MXPA00000225A - Lineage-restricted neuronal precursors - Google Patents

Abstract

A self-renewing restricted stem cell population has been identified in developing (embryonic day 13. 5) spinal cords that can differentiate into multiple neuronal phenotypes, but cannot differentiate into glial phenotypes. This neuronal-restricted precursor (NRP) expresses highly polysialated or embryonic neural cell adhesion molecule (E-NCAM) and is morphologically distinct from neuroepithelial stem cells (NEP cells) and spinal glial progenitors derived from embryonic day 10.5 spinal cord. NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin 3 (NT-3) and express a characteristic subset of neuronal epitopes. When cultured in the presence of RA and the absence of FGF, NRP cells differentiate into GABAergic, glutaminergic, and cholinergic immunoreactive neurons. NRP cells can also be generated from multipotent NEP cells cultured from embryonic day 10.5 neural tubes. Clonal analysis shows that E-NCAM immunoreactive NRP cells arise from an NEP progenitor cell that generates other restricted CNS precursors. The NEP-derived E-NCAM immunoreactive cells undergo self renewal in defined medium and differentiate into multiple neuronal phenotypes in mass and clonal culture. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at embryonic day 13.5 in the spinal cord. Methods for treating neurological diseases are also disclosed.

Description

NEURAL PRECURSORS OF RESTRICTED LINEAGE CROSS REFERENCE TO RELATED REQUESTS This request is a continuation in part of the Serial request No. 08 / 909,435, filed July 4, 1997.

DECLARATION REGARDING RESEARCH OR FEDERALLY SPONSORED DEVELOPMENT This invention was made with the support of the government under a first grant and a Multidiculture and Basic Cancer Research Training Grant Graduate Fellowship of the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION This invention relates to intermediate precursor cells of restricted lineage and methods for making and using same. More particularly, the invention relates to neuronal restricted precursors (NRP's) isolated from mammalian embryos, neuroepithelial stem cells (NEP), embryonic stem cells (ES). These neuronal restricted precursors are capable of self-renewal and differentiation in neurons, but not in glia, that is, astrocytes and oligodendrocytes. It also describes methods to generate, isolate, culture, transfect and transplant said neuronal restricted precursor cells. Multipotent cells with the characteristics of stem cells have been identified in several regions of the central nervous system and in various stages of development. F. H. Gage et al., Isolation, Characterization and Use of Stem Cells from the CNS, 18 Ann. Rev. Neurisci. 159-92 (1995); M. Marvin & R. McKay, Multipotential Stem Cells in the Vertébrate CNS, 3 Semin. Cell. Biol. 401-11 (1992); R. P. Skoff, The Linages of Neuroglial Cells, 2 The Neuroscientist 335-44 (1996). These cells, usually called neuroepithelial stem cells (NEP cells), have the ability to undergo self-renewal and differentiate into neurons, oligodendrocytes and astrocytes, thus representing multipotent stem cells. A. A. Davis & S. Temple, A Self-Renewing Multipotential Stem Cells in Embryonic Brain Rat Cortex, 362 Nature 363-72 (1994); A. G. Gritti et al., Multipotential Stem Cells from the Adult Mouse Brain Proliferate and Self-Renew in Response to Basic Fibroblast Growth Factor, 16 J. Neurosci. 1091-1100 (1996); B. A. Reynolds et al., A Multipotent EGF-Responsive Striatal Embryonic Progenitor Cell Produces Neurons and Astrocytes, 12 J. Neurosci. 4565-74 (1992); B. Reynolds & S. Weiss, Clonal and Population Analyzes Demonstrate that an EGF-Responsive Mammalian Embryonic CNS Precursor is a Stem Cell, 175 Developmental Biol. 1-13 (1996); B. P. Williams and others, The Generation of Neurons and Oligodendrocytes from a Common Precursor Cell, 7 Neuron 685-93 litlÉifiíiij ^ (1991). The nervous system also contains precursor cells with restricted differentiation potentials. T. J. Kilpatrick & P. F. Bartlett, Cloned Multipotential Precursors from the Mouse Cerebrum Require FGF-2, Glial Restricted Precursors are Stimulated with Either FGF-2 or EGF, 15 J. Neurosci. 3653-61 (1995); J. Price et al., Linage Analysis in the Vertébrate Nervous System by Retrovirus-Mediated Gene Transfer, 84 Developmental Biol. 156-60 (1987); B. A. Reynolds et al., Supra; B. Reynolds & S. Weiss, supra; B. Williams, Precursor Cell Types in the Germinal Zone of the Cerebral Cortex, 17 BioEssays 391-93 (1995); B. P. Williams and others, supra. The relationship between multipotent stem cells and restricted lineage precursor cells remains unknown. In principle, cells of restricted lineage can be derived from multipotent cells, but there is still a hypothetical possibility in the nervous system without any direct experimental evidence. In addition, no method for purifying said precursors from multipotent cells has been described. As shown in the patent application of E.U.A. Copendent Series No. 08 / 852,744, entitled "Generation, Characterization, and Isolation of Neuroepithelial Stem Cells and Lineage Restricted Intermediate Precursor" (Generation, Characterization and Assimilation of Neuroepithelial Stem Cells and Intermediate Precursor of Restricted Lineage), presented on May 7 1997, incorporated herein by reference in its entirety, the NEP cells grow in fibronectin and require fibroblast growth factor (FGF) and an as yet uncharacterized component present in chicken embryo extract (CEE) to proliferate and maintain an undifferentiated phenotype in culture. The growth requirements of NEP cells are different from neurospheres isolated from cortical ventricular zone cells E14.5. B. A. Reynolds et al., Supra; B. Reynolds & S. Weiss, supra; WO 9615226; WO 9615224; WO 9609543; WO 9513364; WO 9416718; WO 9410292; WO 9409119. The neurospheres grow in a suspension culture and do not require CEE or FGF, but are dependent on epidermal growth factor (EGF) to survive. The same FGF is not sufficient for the long-term growth of neurospheres, although FGF can support its growth in a transient manner. NEP cells, however, grow in adherent culture, are dependent on FGF, do not express detectable levels of EGF receptors, and are isolated at a stage of embryonic development before it is possible to isolate the neurospheres. In this way, NEP cells can represent a multipotent precursor characteristic of the brain stem and spinal cord, while neurospheres can represent a stem cell more characteristic of the cortex. However, NEP cells provide a model system for studying the lineage restriction principles of multipotent cells or precursor cells of the central nervous system. It is expected that the principles produced from the study of NEP cells will be widely ^^^ ^^^^^^^^^^ & s ^^ s ^^^^^^^^^^^^ i ^ tt. ^ s' - ?? i-sÁssewíüsi ?: applicable to all CNS precursor cells sufficiently multipotent to generate both neurons and glia. Thus, the present application is intended to be applicable to any of the CNS precursor cells without considering their derivation site as long as they are capable of differentiating both cells and glial cells. The patent of E.U.A. No. 5,589,376, by DJ Anderson and DL Stemple, describes mammalian neural crest stem cells and methods for isolation and clonal propagation thereof, but fails to describe cultured NEP cells, restricted lineage precursor cells and methods for generation, isolation and cultivation of the same. The neural crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), while the neuroepithelial stem cells differentiate into neurons and central nervous system (CNS) glia. The patent of E.U.A. No. 08 / 909,435, filed July 4, 1997, for "Isolation of Lineage Restricted Neuronal Precursors", incorporated herein by reference in its entirety, describes neuronal restricted precursor cells (NRP) that they are able to differentiate neurons, but not glial cells. It has been shown that NRP cells can be isolated from NEP cells, as well as directly from embryonic spinal cords. The patent of E.U.A. No. 08 / 980,850, filed on November 29, 1997, for "Lineage Restricted Glial Precursors from ñilf ifif rí ^^. the Central Nervous System "(Glycemic Precursors of Restricted Lineage of the Central Nervous System), incorporated herein by reference in its entirety, discloses glial restricted precursor cells (GRP) that are capable of differentiating into oligodendrocytes, astrocytes carrying the A2B5 * process, and astrocytes of fibroblast type A2B5 *, but not in neurons GRP cells can be isolated from differentiation NEP cells, as well as from CNS tissue, and differ from astrocyte progenitor cells (O-2A) of type 2 oligodendrocyte in requirements of Growth Factor, Morphology and Progeny In US Patent Application Serial No. 09 / 073,881, filed May 6, 1998, for "Common Neural Progenitor for CNS and PNS" (Common Neural Progenitor for CNS and PNS), incorporated herein by reference in its entirety, it has been shown that NEP cells can be induced to differentiate into neural crest cells as well as other CNS and PNS cells. The restricted neuron precursor cells described herein are distinct from NEP cells, GRP cells, neurospheres and neural crest stem cells that have already been described. NEP cells are capable of differentiating into neurons or glia, whereas NRPs can differentiate into neurons, but not glial, and NEP and NRPs display different cell markers. GRP cells can differentiate into glia, but not neurons. As mentioned above, the neurospheres grow in a suspension culture and do not require CEE or FGF, but they are e ^^ & ^^^ g EGF-dependent for survival, while NRP cells grow in an adherent culture and do not express detectable levels of EGF receptors. In addition, neuronal crest cells differentiate into neurons and glia of the peripheral nervous system (PNS), while NRP cells differentiate into neurons of the central nervous system (CNS). NRP cells express the polysialated or embryonic neural cell adhesion molecule (E-NCAM), but NEP cells, neurospheres, GRP cells, and neural crest cells do not. Therefore, NRP cells are different in their potential proliferative requirements, expression of cell and nutritional markers of these other cell types. The ability to isolate and develop mammalian neuronal restricted precursor cells, in vitro, allows us to use pure populations of neurons for transplantation, discovery of specific genes for selected stages of development, generation of specific antibodies in the cell for therapeutic and diagnostic uses such for target gene therapy, and the like. In addition, NRP cells can be used to generate sub-populations of neurons with specific properties, ie motor neurons and other neuronal cells to analyze neurotransmitter functions and small molecules in high production assays. Additionally, methods to obtain NRP cells from NEP cells or embryonic stem cells (ES) provide a source of a large number of neurons post- i, aS & Faith? Bi'r -. ^ '»S ^^ mitotic Post-mitotic cells obtained from a tumor cell line are already commercially available (e.g., Clontech, Palo Alto, CA). The present invention is also necessary to understand how multipotent neuroepithelial stem cells become restricted to the various neuroepithelial derivatives. In particular, culture conditions that allow the growth and self-renewal of mammalian neuronal restricted precursor cells are desirable, so that particular aspects of the development of these mammalian stem cells can be ascertained. This is desirable since a number of neuroepithelial derivative tumors exist in mammals, particularly humans. Knowledge of the development of the mammalian neuroepithelial stem cell, therefore, is necessary to understand these disorders in humans. In view of the foregoing, it will be appreciated that isolated populations of restricted lineage neuronal precursor cells and methods for generating, isolating, culturing, transfecting and transfecting said cells could be important advances in the art.

