spinal cord-derived neural stem cells into a plurality of areas of the brain tissue of the subject.

19. The method of claim 18, wherein the areas of the brain tissue include cerebral hemispheres, cerebral cortex, subcortex, motor cortex, basal ganglia, striatum, internal capsule, thalamus, hypothalamus, hippocampus, corpus callosum, midbrain, substantia nigra, brainstem and cerebellum.

20. The method of claim 1 , wherein the spinal cord-derived neural stems are injected into the hippocampus.

21 . A method for treating a subject with a brain cancer, the method comprising, administering radiation therapy to the brain of the subject, and introducing a therapeutically effective amount of spinal cord-derived neural stem cells to the subject's brain, wherein the therapeutically effective amount of spinal cord-derived neural stem cells is effective to treat a cognitive dysfunction.

22. The method of claim 21 , wherein the cognitive dysfunction includes speech impairment, confusion, disorientation, loss of memory, learning, perception, judgment, initiative, attention, planning, multitasking, spatial or analytical skills, reasoning ability, or combinations thereof.

23. The method of claim 21 , wherein the spinal cord is from a gestation age of about 5 to about 20 weeks.

24. The method of claim 21 , wherein introducing the therapeutically effective amount of the expanded neural stem cell population includes injecting at least a portion of the therapeutically effective amount into a plurality of areas of the brain tissue of the subject.

25. The method of claim 24, wherein the areas of the brain tissue include cerebral hemispheres, cerebral cortex, subcortex, motor cortex, basal ganglia, striatum, internal capsule, thalamus, hypothalamus, hippocampus, corpus callosum, midbrain, substantia nigra, brainstem and cerebellum.

26. The method of claim 21 , wherein the spinal cord-derived neural stems are injected into the hippocampus.