COMPENDIUM OF THE INVENTION It is an object of the present invention to provide isolated (pure) populations of mammalian neuronal restricted precursor cells and their progeny. td ^^^ i ^^? iáí ^ & ^^^^^, It is another object of the invention to provide methods for generating, isolating, culturing, and regenerating neuronal precursor cells of restricted mammalian lineage and their progeny. It is a further object of the invention to provide a method for the generation of restricted lineage neuronal precursor cells from a multipotent CNS precursor cell capable of generating both neurons and glia. It is yet another object of the invention to provide pure differentiated populations of neuronal cells derived from neuronal precursor cells of restricted lineage. It is another object of the invention to provide methods for transfecting and transplanting said neuronal restricted precursor cells. These and other objects can be achieved by providing an isolated, pure population of neuron precursor cells restricted from the mammalian CNS. Preferably, said neuron-restricted precursor cells are capable of self-renewal, differentiation into neuronal cells of CNS, but not glial cells of the CNS, and expressing the embryonic neuronal cell adhesion molecule (E-NCAM), but not expressing a ganglioside recognized by the A2B5 antibody. These restricted neuron precursor cells may or may not express nestin or tubulin β-III. In this way, the embryonic neural cell adhesion molecule (E-NCAM) is a defining antigen for these cells. NRP cells are able to differentiate into neurons that are capable of t ^ WT '-' ^ U ^ U ^^^ "^ -" release and respond to neurotransmitters. These neurons can show receptors for these neurotransmitters, and these cells are capable of expressing neurotransmitter-synthesis enzymes. NRP cells are also capable of differentiation into neurons that can form functional synapses and / or develop electrical activity. NRP cells are also capable of stably expressing at least one material selected from the group consisting of growth factors for said cells, differentiating factors for said cells, maturation factors for said cells, and combinations of any of these. In addition, the restricted neuron precursor cells of the present can be selected, chosen and isolated from human primates, non-human primates, equines, canines, felines, bovines, swine, sheep, lagomorphs and rodents. A method for isolating a pure population of neuron precursor cells restricted to the mammalian CNS comprises the steps of: (a) isolating a population of stem cells from the mammalian multipotent CNS capable of generating both neurons and glia; (b) incubating the stem cells of the multipotent CNS in a medium configured to induce said cells to begin differentiation; (c) purifying from the differentiation cells a sub-population of cells expressing a selected antigen that defines precursor cells of restricted neuron; and (d) incubating the purified sub-population of cells in a medium configured to support their adherent growth. A preferred selected antigen that defines neuron-restricted precursor cells is an embryonic neural cell adhesion molecule. Preferably, the step of purifying the NPR cells comprises a method selected from the group consisting of specific antibody capture, fluorescence activated cell sorting, and magnetic bead capture. The capture of specific antibody is especially preferred. In a preferred embodiment, the stem cells of the mammalian multipotent CNS are neuroepithelial stem cells. A preferred method for isolating a population of neuroepithelial stem cells from the CNS comprises: (a) removing a CNS tissue from a mammalian embryo at an embryonic stage of development after neural tube closure, but before differentiating cells in the neural tube; (b) dissociating cells comprising the neural tube removed from the mammalian embryo; (c) plating the dissociated cells in an independent cell-feeder culture on a sub-stratum and in a medium configured to support the adherent growth of the neuroepithelial stem cells comprising effective amounts of the fibroblast growth factor and extract of embryo chicken; and (d) incubating the plated cells at a temperature and atmosphere that lead to the growth of the neuroepithelial stem cells. Preferably, the mammalian embryo is selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, swine, sheep, lagomorphs, and rodents.

It is also preferred that the sub-stratum be selected from the group consisting of fibronectin, vitronectin, inina and RGD peptides. In a preferred embodiment, the medium comprises effective amounts of fibroblast growth factor and neurotrophin 3 (NT-3). A method for isolating a pure population of mammalian CNS-restricted neuron precursor cells comprises the steps of: (a) removing a tissue sample from the CNS from a mammalian embryo to an embryonic development stage after tube closure neural, but before the differentiation of glial and neuronal cells in the neural tube; (b) dissociating cells comprising the sample of the CNS tissue removed from the mammalian embryo; (c) purifying from the dissociated cells a sub-population that expresses a selected antigen that defines precursor neuron-restricted cells; (d) plating the purified sub-population of cells in an independent cell-feeder culture over a sub-cell. i ^ e ^^ ü ^ tm ^ i < ^ »^^ ^ t mát ^ l? Á? I? M stratum and in a medium configured to support the adherent growth of the neuron-restricted precursor cells; and (e) incubating the cells plated at a temperature and atmosphere that lead to the growth of the neuron-restricted precursor cells. Preferably, the selected antigen that defines neuron-restricted precursor cells is an embryonic neural cell adhesion molecule. It is also preferred that the step of purifying comprises a method selected from the group consisting of specific antibody capture, fluorescence-activated cell sorting and magnetic bead capture. The capture of specific antibody is especially preferred. It is further preferred that the mammalian embryo be selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, swine, sheep, lagomorphs and rodents. A method for obtaining post-mitotic neurons comprises: (a) providing restricted neuron precursor cells and culturing the neuron-restricted precursor cells under proliferation conditions; and (b) changing the culture conditions of the neuron precursor cells restricted from proliferation conditions to a differentiation condition, thereby causing the neuron precursor cells restricted to differentiate into post-mitotic neurons. The change of culture conditions preferably includes adding retinoic acid to the basal medium or removing a mitotic factor from the basal medium. Said mitotic factor is the fibroblast growth factor. Changing culture conditions may also include adding a neuronal maturation factor to the basal medium. The preferred neuronal maturation factors are selected from the group consisting of sonic hedgehog, BMP-2, BMP-4, NT-3, NT-4, CNTF, LIF, retinoic acid, brain-derived neurotrophic factor (BDNF), and combinations of any of the above. Another preferred embodiment of the invention comprises an isolated cellular composition comprising the mammalian CNS restricted neuron cells described herein. Another preferred embodiment of the invention comprises a pharmaceutical composition comprising a therapeutically effective amount of said composition and a pharmaceutically acceptable carrier. A method for treating a neuronal disorder in a mammal comprises administering to said mammal a therapeutically effective amount of the isolated cellular composition comprising the mammalian CNS restricted neuron cells described herein. Another method of treating a neuronal disorder in a mammal comprises administering to said mammal a therapeutically effective amount of said pharmaceutical composition and a pharmaceutically acceptable carrier. Said composition can be administered through a route selected from the group consisting of intramuscular administration, intraheal administration, administration intraperitoneal, intravenous administration and combinations of any of the above. This method may also include administration of a member selected from the group consisting of differentiation factors, growth factors, cell maturation factors and combinations of any of the foregoing. Said differentiation factors are preferably selected from the group consisting of retinoic acid, BMP-2, BMP-4 and combinations of any of the foregoing. A method for treating neurodegenerative symptoms in a mammal comprises the steps of: (a) providing a pure population of neuronal restricted precursor cells; (b) genetically transforming said neuronal restricted precursor cells with a gene encoding a growth factor, neurotransmitter, neurotransmitter synthesis enzyme, neuropeptide synthesis enzyme, or substance that provides against free radical mediated damage, thus resulting in a transphorea population of glial restricted precursor cells expressing said growth factor, neurotransmitter, neurotransmitter synthesis enzyme, neuropeptide, neuropeptide synthesis enzyme, or substance that provides protection against free radical mediated damage; and (c) administering an effective amount of said transformed population of neuronal restricted precursor cells to said mammal. A method for classifying compounds for neurological activity comprising the steps of: (a) providing a pure population of neuronal restricted precursor cells or derivatives thereof or mixtures thereof cultured in vitro; (b) exposing said cells or their derivatives or mixtures thereof to a selected compound at varying doses; and (c) verifying the reaction of said cells or their derivatives or mixtures thereof to said selected compound during selected periods. A method for treating a neurological or neurodegenerative disease comprises administering to a mammal in need of such treatment an effective amount of neuronal restricted precursor cells or their derivatives or mixtures thereof. Said neuronal restricted precursor cells or their derivatives or mixtures thereof can be either a heterologous donor or an autologous donor. The donor can be a fetus, young or adult. A method for isolating a pure population of mammalian CNS-restricted neuron precursor cells comprises the steps of: (a) providing a sample of mammalian embryonic stem cells; (b) purify from mammalian embryonic stem cells a sub-population that expresses a selected antigen that defines '"* ^! ^^! --- ^^^^^^^^ i ^^ * ^ = restricted neuron precursor cells; (c) plating the purified sub-population of cells in a cell-feeder independent culture on a sub-stratum and in a medium configured to support the adherent growth of the neuron-restricted precursor cells; and (d) incubating the cells plated at a temperature and atmosphere that lead to the growth of the neuron-restricted precursor cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a summary of the immuno-reactivities of NEP cells and their progeny, including NRP cells. Figure 2 shows results of the RT-PCR amplification of the total RNA isolated from rat E-NCAM + cells to determine the expression of choline acetyl transferase (ChAT), p75,? Slet-1 (lsl-1), clabindin, decarboxylase of glutamic acid (GAD), glutaminase and cyclophilin (a driving gene). Figure 3 shows a bar graph of the number of cells responsive to neurotransmitters in acutely dissociated (non-shaded) and differentiated (shaded) cells as measured by calcium fura-2 ion imaging; GABA (α-aminobutyric acid, Gly (glycine), DA (dopamine), Glu (glutamate), Ach (acetylcholine), RR (solution of rat Ringers), 50 mM of RR (modified rat Ringers solution replacing ^? ^ t ^^? ^ ß Sáitía Na + with K +). Figure 4 shows an illustrative graph of the (1340 / 13β) ratio of Ca2 + responses with time from a sharply dissociated E-NCAM * cell. Figure 5 shows an illustrative graph of the relationship (134o I 3o) of Ca2 + responses with time from a differentiated E-NCAM + cell. Figure 6 shows the results of PCR analysis of an individual E-NCAM + clone for the expression of markers of mature neurons. Figure 7 shows a bar graph of the percentage of cells of four E-NCAM + clones that respond to neurotransmitters as measured by calcium fura-2 ion image formation: GABA (α-amino butyric acid), Gly (glycine) ), DA 15 (dopamine), Glu (glutamate), Ach (acetylcholine), RR (solution of rat Ringers), 50 mM of K RR (solution of Ringers of rat modified replacing Na + with K +). Figures 8 and 9 show illustrative footprints of the ratio (I340 / I380) of Ca2 + responses of two registered cells of an E-NCAM + clone. Figure 10 shows the effect of bone morphogenetic protein 2 (BMP-2) is the cell division of E-NCAM + cells as measured through incorporation of BRDU. Figure 11 shows the effect of sonic hedgehog (Shh) in the cell division of E-NCAM + cells as measured through "-ff¡ ^" = pB && fMfafth ^^ BRDU incorporation. Figure 12 shows the results of the RT-PCR amplification of total RNA isolated from mouse E-CAM + cells to determine the expression of (from left to right after the molecular weight markers on the left) p75, lsl-1 , ChAT, calbindin, GAD and glutaminase. Figure 13 shows the results of the RT-PCR amplification of total RNA isolated from differentiated mouse ES cells to determine the expression of (from left to right) nestin, N-CAM, neurofilament-M (NF-M), protein associated with microtubule 2 (Map-2), GFAP, DM-20 / PLP. Figure 14 shows the results of the RT-PCR amplification of total RNA isolated from differentiated mouse ES cells to determine the expression of (from left to right) ChAT, p75, islet-1, calbindin, GAD and glutaminase.

DETAILED DESCRIPTION Before describing and explaining neuronal restricted precursor cells and methods for making and methods for using same, it should be understood that this invention is not limited to the particular configurations, process steps, and materials described herein since such configurations, Process steps, and materials may vary a bit. It should also be understood that the terminology used here is used with the ^^^ SS S ÁSS ^^^^^^^^^^^^^^^^^^^ M ^^ purpose of describing only particular embodiments and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and their equivalents. It should be noted that, as used in this specification and in the appended claims, the singular forms, "a," "one," and "the," include plural references, unless the context clearly dictates otherwise. Thus, for example, the reference to "an embryo" includes the reference to two or more embryos, the reference to "a mitogen" includes the reference to a mixture of two or more mitogens, and the reference to "one factor" includes reference to a mixture of two or more factors. To describe and claim the present invention, the following terminology will be used in accordance with the definitions set forth below. As used herein, "self-renewal" refers to, for example, the ability of a neuroepithelial stem cell to divide to produce two daughter cells, at least one of which is a multipotent neuroepithelial stem cell, or to the ability of a neuronal restricted precursor cell to divide to produce two daughter cells, at least one of which is a neuronal restricted precursor cell. As used herein, "clonal density" and similar terms means a sufficiently low density to result in the isolation of individual non-shock cells, when they are placed in plates in a selected culture box. As an illustrative example of said clonal density is a culture box of approximately 225 cells / 100 mm. As used herein, "cell-feeder-independent adherent culture" and similar terms means the growth of cells in vitro in the absence of a layer of different cells that are usually first plated in a culture box to which tissue cells of interest are added. In feeder cell cultures, the feeder cells provide a sub-stratum for the union of cells of the tissues of interest and further serve as a source of mitogens and survival factors. The independent cell-feeder adherent cultures of the present use a chemically defined sub-stratum, for example, fibronectin, and mitogens or survival factors are provided by supplementation of the liquid culture medium with either purified factors or crude extracts from other cells or tissues. Therefore, in cell-feeder independent cultures, the cells in the culture box are mainly cells derived from the tissue of interest and do not contain other types of cells required to support the growth of cells derived from the tissue of interest. As used herein, "effective amount" means an amount of a growth factor or survival factor or other factor that is non-toxic but sufficient to provide the desired effect and function. For example, an effective amount of & í mi¡¿k r FGF, as used herein, means a quantity selected in order to support self-renewal and proliferation of NEP cells when used in combination with other essential nutrients, factors and the like. An effective amount of NRP cells or their derivatives or their mixtures for transplantation refers to an amount or number of cells sufficient to obtain the selected effect. NRP cells will generally be administered at concentrations of approximately 5-50,000 cells / microliter. The transplant will usually occur in a volume of up to approximately 15 microliters per injection site. However, the transplant subsequent to surgery in the central nervous system may involve volumes as many times as large. In this way, the number of cells used for the transplant is limited only by utility, and such numbers can be determined by a person skilled in the art without undue experimentation. As used herein, "derivative" of a NRP cell means a cell derived from an NRP cell in vitro through genetic transduction, differentiation or similar processes. As used herein, "administering an NRP cell to a mammal" means transplanting or implanting said NRP cell into a CNS tissue or adjacent said CNS tissue of a recipient. Said administration can be carried out by any method known in the art, such as surgery, with a cannula of infusion, needle and the like.

As used herein, "heterologous" refers to individuals, tissues, or cells other than a transplant recipient. The transplant donor can be of the same species or a different species from the transplant recipient. For example, a heterologous donor of NRP cells for transplantation may be of a different species from the transplant recipient. As used herein, "autologous" refers to self-generated or origin within the body. Thus, for example, an autologous donor of tissue or cells for transplantation is the same individual who receives the transplant. By way of another example, autologous cells are cells that arise, are transferred or transplanted into an individual. In vitro manipulation can take place between harvesting the cells and transplanting said cells or derivatives thereof, but it is not required before transplantation. As used herein, "transform", "transduce", "transfection" and similar terms mean the insertion or transfer of a gene or genes to NRP cells without considering the method of insertion or transfer. In this way, transformation can be achieved through calcium phosphate transfection, DEAE-dextran transfection, polybrene transfection, electroporation, lipofection, virus infection, and the like, and any other methods known in the art. The present invention is illustrated using restricted neuron precursor cells isolated from rats, mice, and humans.

However, the invention encompasses all mammalian neuronal restricted precursor cells and is not limited to neuronal restricted precursor cells of rats, mice, and humans. The neuron precursor cells restricted from mammals can be isolated from human and non-human primates, equines, canines, felines, bovines, swine, sheep, lagomorphs, and the like. The pluripotent cells in the central nervous system can generate differentiated neurons and glia either directly or through of the generation of intermediate precursors of restricted lineage. In the development of the retina, it seems that multipotent retinal precursors can generate any combination of cells differentiated even in their final division, indicating that intermediary precursors do not exist. In other regions of the nervous system In contrast, retroviral labeling studies have suggested the existence of restricted lineage precursors that generate only one type of cell or a limited number of cell types. Intermediate stage precursors, such as the bipotential oligodendrocyte type 2 astrocyte precursor (O-2A) and a neuronal precursor have also been described in tissue culture studies. Still, the generation of restricted lineage precursors intermediates of embryonic stem cells or pluripotent adult cells or other stem cells capable of differentiation into neurons and glia has not been observed until recently, that is, the patent application of E.U.A. Series No. ^^^ w ^ m ^^^ j ^^^^^^ ft ^ j ^^^ i ^^^^^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^ MY | 08 / 909,435, filed July 4, 1997. Thus, the linear relationship between pluripotent stem cells identified in the culture and the committed precursors identified in vivo and in vitro has hitherto been unknown. Possible developmental models have included, (1) pluripotent and more involved stem cells representing linearly related cells, or (2) cells representing independent differentiation trajectories. The development of rat spinal cord represents an ideal model to study this differentiation. On embryonic day 10.5 (E10.5), the caudal neural tube appears as a homogeneous population of nesting immune-reactive cells to nestin in vivo and in vitro. These initially homogenous cells are designed for several days to generate neurons, oligodendrocytes, and astrocytes in a characteristic spatial and temporal profile. Neurogenesis occurs first in a ventro-dorsal gradient, with the earliest neurons becoming post-mitotic in E13.5 in rats. Neurogenesis continues for two additional days followed by differentiation of oligodendrocyte precursors and the subsequent differentiation of astrocytes. Methods for growing neuroepithelial stem cells (NEP) isolated from E10.5 rat embryos as undifferentiated cells during extended periods in vitro have been described in the application Serial No. 08 / 852,744, and further shown that these populations were able to generate all three main cells in the CNS. In this way, NEP cells represent a dividing multipotent stem cell that can be differentiated into neurons either through an intermediary neuroblast or directly as a part of their differentiation terminal. To determine whether neurons differentiate from NEP cells through more restricted, intermediate precursors, a variety of immunologically-defined populations of NEP cell differentiation cultures was isolated and characterized. It was shown in the present that the cells morphologically and phenotypically identical to NRP's can be isolated from NEP cell cultures. The clonal analysis showed that individual NEP cells generate neurons via the generation of neuronal precursors and that individual NEP cells can generate precursors of restricted neuron and restricted glia. It was also shown that the cells E-NCAM * (embryonic neural cell adhesion molecule positive) are present in E13.5 neural tube cultures and that these cells are mitotic, self-renewing stem cells that can generate multiple neuronal phenotypes, but not astrocytes or oligodendrocytes. In this way, the neuron precursors Restricted (NRPs) are an identifiable step in the in vivo differentiation of neurons. In addition, it was shown that NRPs can be isolated and cultured from mouse embryos, mouse embryonic stem cells (ES), and from human embryonic bone marrow. These data provide a demonstration of a linear relationship direct between multipotent and restricted neuron stem cells and suggest that neural differentiation implies the progressive restriction in the destiny of development. Figure 1 presents a model for spinal cord differentiation. This model is similar to that proposed for hematopoiesis and for neural crest differentiation (see review by D. J. Anderson, The Neural Crest Lineage Problem: Neuropoiesis ?, 3 Neuron 1-12 (1989)). According to this model, NEP 10 cells represent a homogeneous population of cells in the caudal neural tube that express nestin (ie, nestin +), but not another lineage marker (lin). These cells divide and self-renew in culture and generate differentiated phenotypes. The previous data have suggested intermediary division precursors with a more restricted potential. Such precursors include restricted glia precursors 14 that generate oligodendrocytes 18 and astrocytes 22, as well as neuronal progenitors 26 that generate various types of neurons 30, 34. The model also shows that cells of the neural crest stem 38, which can differentiate into PNS neurons 42, Schwann cells 46, and smooth muscle cells 50, also descend from NEP cells. The model, therefore, suggests that multipotent precursors (NEP cells) generate differentiated cells (ie, oligodendrocytes, type 2 astrocytes, type 1 astrocytes, neurons, motoneurons, PNS neurons, Schwann cells, and smooth muscle) through intermediary precursors. Consistent with this model are the results presented here showing the existence of cells with ^^^ igjÜÉÉ¿ a proliferative potential of restricted neuron. NEP cell cultures provide a large source of transient cells that can be classified to obtain differentiated cell types. The results described here provide direct evidence to support a model that describes initially multipotent cells that undergo progressive restriction in the development potential under extrinsic influence to generate different phenotypes within the central nervous system. Evidence is provided that the NEP cells initially multipotent generate neuron precursors restricted in vitro and said restricted neuron precursors are also present in vivo. It was also shown that the NRPs satisfy the criteria of reticular cells and that there is a direct linear relationship between the multipotent stem cells and the NEP cells. more restricted. The results presented here support that the E-NCAM immunoreactive cells are restricted in their development potential. E-NCAM + cells failed to differentiate into oligodendrocytes or astrocytes under any culture condition tested. In contrast, NEP cells differentiated into neurons, astrocytes, and oligodendrocytes, and the immunoreactive A2B5 cells differentiated into oligodendrocytes under identical conditions. For these reasons, the E-NCAM immunoreactive cells are described herein as restricted neuron precursors or NRPs. 25 Immunological panning and dialing data double shows that E-NCAM can be used to identify a specific neuronal sub-lineage that is generated from multipotential NEP cells. However, similar markers for intermediate precursors in the hematopoietic system and the neural crest, E-NCAM, and the glial precursor marker A2B5 are also not unique to the intermediate precursors. It has been shown that E-NCAM marks some astrocytes. Similarly, it has been shown that A2B5 recognizes neurons in some species and is transiently expressed by astrocytes in some culture conditions. However, under specific culture conditions defined herein, these markers can be used to select intermediate precursors and, therefore, represent the first cell surface epitopes that are co-expressed according to a restriction on the development potential. The basal medium (medium of NEP) used in the experiments described herein comprises DMEM-F12 (GIBCO / BRL, Gaithersburg, MD) supplemented with 100 μg / ml of transferrin (Calbiochem, San Diego, CA), 5 μg / ml of insulin (Sigma Chemical Co., St. Louis, MO), 16 μg / ml putrescine (Sigma), 20 nM progesterone (Sigma), 30 nM selenious acid (Sigma), 1 mg / ml bovine serum albumin (GIBCO / BRL), plus B27 additives (GIBCO / BRL), 20 ng / ml basic fibroblast growth factor (bFGF), and 10% chicken embryo extract (CEE). In general, these additives were stored as 100X concentrates at -20 ° C until use.

Normally, 200 ml of the NEP medium was prepared with all the < »J? Ri¡Cas ^?» Ft »H?« »Iha6at¿S < flt? = * ^ '^^ m ^ t ^^^^^^ É ^ additives, except EEC, and were used after two weeks of preparation. The CEE was added to the NEP medium at the time of the feeding of cultured cells. FGF and CEE were prepared as described by D. L Stemple & D. J. Anderson, supra; M. S. Rao & D. J. Anderson, supra, L. Sommers et al., Cellular Function of the bHLH Transcription Factor MASH1 in Mammalian Neurogenesis, 15 Neuron 1245-58 (1995), incorporated herein by reference. FGF is also commercially available (UBI). In summary, the CEE was prepared as follows. Chicken eggs were incubated for 11 days at 38 ° C in a humid atmosphere. The eggs were washed and the embryos were removed and placed in a Petri dish containing the sterile Minimum Essential Medium (MEM with glutamine and Earle salts) (GIBCO / BRL) at 4 ° C. Approximately 10 embryos each were macerated by passing through a 30 ml syringe into a 50 ml test tube. This procedure typically produced about 25 ml of the medium. To each 25 ml was added 25 ml of MEM. The tubes were shaken at 4 ° C for 1 hour. Sterile hyalurinidase (1 mg / 25 g of embryo) (Sigma) was added, and the mixture was centrifuged for 6 hours at 30,000 g. The supernatant was collected, passed through a 0.45 μm filter and then through a 0.22 μm filter, and stored at -80 ° C until used. Laminin (Biomedical Technologies Inc.) was dissolved in distilled water at a concentration of 20 mg / ml and applied to plates of ^^^^^^^^^^^^ agÉH ^ tissue culture (Falcon). Fibronectin (Sigma) was resuspended at a stock concentration of 10 mg / ml and stored at -80 ° C and then diluted to a concentration of 250 μg / ml in D-PBS (GIBCO / BRL). The fibronectin solution was applied to tissue culture dishes and immediately removed. Subsequently, the laminin solution was applied and the plates were incubated for 5 hours. The excess laminin was removed, and the plates were allowed to air dry. Then the coated plates were rinsed with water and allowed to dry again. Fibronectin was chosen as a growing substrate for NEP cells, since NEP cells did not adhere to collagen or poly-L-lysine (PLL) and poorly adhered to laminin. Thus, all subsequent experiments to maintain NEP cells in culture were performed on dishes covered with fibronectin. The dishes covered with laminin were used, however, to promote cell differentiation of the NEP stem. For clonal analysis, cells harvested through trypsinization were plated at a density of 50-100 cells per 35 mm dish. The individual cells were identified and placed on the plate marking the position with a grease pencil. The cells grew in DMEM / F12 with additives, as described above, for a period that varied from 10-15 days. The cells of the present invention can be used in the preparation of compositions, including compositions ma aaáasE ^ aÉfe ^ Al * ^ pharmaceuticals, which can be appropriately formulated and administered to treat and correct deficiencies, debilitations and other malfunctions that may result from damage, disease, or other degeneration of the relevant neural tissue. By way of non-limiting examples, suitable cells prepared according to the present invention can be administered, for example, via implant, as a means to effect cell replacement therapy, to treat cases where cell damage or impairment has occurred. In this way, for example, the cells can be prepared in an appropriate growth medium, such as one for the promotion of growth and differentiation. The suitable medium may include, for example, growth or differentiation factors, for example, retinoic acid, BMP-2, BMP-4, or one or more members of neurotrophins such as NT-3, NT-4, CNTF, BDNF. and similar. The cells thus conveniently prepared in said medium could be introduced either intrahecally, I.V., I.P., or through any means by which the introduction of the cell preparation to the target site can be better achieved. The particular aspects of administration of this type may vary and could be within the experience of the physician or practitioner. The cells of the present invention are also useful in a variety of diagnostic applications and, for example, can be prepared for use in a screening assay, for example, for the identification of neuronal and other markers.

'^^ SU ^? ^ Í ^ ah ^ & ^ s ^^^^ binding patterns or ligands, modulators or other factors that can function as modulators of cell growth and / or differentiation. The cells of the present invention can also be used as, for example, a positive control in an assay to identify deficiencies in cell growth and differentiation, and the factors that may be the cause thereof. The cells of the present invention can be used in a variety of therapeutic applications, including in the preparation of pharmaceutical compositions and appropriate vehicles, for administration to individuals in need of such therapy, for treating various cellular impairments, disorders or other irregularities or abnormalities associated with genetically caused damage, disease or neuronal deficits. The ailments or conditions contemplated herein include Parkinson's disease, Huntington's disease, Alzheimer's disease, malfunctions resulting from injury or trauma, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), and anencephaly.

EXAMPLE 1 To determine whether a split-restricted neuron precursor is normally present in vivo, sections of rat spinal marrow E13.5 were analyzed with a panel of markers 'r-ytf trfrj? iiSjSiii f? ijlp ^ early neuronal The sections were cut from fresh embryos, frozen, at a gestation of 13.5 days and then marked by immunocytochemistry. Staining procedures were performed according to methods well known in the art. Cells were double-labeled with antibodies against E-NCAM (Developmental Studies Hybridoma Bank, Iowa) and β-III tubulin (Sigma Chemical Co., St. Louis, Missouri) or stained with E-NCAM and counteracted with DAPI, a marker nuclear to identify all the cells. All secondary monoclonal antibodies were from Southern Biotechnology. The polysialated or embryonic N-CAM (E-NCAM) appeared to be probably a marker for neuronal precursors. The immuno-reactivity of E-NCAM was first detected in E13.5 The immunoreactive cells of E-NCAM could be seen in the margins of the neural tube, but not in the ventricular proliferation zone. Double labeling with β-III tubulin indicated that most of the E-NCAM immunoreactive cells co-expressed this neuronal marker. A small portion of cells present more medially were E-NCAM +, but did not express immuno-reactivity of β-lll tubulin, suggesting that E-NCAM may be an early and specific marker of differentiation to neuronal precursors that is expressed before the β -lll tubulina. -... »» 3 »fa- * é¿ sajrf ^ SjalMajJ, ^^^^ EXAMPLE 2 To characterize E-NCAM immunoreactive cells, E13.5 spinal cords were dissociated and the E-NCAM immunoreactive cells were stained with a panel of antibodies (Table 1). Sprague-Dawley rat embryos were removed on embryonic day 13.5 and placed in a Petri dish containing balanced salt Hanks solutions (HBSS, Gibco). The trunk segments of the embryos were dissected using tungsten needles, rinsed and then transferred to fresh HBSS. The spinal cords were mechanically dissected free of the surrounding connective tissue using sharp No. 5 forceps. The isolated spinal cords were incubated in a 5% solution of trypsin / EDTA for 20 minutes. The trypsin solution was replaced with fresh HBSS, containing 10% fetal bovine serum (FBS). The segments were moderately titrated with a Pasteur pipette to dissociate the cells. Dissociated cells through titration were plated on 35 mm plates covered with PLL / laminin (Nunc) at high density and stained after 24 hours. Staining for cell surface markers, such as A2B5 and a-GalC, was performed with cultures of living cells. To stain cells with antibodies against internal antigens such as GFAP, which specifically recognize astrocytes (A. Bignami et al., Localization of the Glial Fibrillary Acidic Protein in Astrocytes, by Immunofluorescence, 43 Brain Res. 429-35 (1972)), ß-III tubuhna (DAKO) and RT-97, which stain neurons (E. Geisert &A. Frankfurter, The Neuronal Response to Injury as Visualized by Immunostaining of Class ß-tubulin in the Rat, 102 Neurosci.Lett 137-41 (1989), nestin, which is a marker for undifferentiated stem cells (U. Lendahl et al., CNS Stem Cells Express to New Class of Intermediate Filament protein, 60 Cell 585-95 (1990)), or 5-bromodeoxyuridine (BrdU, Sigma), which is a marker to determine the number of dividing cells, the cultures were fixed in ice-cooled methanol. or triple labeling simultaneously incubating cells in appropriate combinations of primary antibodies followed by cross-reactive secondary antibodies, for example, M. Mayer et al., Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor Promote the Generation, Maturation and Survival of Oligodendrocytes, 120m D evelopment 142-53 (1994), incorporated herein by reference. In the triple labeling experiments, the cultures were incubated with the primary antibody in blocking pH regulator for a period of 1 hour, rinsed with PBS and incubated with a specific species-specific secondary antibody in blocking pH regulator for 1 hour. hour. The cultures were rinsed three times with PBS and examined under a fluorescence microscope. For labeling with 4 antibodies simultaneously, the live cells were first incubated with the surface antibodies A2B5 and a-GalC without the secondary layers.

"^^ • S ^^^^^^ 3 ^ 8 - ^ - *" The cells were then fixed in ice-cold methanol for 10 minutes and stained with α-β-lll tubulin and the appropriate secondary antibody. After classification of the results of this staining, which was usually negative, the clones were stained with GFAP and the secondary layer for the first group of surface antibodies. Finally, the secondary antibody for GFAP was added. This procedure allowed staining with four antibodies using only three fluorescent-colored conjugated secondary antibodies. The E-NCAM immunoreactive cells constituted 60% A3% of all cells present in the dissociated culture 24 hours after placement in boxes. The majority of the remaining cells were A2B5 +. In the patent application of E.U.A. Series No. 08 / 852,744 it was shown that at this stage of development, the immunoreactive A2B5 cells are glial precursor cells. According to these results, ß-III tubulin or E-NCAM immunoreactive cells did not co-express A2B5. the vast majority of cultured E-NCAM immunoreactive cells (85% ± 8%) coexpressed the immuno-reactivity of β-lll tubulin as well as the immuno-reactivity of nestin, but not the characteristic markers of precursor immuno-reactivity of glia. Approximately 20% of the E-NCAM + cells were divided in a 24-hour period. The majority of the dividing E-NCAM + cells did not co-express β-lll tubulin, indicating that this population of cells may represent a division neuroblast. I do not know yet it knows if a higher percentage of the cells can be observed to divide under these conditions with longer marking periods. However, even if this population were to include a subgroup of cells sufficiently committed to neuronal differentiation in order not to fit more into the division, these compromised neurons could be eliminated from the population with expansion and division in tissue culture. . Table 2 summarizes the results of the antigenic profile of the cells, showing the percentages of E-NCAM + cells from E13.5 embryos expressing several other antigens. These results show that the E-NCAM + cells of the spinal cord E13.5 express neuronal but not glial markers. b Sigma Chemical Co., St. Louis, MO c Boehringer Mannheim Biochemicals, Gaithersburg, MD Accurate, Westbury, NY e Chemicon, Temecula, CA EXAMPLE 3 To determine the differentiation potential of E-NCAM immunoreactive cells, E-NCAM + cells were purified through immunological panning and placed in plates at a clonal density in dishes with gratings. Cells were prepared E13.5 according to the procedure of Example 2. A population of E-NCAM + cells was purified from these E13.5 cells using a specific antibody capture technique according to the procedure of L. Wysocki & V. Sato, "Panning" for Lymphocytes: A Meted for Cell Selection, 75 Proc. Nat'l Acad. Sci.

USA 2844-48 (1978); M. Mayer et al., Supra, incorporated herein by reference. Briefly, the cells were trypsinized and the resulting cell suspension was plated on a dish coated with the A2B5 antibody to allow the binding of all A2B5 + cells on the plate. The supernatant was removed, and the plate was ^? ^ s ^? ^^ ^ washed with DMEM supplemented with additives described by J. Bottenstein and G. Sato, Growth of a Rat Neuroblastoma Cell Line in Serum-free Supplemented Medium, 76 Proc. Nat'l Acad. Sci. USA 514-17 (1979), incorporated herein by reference, (DMEM-BS). The supernatant was then plated onto a dish coated with the E-NCM antibody to allow the binding of the E-NCAM immunoreactive cells. The bound cells were scraped from the plate and replated onto plates on glass coverslips coated with fibronectin / laminin in 300 ml of DMEM-BS ±. growth factors of 5000 cells / cavity. The A2B5 and E-NCAM antibodies to coat the plates were used at concentrations of 5 μg / ml. The cells were allowed to attach to the plate for 20-30 minutes in an incubator at 37 ° C. Growth factors were added every third day to a concentration of 10 ng / ml. BFGF and neutrophin 3 (NT-3) were purchased from PeproTech and retinoic acid (RA) was obtained from Sigma. After 24 hours, some of the E-NCAM + immunological panning cells were analyzed through immunocytochemistry according to the procedure of Example 2. More than 95% of the cells were E-NCAM + at that time. The purified and stained cells were plated on clonal dishes with gratings, and the individual E-NCAM + cells were identified and followed for a period through immunocytochemistry according to the procedure of Example 2.

Of all the cytokines tested, optimal growth was observed when the cells were cultured in FGF (10 ng / ml) and NT-3 (10 ng / ml). In the presence of FGF and NT-3, the individual E-NCAM + cells were divided in culture to generate colonies varying from one to several hundred cells. On day 5, the majority of the colonies contained between 20 and 50 daughter cells that continued to express immunoreactivity of E-NCAM. The daughter cells appeared as bright phase and had short processes. At this stage, most of the E-NCAM-positive cells did not express β-0 lll tubulin or neurofilament-M immuno-reactivity. To promote the differentiation of E-NCAM + clones, the medium containing FGF and NT-3 was replaced with a medium containing retinoic acid (RA) and from which the mitogen, bFGF, was stopped. In this differentiation medium, the NCAM + E-5 cells stopped dividing and elaborated extensive processes and began to express neuronal markers. The quadruple labeling of clones with neuronal and glial markers and the DAPI histochemistry, to identify all the cells, showed that all the clones contained ß-III or tubulin immunoreactive cells and neurofilament-M immunoreactive cells (NF- M) and that none of the E-NCAM + clones differed in oligodendrocytes or astrocytes. Table 3 summarizes the results obtained by the quadruple labeling of clones 124 E-NCAM * with DAPI, oc- ß- 111 5 tubulin, A2B5 and a-GFAP.

^ ¿MJSMS ^^ EXAMPLE 4 In this example, A2B5 * immunological panning cells derived from E13.5 spinal cords dissociated according to the procedure of Example 2 were cultured in a neuron promoter medium, i.e., a basal medium plus FGF and NT-3. cultures were grown for 5 days and then switched to a medium containing RA as described in Example 3, and the sister plates were stained for immunoreactivity for both E-NCAM and A2B5. No A2B5 immunological panning cell expressed E-NCAM immunoreactivity when grown under conditions that promote the growth of neuronal cells. All A2B5 immunological panning cells, however, continued to express A2B5 immunoreactivity, indicating that neuron-promoting conditions do not affect the survival and proliferation of glia precursor cells. In this way, the inability to detect oligodendrocyte and astrocyte differentiation in Example 3 was remotely due to the death in neuronal cultures of oligodendrocytes and astrocytes that could have been differentiated from E-NCAM * precursors, since the cells purified A2B5 glia precursors that grew in parallel in the presence of FGF and NT-3 continued to express A2B5 without apparent cell death and generated healthy oligodendrocytes and astrocytes after 10 days in culture. In addition, A2B5 * cells never generated neurons in the presence of FGF and NT-3 and did not show E-NCAM expression at any time tested. Thus, the E-NCAM immunoreactive cells, unlike the immuno-reactive glial restricted precursors A2B5, can not be differentiated to oligodendrocytes and appeared limited to neuronal differentiation when compared with multipotential E10.5 neuroepithelial cells.

EXAMPLE 5 Although it has been clearly shown in the present system that E-NCAM identifies neuronally restricted precursor cells, it has been reported that certain glial precursors, at later stages of development, can also express E-NCAM immunoreactivity. From this observation arises the possibility that some E-NCAM * cells identified by the methods currently described may be bi-potential. To test this possibility, E-NCAM * cells were plated clonally in either a neuron promoter medium (FGF + NT-3) or in a glial promoter medium (FGF + 10% fetal bovine serum) and They were compared for their development. The medium containing FGF with % of fetal bovine serum was chosen for glia differentiation, because this medium promotes astrocyte differentiation of both spinal cord NEP cells and immunoreactive A2B5 glia precursor cells, A2B5, as shown in the application of US patent Series No. 08 / 852,744. All E-NCAM * clones (24/24) that grew in the neuron-promoting medium only contained β-III tubulin cells after 8 days, whereas clones that grew in a medium containing serum did not generate astrocytes or proliferated. From a total of 97 E-NCAM * cells developed under glial-promoting conditions, 90 clones (92%) consisted of a single dead cell after 24 hours, while the remaining 7 clones (8%) contained 1 or 2 dead cells after 48 hours. In this way, the E-NCAM immunoreactive cells, in contrast to the glia precursor cells, failed to proliferate or differentiate under astrocyte promoting conditions.

EXAMPLE 6 To determine whether the restriction of E-NCAM * cells for the generation of neurons also includes a restriction for the generation of certain sub-types of neurons, the E-NCAM * clones developed in RA and NT-3 in the absence of FGF were examined for the expression of different neurotransmitters. The antibodies useful in this example are described in Table 4.

These results indicate that individual clones can generate GABA-energetic, glutaminergic and cholinergic neurons. Of the ten clones tested, all contained glutaminergic, GABA-energetic and cholinergic neurons. In this way, E-NCAM immunoreactive cells, although limited to differentiate neurons, are capable of generating excitatory, inhibitory and cholinergic neurons.

EXAMPLE 7 Primary clones of E-NCAM * cells grown in FGF and NT-3 according to the procedure of Example 5 were grown to sizes of several hundred cells after 7 to 10 days in culture, indicating some degree of self-renewal. To demonstrate the prolonged self-renewal of the E-NCAM * population, the selected clones were followed by secondary and tertiary sub-cloning. Individual E-NCAM * cells from spinal cord E13.5 were plated on fibronectin / laminin and ^^^? ^^ .-- U • expanded for 7 days in the presence of FGF and NT-3. Five individual clones were randomly selected and plated back to a clonal density using the same expansion conditions. The number of secondary clones was counted, and the large clones were selected and plated. The number of tertiary clones obtained was counted, and the clones were then induced to differentiate into post-mitotic neurons by replacing FGF and RA. All the clones examined generated numerous child clones that subsequently generated tertiary clones. Small clones and very large clones showed similar self-renewal potential. When the tertiary clones were switched to a medium containing RA and lacking FGF, most of the cells in a clone were differentiated to post-mitotic neurons expressing β-lll tubulin. In this way, E-NCAM * cells capable of prolonged self-renewal can generate multiple daughter cells capable of generating neurons. These results suggest that the immuno-reactivity of E-NCAM identifies a neuroblast cell that can differentiate into multiple multiple neuronal phenotypes in culture, even after multiple passages. NT-3 and FGF are required to maintain the reticular cell in a proliferative state, whereas RA promotes differentiation.

., - ** *? »B? NU? AA? ^ SJj? ^ TgMSßS ^^ EXAMPLE 8 It has previously been shown that the individual NEP spinal cord E10.5 cells are a population of E-NCAM immunonegative, multipotent, self-renewal cells that can generate neurons, astrocytes and oligodendrocytes (US Patent Application Series No. 08). / 852,744). To determine whether neuronal differentiation of NEP precursors involved the generation of a precursor, neuronal, intermediary, E-NCAM * cell, NEP cell cultures that were induced to differentiate in vitro were examined for the presence of E-reactive immune cells. NCAM *. The NEP cells were prepared according to the method described in Patent Series No. 08 / 852,744. Briefly, Sprague-Dawley rat embryos were removed in E10.5 (13-22 somites) and placed in a Petri dish containing a balanced salt solution of Ca / Mg free Hanks (HBSS, GIBCO / BRL). The trunk segments of the embryos (last 10 somites) were separated using tungsten needles, rinsed and then transferred to fresh HBSS. The trunk segments were incubated at 4 ° C in a 1% trypsin solution (GIBCO / BRL) for a period of 10 to 12 minutes. The trypsin solution was replaced with fresh HBSS containing 10% fetal bovine serum (FBS, GIBCO / BRL). The segments were moderately titrated with a Pasteur pipette to release m¡jz ** .- x &Bt £ lsX? * é *. ... ^., ...; «« i, J »isei." ".. neural tubes free of surrounding somites and connective tissue. The isolated neural tubes were transferred to a 0.05% trypsin / EDTA solution (GIBCO / BRL) for an additional 10 minutes. The cells were dissociated through titration and plated at a high density in plates coated with 35 mm fibronectin in a NEP medium. The cells were maintained at 37 ° C in 5% CO2 / 95% air. The cells were again plated at low density, i.e., < 5000 cells per 35 mm plate, one to three days after plating. The cells of several dishes were then harvested through trypsinization (0.05% solution of trypsin / EDTA for two minutes). Then the cells were formed into pellets, they were resuspended in a small volume and counted. Approximately 5000 cells were plated in a 35 mm dish (Corning or Nunc). The NEP cells derived from E10.5 embryos were expanded in the presence of FGF and CEE for 5 days and differentiated by replacing them on plates on laminin in the presence of CEE. NEP differentiation cells were labeled in triplicate with antibodies to E-NCAM, GFAP and GalC. This showed that the E-NCAM immunoreactive cells that differentiated from NEP cells did not express astrocytic (GFAP) or oligodendrocytic (GalC) markers. A sister plate was double labeled with antibodies to E-NCAM and nestin. This showed that the E-NCAM immunoreactive cells that differentiated from cells NEP co-expressed nestina. NEP differentiation cells were incubated for 24 hours with BrdU and subsequently double-labeled with an antibody against BrdU and E-NCAM. This showed that most of the E-NCAM immunoreactive cells were divided in 24 hours. This higher speed of marking may reflect differences in the isolation procedure as compared to the previous example. Table 5 summarizes the antigenic profile of E-NCAM * cells derived from E10.5 cells. Note that E-NCAM * cells derived from NEP are antigenically similar to E-NCAM E13.5 cells and, as E-NCAM E13.5, do not express any of the glia markers examined.

* A sub-group of cells expressing this marker In this way, induced NEP cultures comprise 15 multiple phenotypes, including E-NCAM * cells. Like E-NCAM * E13.5 cells, E-NCAM * cells derived from NEP did not express glial markers, but co-expressed immuno-reactivity of β-III tubulin (20-30%) and nestin (70-80). %). Ninety percent of E-NCAM * cells from immunological panning incorporated 20 BrdU in culture and generated neurons after the addition of RA or NT-3 and thus appeared similar to the immunoreactive E-NCAM E13.5 cells. EXAMPLE 9 To determine if the individual NEP-derived E-NCAM * cells were also restricted to neurons in their potential differentiation, the cells were studied in a clonal culture. NEP cells were induced to differentiate by replacing plates on laminin and EEC removal, as described in the patent application of E.U.A. Series No. 08 / 852,744. The NEP cells derived from E10.5 embryos were expanded in the presence of FGF and CEE for 5 days and differentiated by replacing them on plates on laminin in the absence of CEE. Afterwards, the E-NCAM immune-reactive immune imaging cells were plated on clonal grid dishes (Greiner Labortechnik) covered with fibronectin / laminin, and the individual cells were followed in culture. After 5 days, the clones were switched to RA and FGF was removed. The clones were allowed to grow for 3 more days, fixed with paraformaldehyde, and labeled in triplicate with A2B5 and antibodies against GFAP and β-lll tubulin. In addition, the cells were counter-stained with DAPI to show individual cell nuclei. Table 6 summarizes the results of the staining of the 47 clones studied (8 of 47 clones did not survive the placement . * S¿i¿a¿í & amp; ^^ ^ i & ^^ S? M iSÍM of additional plate). Note that no clones contained astrocyte cells (GFAP *) or glia precursor cells (A2B5 *).

After 48 hours, the cells were induced to differentiate, 10-30% of the cells began to express E-NCAM immunoreactivity. The E-NCAM * cells derived from NEP cells were selected through immunological panning according to the procedure of Example 3, and the individual E-10 NCAM * cells were plated in a medium containing FGF and NT-3 and the clones were analyzed after 10 days. All clones contained only E-NCAM * / β-lll-tubulin cells, but not GFAP p A2B5 immunoreactive cells. Further, individual E-NCAM * cells failed to differentiate into oligodendrocytes or astrocytes under culture conditions that promote astrocytic or oligodendroglial differentiation of the NEP cell population of origin. E-NCAM * cells can be maintained as dividing precursor cells in a medium defined in the presence of high concentrations of FGF (10 ng / ml) and NT-3 (10 ng / ml). E-NCAM * cells maintained for up to three months could easily be differentiated to mature neurons of β-III tubulin that expressed a variety of neurotransmitter phenotypes when exposed to the growth of RA on laminin. In this way, E-NCAM * cells are similar to neuronal precursors E13.5 in their differentiation potential, antigenic profile, and optimal conditions for extended growth as a population of dividing precursor cell.

EXAMPLE 10 The differentiation of the E-NCAM * population from a nesting NEP cell population of nestin * / E-NCAM, "apparently homogeneous, suggests a progressive restriction on the fate of development." It was thought possible, but remote, that individual NEP cells can be pre-committed to generate neuroblasts or glioblasts To fix this possibility, the individual NEP clones were examined for their ability to generate immunoreactive E-NCAM cells and A2B5 immunoreactive cells.A2B5 and E-NCAM cells were selected , since previously it has been shown that the immuno-reactivity of A2B5 is unique to the oligodendrocyte-astrocyte precursors at this stage of development.The NEP cells derived from E10.5 embryos were expanded in the presence of FGF and CEE for 5 days, harvested through trypsinization, and plated back to a clonal density in clonal dishes with grid.After 7 days in culture, the individual clones were double-labeled with antibodies against E- ^^^^^^^ M ^^ m ^ ^^^^^^ NCAM and A2B5 according to the procedure of Example 2. Of the 112 NEP clones that remained in culture, 83% generated immunoreactive cells from both A2B5 and E-NCAM. 5% of the clones consisted only of A2B5 immunoreactive cells, and 12% of the clones did not show convincing staining nor for immuno-reactivity of A2B5 as of E-NCAM. In all the tested clones, e-NCAM and A2B5 were expressed in non-overlapping populations. That is, no cell co-expressed both markers. Table 7 summarizes the results obtained with 112 clones.

In this way, most NEP cells appear to be capable of generating precursors for glia restricted cells as well as neuronal restricted precursors.

EXAMPLE 11 To test whether neurons were generated via an E-NCAM * intermediate neuroblast, complement-mediated cell lysis was used to selectively kill E-NCAM * cells.

After 24 hours of replacing the NEP cells in differentiation conditions, the immunoreactive cells E- ^^^^^^^^ MB ^^^^ NCAM were annihilated using an IgM antibody to E-NCAM and a guinea pig complement from India. In sister plates, the glia precursors were annihilated using an anti-A2B5 IgG antibody and complement. At this stage of development, the majority of E-NCAM * cells do not express ß-III tubulin. The treated plates were allowed to differentiate for 3 more days, and the development of neurons was verified. E-NCAM-mediated lysis significantly reduces the number of ß-III tubulin immunoreactive cells that developed when compared to cultures treated with A2B5 (219A35 against 879 + 63, respectively) suggesting that neuronal differentiation of NEP cells in vitro requires a transition through an immunoreactive E-NCAM state.

EXAMPLE 12 15 E-NCAM * cells can be distinguished from Acutely Dissociated NRP Cells ENCAM * cells were isolated by immunological panning according to the procedure of Example 3, placed on plates in 35 mm plates, and allowed to grow for 24 hours (sharply dissociated) or 10 days (differentiated). Afterwards, the cultured cells were analyzed for cell division through the incorporation of BRDU, expression of E-NCAM, expression of NF-M and expression of synaptophysin according to the The procedure of Example 2. Approximately 70% of the cells ^^ M ^^^^^^^^ te ^^^^^^ a ^^ g »^ rag > ^^^^^^^^^^^^^^ - E-NCAM * acutely dissociated incorporated BRDU, showing that said cells were divided in culture, whereas after 10 days in the middle of differentiation promotion, some or no cells incorporated BRDU, and, therefore, they stopped dividing. The double labeling for the immuno-reactivity of E-NCAM and NF-M showed that very few sharply dissociated cells expressed NF-M, whereas all the differentiated cells expressed this protein. Similarly, synaptophysin, a protein specifically associated with synaptic vesicles and functional synapses, see, TC Sudhof, The Synaptic Vesicle Cycle: A Cascade of Protein-Protein Interactions, 375 Nature 645-653 (1995), was expressed through ENCAM cells * differentiated but not sharply dissociated. Although the expression of the synaptophysin protein was associated with synaptic vesicles, early expression can also be detected in the cell body and through the sizes of the processes, where it was initially expressed during neurogenesis. M. Fujita et al., Developmental Profiles of Synaptophysin in Granule Cells of Rat Cerebellum: An Immunocytochemical Study, 45 J. Electron Microsc. Tokyo 185-194 (1996); D. Grans et al., Differential Expression of Synaptophysin and Sinaptoporin during Pre- and Postnatal Development of the Rat Hippocampal Network, 6 Eur. J. Neurosci. 1765-1771 (1994). These results show that sharply dissociated E-NCAM * cells are dividing, immature cells that mature in culture. These results suggest that if the ^^^^^ ata t? fcf ra? a? iÉ? rrifr ^ - ¡2 ^^ - ^^ -'-- jrs ^ & --- NRP cells are induced to differentiate through RA and mitogen removal, acquire many morphological and immunological properties of mature neurons.

EXAMPLE 13 The numerous neuronal phenotypes can be detected in E-NCAM cells * Differentiated but not sharply dissociated It has been shown previously that NRP cells can differentiate into post-mitotic neurons, but not in oligodendrocytes or astrocytes. To determine if NRPs can differentiate into all major neuronal phenotypes present in the spinal cord, or if they are more limited in their differentiation potential, the expression of neurotransmitter synthesis enzymes and cell-type specific markers for mature neurons was examined after of inducing NRPs for differentiation. In addition, the expression of p75, Q. Yan & EJ Johnson, An Immunocytochemical Study of the Nerve Growth Factor Receptor in Developing Rats, 8 J. Neurosci, 3481-3498 (1988), and lslet-1, T. Tsuchida et al, Torographic organization of Embryonic Motor Neurons Defined by Expression of LIM Homebox Genes, 79 Cell 957-970 (1994), which is characteristic of motoneurons in the spinal cord, and calbindin, which is usually co-expressed with GABA, C. Batini, Cerebellar Localization and Colocalization of GABA and Calcium Binding Protein-D28K, 128 Arch.ltal. Biol. 127-149 81990), were examined. The E-NCAM * cells of the E13.5 rat neural tube were isolated by immunological panning according to the procedure of Example 3, plated on 35 mm plates, and cultured in a differentiation promoter medium. After 10 days in culture, the total RNA was isolated from these cells and the ability to synthesize the neurotransmitters acetylcholine (Ach), GABA, and glutamate was analyzed through the expression of their synthesis enzymes through RT-PCR. Total RNA was isolated from whole cells or tissues through a modification of the guanidine-phenol-chloroform isothiocyanate extraction method (TRIZOL, Gibco / BRL). For cDNA synthesis, 1-5 μg of total RNA was used in a 20 μl reaction using SUPERSCRIPT II (Gibco / BRL), a reverse transcriptase (RT) of modified Maloney murine leukemia virus, and oligo primers (dT) ) 12-18 according to the Gibco / BRL protocol. For the amplification by PCR of the cDNA, aliquots of cDNA, equivalent to 1/20 of the reverse transcriptase reaction, were used in a reaction volume of 50 μl. PCR amplification was performed using the ELONGASE polymerase (Gibco / BRL). The sequences of the initiator and the temperatures of cyclization used for the PCR amplification of receptors are shown in Table 8. The reactions were performed during 35 cycles, and a 10 minute incubation at 72 ° C was added at the end. j ^^^ ^ | ggg ^ ¡^^^^^ j ^ £ | g ^ to ensure a full extension. The PCR products were purified using the ADVANTAGE PCR-PURE equipment (Clontech, palo Alto, CA) and sequenced to confirm their identities.

As shown in Figure 2, all of these were present in differentiated cells (marked "D"). In contrast, none of these markers of neurotransmitter phenotypes could be detected from cells that were examined in 24 hours of isolation (referred to as "acutely dissociated", marked as "AD" in Figure 2), although the expression of the driving gene, cyclophilin, could be easily detected from both cell populations. These data show that NRP cells mature in culture and that NCAM expression and determination of neuronal fate precedes neurotransmitter synthesis. The expression of neurotransmitter synthesis enzymes was also examined through immunocytochemistry to ^^^ * fa ^^^^ ai > a'¿' ^ ^ as determine whether all cells, or only a sub-group of differentiated cells, express these markers. The cells were grown in a culture for 10 days and allowed to differentiate, fixed, and processed by immunocytochemistry according to the procedure of Example 2 to detect the expression of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) , Tyrosine hydroxylase (TH), glycine and glutamate. Antibodies were obtained for ChAT, TH and GAD from Chemicon; the antibodies for glutamate and glycine were from Signature Immunologicals. Virtually 100% of the differentiated cells expressed detectable levels of glutamate. A much smaller percentage expressed glycine and GAD. The exact percentages varied between the 10-50% experiments. The percentage of ChAT and TH * cells were even smaller and ranged from 1-5%. However, substantially larger numbers could be seen altering the culture conditions. Since virtually 100% of the cells synthesized glutamate, it is likely that at least some of the cells synthesized more than one neurotransmitter. However, these results clearly show that after differentiation, E-NCAM * cells are able to mature in a heterogeneous population with respect to their neurotransmitter phenotype. In contrast to the results obtained with differentiated cells, neither ChAT, GAD, TH nor glycine could be detected in acutely dissociated cells. The glutamate was detected in a small subgroup of said cells (less than 10%). Nevertheless, ^ Sá & ^ £ i? ^^^ s ^ l ££ & ^ a ^^ M glutaminase could not be detected in these cells through RT-PCR (Figure 2), which suggests that glutamate was taken by these cells from the medium.

EXAMPLE 14 The E-NCAM Cell Neurotransmitter Receptor Profile changes with Maturation Another important feature of mature neurons is their ability to respond to multiple neurotransmitters by expressing appropriate neurotransmitter receptors on their surfaces. To examine the ability of differentiated E-NCAM * cells to respond to glutamate, glycine, dopamine, and acetylcholine, fura-2 Ca2 * imaging techniques were used. E-NCAM * E13 cells were grown in a culture for 10 days and allowed to differentiate. They were then loaded with fura-2, and the depolarization response to the neurotransmitter application was verified. Cells were loaded with 5 μM Fura-2 / AM, D. Grykiewicz et al., A New Generation of Calcium Indicators with Greatly Improved Fluorescence Properties, 260 J. Biol. Chem. 3440-3450 (1985), incorporated herein by reference, plus PLURONIC F127 (80 μg / ml) in rat Ringers (RR) at 23 ° C in the dark for 20 minutes followed by 3 washes in RR and a deesterification of 30 minutes. The relative changes in the concentration of tMf ^ - ^ Aaf ^^ intracellular calcium from the background-corrected ratio of the fluorescence intensity through excitation at 340/380 nm. The response was defined as a minimum increase of 10% of the baseline value provided. A Zeiss-Attofluor imaging system and software (Atto Instruments Inc., Rockville, MD) were used to acquire and analyze the data. The data points were sampled at 1 Hz. Neurotransmitters were made in RR and supplied through a batch exchange, using a small volume loop injector (200 μl). The RR contained 140 mM NaCl, 3 mM KCl, 1 mM MgCl 2, 2 mM CaCl 2, 10 mM HEPES and 10 mM glucose. In addition, 500 μM of ascorbic acid was added to the dopamine solutions to prevent oxidation. The application of a 500 μM control of ascorbic acid had no effect. The pH of all solutions was adjusted to 7.4 with NaOH. In addition, 50 mM of K * RR was made by substituting equimolar K * for Na * in the normal RR. Figure 3 shows a bar graph of the number of cells that respond to the application of the indicated neurotransmitter in acutely dissociated and differentiated cells. In general, the number of cells that respond to the neurotransmitters and the amplitude of the Ca2 * responses induced by the neurotransmitter were increased in the differentiated cells. The most striking example is dopamine, where only 10% of the acutely dissociated cells responded to 500 μM of dopamine with increases in internal Ca2 * compared to 76% of cells Stt? Fe ^^? AaaiateB-i ^ iia ^ ai ^ A ^ t ^ Jaa ^ ^^. ^? ^^^ & sm differentiated, a net increase of 66%. Similar, but less surprising, changes in the number of cells that responded were seen for other excitatory neurotransmitters. The exceptions to this trend were the responses of Ca2 * to GABA and glycine. Of interest, 46% of the acutely dissociated cells responded to GABA compared only to 8% of the differentiated cells. Similarly, the flow of Ca2 * in response to glycine was reduced from 20% in cells sharply dissociated to 0% in differentiated cells. This change in the inhibitory neurotransmitter profile probably reflects the reduction in the concentration of internal chloride ion with maturation that represents the displacement of the depolarization responses to hyperpolarization of GABA to glycine. W. Wu et al., Early Development of Glycine and GABA-Mediated Synapses n Rat Spinal Cord, 12 J. Neurosci. 3935-3945 (1992). The possibility can not be excluded, however, that chloride ion levels remain high and the few GABA and glycine receptors are expressed in differentiated cells. The representative graphs of the relationship (13o l38o) and Ca2 * responses over time of an acutely dissociated and differentiated cell are shown in Figure 4 and Figure 5, respectively. The sharply dissociated cell responded to GABA and glutamate, whereas the differentiated cell of the same embryo responded to dopamine, glutamate and acetylcholine, but not to GABA or glycine. The comparison of Ca2 * responses to the various transmitters in adjacent cells revealed that there is heterogeneity in the fe ^^^ a ^^^ & iaS ^^^^ t ^^^^ a ^ .. response profiles between cells, indicating that not only heterogeneous E-NCAM * cells have the ability to synthesize neurotransmitters, but they are also selected in terms of transmitter receptor expression. In addition to the neurotransmitters, a high level of K * in rat Ringers (50 mM of K * RR) was applied to depolarize the cells and allow the entry of Ca2 * through the voltage gate channels. In acutely dissociated cells, 49% responded to 50 mM of K * RR compared to 85% of differentiated cells, suggesting that more of the differentiated cells were electrically competent than the cells were acutely dissociated. In this way, the contrast between the various properties of E-NCAM * sharply dissociated cells and the fully differentiated E-NCAM * cells, which are summarized in Table 9, is surprising. Immature cells are mitotically active, but differentiated cells are not. Immature cells do not express any mature neuronal protein such as NF-M, synaptophysin, or synthetic neurotransmitter enzymes, while all these can be detected in differentiated cells. In addition, acutely dissociated cells are all less responsive than cells differentiated to Ca2 + responses induced by neurotransmitter.

. * - ^ ^ ^ ^ ^^^^^^^^^^^^^^^^^ EXAMPLE 15 Individual E-NCAM * Cells can generate Multiple Neurotransmitter Phenotypes The mass culture experiments described above showed that the E-NCAM * population can generate multiple neurotransmitter phenotypes. There is a possibility, however, that individual cells can be pre-committed to generate specific neuronal phenotypes. To determine if the potential for differentiation of NRPs in the mass culture reflected the potential of an individual NRP, a clonal analysis of E-NCAM * cells was performed. ^^? t, ^ ¿¿i., > ^, »,., > .n¿ * »- t. ¡Gjj ^ ll E-NCAM * cells were immunoselected according to the procedure of Example 3, plated at a clonal density, and conditions that promote proliferation were developed in FGF and NT-3. The clones grew to sizes of several hundred cells after 10 days in culture, after which their differentiation was promoted through the removal of FGF and the addition of RA in the medium. Three different techniques were used to determine whether the clones generated from individual NRP cells were composed of heterogeneous populations of neurons: RT-PCR according to the procedure of Example 13, immunocytochemistry according to the procedure of Example 2, and imaging with calcium according to the procedure of Example 14. 6 clones were examined through RT-PCR analysis. Five of the six clones expressed multiple neurotransmitter phenotypes: one clone expressed the tested markers, 3 clones expressed four markers, and 1 clone expressed three markers. Therefore, all, but one clone, were composed of heterogeneous populations of cells. One clone expressed detectable levels only of p75 and Isl-1, but not of ChAT. This probably represented an immature clone that was not totally differentiated. Figure 6 shows the results of a representative clone that expressed all neurotransmitter markers tested. These results demonstrate that individual clones express multiple synthetic neurotransmitter enzymes or other phenotypic markers, and that most of the clones were composed of a heterogeneous population. To confirm the PCR results and to show the heterogeneity at the protein level, the clones were analyzed for the expression of p75. No clone (0/17) consisted of exclusively p75 immunoreactive cells, but all clones (17/17) contained p75 immunoreactive cells as well as other neurons. Similarly, the staining for the immuno-reactivity of both glutamate and glycine showed that each transmitter was expressed only by a sub-group of cells in the same clonal population, indicating that the clones are from a heterogeneous population. The heterogeneity was demonstrated not only through the synthesis of different neurotransmitters, but also through the heterogeneity in the receptors expressed by the cells. The response profiles of differentiated clonal cells to the application of GABA, glycine, dopamine, glutamate, acetylcholine and 50 nM K + RR, as evidenced by the increased concentrations of intracellular calcium, were examined. Ca2 * measurements were taken from 113 cells of 4 different clones. All the clones examined (4/4) exhibited heterogeneity in their response profiles, which varied a little between individual clones. Figure 7 shows a bar graph of the percentage of cells of the 4 clones that responded to each of the applied neurotransmitters. As with E-NCAM cell mass cultures * - > & ¡p «^. t-« fc * »" ff * ^ Differentiated, the high percentages of clonal cells responded to glutamate (93%), acetylcholine (96%), 50 mM K + RR (70%), and dopamine (50%), while few cells responded to GABA (27%) and glycine (1%). Figures 8 and 9 show fingerprints representative of the ratio (I340 / I380) of Ca2 * responses of two cells registered in a clone. This heterogeneous expression of receptors also suggested a multipotential characteristic of individual NRP cells. In this way, the maturation of clonal populations of cells closely resembled the maturation of cells in the mass culture. Through multiple independent methods, this clonal analysis demonstrates the multipotential characteristic of individual NRP cells. This analysis confirms that the mass culture results clearly define the development potential of the NRP cell. Although committed to generation neurons, the particular phenotypes of this progeny are dictated to some later stage in their development. In this way, the existence of a neuronal precursor cell that can be purified and subsequently manipulated to define the transition between the neuronal precursor of restricted lineage and the differentiated neuronal progeny has been established. ^^^^ ii ^ au.l ^ ?. afa- ^ aiU ^^ i a ^ EXAMPLE 16 Extracellular Signals Influences the Destination of NRP Cells The results described here show that neuronal precursors can be developed in vitro in mature neurons of multiple phenotypes in both mass and clonal cultures and that either the application of RA or the removal of FGF can promote differentiation in multiple phenotypes. However, in normal development, differentiation is spatially and temporally regulated, with motoneurons being generated ventrally and sensory neurons being generated dorsally, suggesting that specific environmental signals can divert the differentiation of neuronal precursors. In this example, the effects of two potentially regulatory molecules that are expressed in the spinal cord at the time of neurogenesis have been shown to bypass cells to either dorsal phenotypes (BMP-2/4, JM Graff, Embryonic Patterning: To BMP or Not to BMP, That is the Question, 89 Cell 171-174 (1997)) or ventral (Shh; MJ Fiets et al., The Hedgehog Gene Family in Drosophila and Vertébrate Development, Development (Suppl.) 43-51 ( 1994)). When BMP-2 was added to the E-NCAM * cell cultures, a dramatic reduction in cell division was seen. The effect of BMP-2 passed over the effect of the mitogen, FGF and even in the presence of FGF, caused a reduction of 60% in the division of JaBAiiiMtafe ^ .... ^^. ^^^ cell (Figure 10). Identical effects were seen with BMP-4. BMP-2 was not a survival factor, since cells growing only in BMP-2 did not survive. The reduction in mitosis was accompanied by the appearance of differentiated cells. The size of the cell increased and the cells shut down the extensive processes. Cells grown in BMP-2 for 48 hours were also examined for neurotransmitter expression. Glutamatergic, GABAergic, dopaminergic and cholinergic neurons were detected. The number of cholinergic neurons was significantly larger than in the untreated controls (5-10% v. 0-1%), however, there seemed to be no deviation towards ventral phenotypes, since the promotion of the other phenotypes was also significantly larger. In this way, BMP-2 acted as an antimitotic agent and promoted the differentiation of NRP cells from E-NCAM *, but did not appear to inhibit ventral destinations. In contrast to the antimitotic effect and promoter of BMP differentiation, Shh appeared to be a mitogen. The mitotic effect of Shh at 100 ng / ml (the maximum response) was three times higher than the controls, but was less than the effect of FGF at 10 ng / ml (Figure 11). Experiments were performed with Shh in the presence of NT-3, which acts as a survival agent, Y. A. Barde, Neurotrophins: A Family of Proteins Supporting the Survival of Neurons, 390 Prog. Clin. Biol. Res. 45-56 (1994), and not as a mitogen, since the same Shh did not seem to be a factor of ^ & ü ^ ^^^ ^ ^ z! & ^ g ^ & t? Survival for E-NCAM * cells, ie, E-NCAM * cells that grew only in Shh did not survive. The effect of Shh on mitosis was only apparent after two days of exposure and was maintained for a period of 5 days of the trial. No difference in cell division was seen during the first 24 hours. Shh did not seem to promote motor neuron differentiation during the 5 days of the trial. The cells continued to proliferate and no immunoreactive neurons could be detected, neither p75 nor ChAT. The failure to see cholinergic neurons was not due to the inability of E-NCAM * cells to differentiate to p75 or ChAT positive cells, as easily differentiated sibile cultures to ChAT and p75 immunoreactive cells when treated with such a differentiation agent. as BMP-2 or RA. In this way, e-NCAM * cells respond to Shh through proliferation. Shh unexpectedly did not promote differentiation of motor neurons, at least during the test time. These results indicate that the extracellular signaling molecules, Shh and BMP, modulate the phenotypic differentiation of E-NCAM * cells. BMP-2 inhibits cell proliferation and promotes differentiation and does not inhibit the differentiation of ventral phenotypes. In contrast, Shh promotes proliferation and inhibits the differentiation of any of the neuronal phenotypes, including p75 and ChAT immunoreactive neurons. 25 A i aMí ^ .3 £ aisiMi ^ EXAMPLE 17 Neural Mouse Tubes containing E-NCAM Immuno-reactive Neural Precursors To determine whether NRPs are present in mouse neural tubes, E11 mouse spinal cords were dissociated and examined for the properties of E-NCAM immunoreactive cells, according to with the procedures of Example 2. A large number of E-NCAM immunoreactive cells was found in E 11, and these cells comprised approximately 60% of the total cell population. E-NCAM positive cells appeared morphologically similar to neurons with extensive processes. At this stage of development, no co-expression of E-NCAM was observed with either Gal-C or GFAP in double-labeling experiments, suggesting that the immuno-reactivity of E-NCAM can identify neuronal precursors. To determine if the mouse E-NCAM positive cells, like their rat counterparts, underwent cell division, the cells were pulsed with BRDU and then double-labeled to detect cells that co-expressed BRDU immunoreactivity and IN CAM. The results showed that E-NCAM positive cells were divided at least three days in culture. The E-NCAM positive cells, in this way, appeared similar to the NRPs previously described in rats. To confirm that the E-NCAM positive cells can generate Multiple neuronal phenotypes, the immuno-selected E-NCAM cells prepared according to the procedure of Example 3, were allowed to differentiate in the culture for 10 days. The plates were then harvested and cDNA prepared according to the procedure of Example 13 to analyze neurotransmitter synthesis. As can be seen in Figure 12, the expression of p75, i s let-1, ChAT, calbindin, GAD and glutaminase was easily detected in differentiated populations. Thus, mouse E-NCAM immunoreactive cells can generate neurons that express cholinergic, excitation and inhibitory phenotypes.

EXAMPLE 18 The E-NCAM Immuno-reactive Neuroblasts can be generated from ES cells. In Example 17 the mouse spinal cords were shown to contain E-NCAM immunoreactive NRPs that are similar to rat NRP cells. To determine if similar restricted lineage precursors can be generated from ES cells, obtained mouse ES cells from Developmental Studies Hybridoma Bank (DSHB; University of Iowa, Lowa City, Iowa) and then developed in the culture and examined for the expression of E-NCAM, A2B5 and other neurogliale markers. As previously described, the undifferentiated ES cells did not express immuno-detectable reactivity for any of the markers tested. In a »at > É ^^. MaiaiaiBa «« ^^ In contrast, when ES cells were plated under conditions of neural differentiation, ES cells altered their morphology and began to express multiple neuronal and glial markers (Figure 13). The ES cells differentiated harvested and the total RNA was prepared for RT-PCR according to the procedure of Example 13. Of particular importance is the early expression of E-NCAM (early neuronal marker) and PLP / DM20 genes (which are known to be expressed by embryonic glia precursors). Consistent with marker detection In the early neural and glial cells through PCR, the highly polyamial NCAM expression cells represented a small percentage of the total cells. Less than 5% of the cells in culture expressed E-NCAM immunoreactivity after 5 days in culture. The percentage of A2B5 immunoreactive cells was significantly higher; about 10% of the differentiated cells expressed this marker. To determine whether the E-NCAM immunoreactive cells represented neuronal precursors, the co-expression of neuronal and glial markers was examined. E-20 NCAM immunoreactive cells co-expressed the immuno-reactivity of MAP-2 and β-III tubulin, but did not express the immuno-reactivity of GFAP and nestin. The E-NCAM positive cells did not express Gal-C or other oligodendrocytic markers. In this way, the E-NCAM immunoreactive cells that were derived from mouse ES cells appeared similar to the positive NRPs E-NCAM derived from spinal cord.

To confirm that ES cell-derived neuronal precursors can generate multiple types of neurons, the E-NCAM immunoreactive cells were immunoselected according to the procedure of Example 3 and said purified cells were allowed to differentiate for 10 days. The cells were then harvested and analyzed by immunocytochemistry and RT-PCR for the expression of phenotypic markers. Figure 14 shows the results of an illustrative PCR experiment, where the expression of ChAT, p75, is let-1, calbindin, GAD and glutaminase was easily detected in differentiated populations. In this way, E-NCAM immunoreactive cells derived from ES cells differentiated into post-mitotic neurons that expressed multiple neurotransmitters, including cholinergic, excitation and inhibitory phenotypes. Therefore, ES cells can be used as a source of restricted lineage NRPs.

EXAMPLE 19 NRPs in Human Neural Tubes To determine if NRPs are present in human neural tubes, human embryonic spinal cords were dissociated and the cell phenotypes, when developed in DMEM / F12 at a high concentration of FGF, were examined according to the procedure of Example 2. Human spinal cord cells (HSCs) at the beginning It appeared morphologically similar to the rat and mouse spinal cords, but rapidly differentiated into cells of fibroblastic appearance with a significant proportion of cells having a neuronal morphology. HSCs continued to divide rapidly and most cells (95%) were immuno-reactive to nestin. In this stage, the cultures did not contain astrocytes, oligodendrocytes or their precursors as detected by the expression of GFAP or O4 / Gal-C. A substantial number of E-NCAM immunoreactive cells was present, however, and constituted approximately 40% of the total population. The E-NCAM immunoreactive cells appeared morphologically similar to neurons, although some flat E-NCAM immunoreactive cells were also present. Both populations of E-NCAM positive cells were immunoreactive to MAS2K and also expressed a variety of other early neuronal markers, as summarized in Table 10.

At this stage of development, no observations were E-NCAM expression with either Gal-C or GFAP in double-labeling experiments, suggesting that the immuno-reactivity of E-NCAM identifies neuronal precursors. That is, the E-NCAM immunoreactive human spinal cord cells expressed neuronal but not non-neuronal antigens. To determine whether the E-NCAM cells, as well as their rat counterparts, underwent cell division, the mixed cultures of HSCs were pulsed with BRDU and then double-labeled to detect cells that co-express BRDU and immuno-reactivity of IN CAM. The results of this experiment showed that the E-NCAM positive cells were divided during at least three days in culture. Consistent with the results in Table 10 that the E-NCAM immunoreactive cells also express NF-H, the BRDU incorporation cells also co-expressed neurofilament-H. Thus, as in the spinal cord cultures of fetal rodents, nesting immunoreactive precursor cells from dividing humans are present and the E-NCAM immunoreactive cells represent a significant fraction of the total precursor population in this stage. The E-NCAM * cells appear similar to the NRPs previously described for rats and mice. The transplanted cells can be administered to any animal, including humans, with abnormal neurological or neurodegenerative symptoms obtained in any form, including as a result of chemical electrolyte lesions, experimental destruction of neural areas, or aging processes. The transplant can be bilateral, or, for example in patients suffering from Parkinson's disease, it can be contralateral to the most affected side. The surgery is preferably performed so that the particular regions of the brain are localized, such as in relation to sutures of the skull, and surgery performed with stereotactic techniques. Alternatively, the cells can be implanted in the absence of stereotactic surgery. The cells can be delivered to any affected neural area using any method of cell injection or transplantation known in the art. In another embodiment of the invention, NRP cells are transplanted into a host and induced to proliferate and / or differentiate in that host through (1) proliferation and / or differentiation in vitro before being administered, or (2) differentiation in vitro before being administered and proliferation and differentiation in vivo after being administered, or (3) proliferation in vitro before being administered and then differentiation in vivo without further proliferation after being administered, or (4) proliferation or differentiation in vivo after being injected directly after being recently isolated. NRP cells can also be used for the delivery of therapeutic compounds or other compounds. Methods for deriving the blood-brain barrier for purposes of therapeutic compound delivery include implanting cells in an encapsulation device according to methods known in the art or directly implanting genetically engineered cells, so that the cells themselves produce the therapeutic compound. Sayings compounds can be small molecules, peptides, proteins, or viral particles. Cells can be genetically transduced through means well known in the art, including calcium phosphate transfection, DEAE-dextran transfection, polybrene transfection, electroporation, lipofection, virus infection, and the like. The cells are first genetically engineered to express a therapeutic substance and then transplanted either as free cells capable of diffusing and incorporating into the CNS parenchyma or contained within an encapsulation device. R. P. 15 Lanza & W. L. Chick, Encapsulated Cell Therapy, Sci. Amer .: Sci. & med. July / August, 16-25 (1995); P. M. Galletti, Bioartificial Organs, 16 Artificial Organs 55-60 81992); A. S. Hoffman, Molecular Engineering of Biomaterials in the 1990s and Beyond; A Growing Liaison of Polymers with Molecular Biology, 16 Artificial Organs 43-209 (1992); B. D. Ratner, New Ideas in Biomaterials Science - A Path to Engineered Biomaterials, 27 J. Biomed. Mat. Res. 837-850 (1993); M. J. Lysaght et al., Recent Progress in Immunoisolated Cell Therapy, 56 J. Cell Biochem. 196-203 (1994), incorporated herein by reference. 25 Transplanted cells can be identified by previous incorporation of trace dyes such as rhodamine or fluorescein-labeled, fast blue, bis-benzamide, or incorporated genetic markers by any genetic transduction method known in the art to allow the expression of said enzymatic markers such as β-galactosidase or alkaline phosphatase . Any expression system known in the art can be used to express the therapeutic compound, as long as it has a promoter that is active in the cell, and internal signals appropriate for initiation, termination and polyadenylation. Examples of suitable expression vectors include recombinant vaccine vectors including pSCII, or vectors derived from viruses such as simian virus 40 (SV40), Rous Sarcoma Virus (RSV), mouse mammary tumor virus (MMTV), adenovirus, herpes simplex virus (HSV), bovine papilloma virus, Epstein-Barr virus, lentivirus, or any other eukaryotic expression vector known in the art. Many of the expression vectors are commercially available. The cells can also be transduced to express any gene encoding a neurotransmitter, neuropeptide, neurotransmitter synthesis enzyme or neuropeptide synthesis enzyme for which expression in the host is desired. The NRP cells and / or their derivatives cultured in vitro can be used for the classification of potential and neurologically therapeutic compositions. These compositions can be gfl ^ g ^^ ¿^^^^^ ¿^^ applied to cells in culture at varying doses, and the response of the cells was verified during several periods. The induction of expression of new or increased levels of proteins such as enzymes, receptors and other cell surface molecules, or of neurotransmitters, amino acids, neuropeptides and biogenetic amines can be analyzed with any technique known in the field that can identify the alteration of the level of said molecules, including protein assays, enzymatic assays, receptor binding assays, linked enzyme immunoassay assays, electrophoretic analysis, high performance liquid chromatography analysis, Western staining and radioimmunoassays. Nucleic acid analysis, such as Northern stains, can be used to examine the levels of mRNA encoding these molecules, or for the enzymes that synthesize these molecules. Alternatively, cells treated with these pharmaceutical compositions can be transplanted into an animal and their survival, ability to form neurons and to express any of the functions of these cell types can be analyzed by any method available in the art. The NRP cells can be cryopreserved through any method known in the art. $ & l t &% 8í ^^ & ^ EXAMPLE 20 Use of NRP Cells and / or their Derivatives for the Treatment of Neurological or Abnormal Neurodegenerative Symptoms 5 NRP cells are isolated through the methods of Examples 2, 3, 8, 18 or 19. The cells were obtained from embryonic CNS or human adult from xenographic sources from which immuno-rejection of cells is not a critical problem, such as pigs genetically engineered with the end of not present a foreign stimulus to the human immune system. The cells harvested from embryos are obtained through tissue dissection of the CNS following routine absorption and tissue collection procedures in a sterile collection apparatus. The post-natal CNS cells are obtained through the digestion of the tissue following the routine autopsy. The tissue is prepared, the cells are immunopurified and the resulting purified cells are cultured as in Example 2. The cells can be transplanted directly or first they can be expanded in vitro before transplantation. The populations Expanded in vitro can also be expanded under conditions that improve the generation of neurons or cells committed to the generation of neurons. Transplantation is performed routinely in cell suspensions of 5-50,000 cells / μl in physiological salt solutions, such as PBS. Cells can be transplanted to or near any a i ^ i ^^ ftitfMif ^ of the CNS regions affected by the disease or condition. The transplantation procedures, with modifications suitable for use in human patients, are, in their essence, similar to those well known to those skilled in the art of transplantation of progenitor cells O-2A, for example, AK Groves et al. Repair of Demyelinated Lesions by Transplantation of Purified O-2A Progenitor Cells, 362 Nature 453-455 (1993), incorporated herein by reference. More specifically, the transplant is performed using a stereotaxic tomographic computerized guide. The patient is operated using any of the methods known in the art. In cases where precisely localized transplantation is desirable, the patient undergo CT scan to establish the coordinates of the region to receive the transplant. The cannula of injection may be of any configuration used by those skilled in the relevant art. The cannula is then inserted into the brain to correct the coordinates, then removed and replaced with a 19-gauge infusion cannula that has been pre-loaded with the cell suspension in a small selected volume. The cells are then slowly infused, at speeds generally of 1-10 ml per minute as the cannula is withdrawn. For some diseases where it is desired to spread the cells over the largest possible area, multiple stereotactic needle steps can be made to through the area. Patients are examined post-operatively ^ aB ^ J ^ i = rtaaJa ^^^^ ia - ^^ A ^^ fff ^, ^. ^ a ^, .., - ,. : -., - ^., ^ ¿^ ^ ». ^ for hemorrhage or edema. Neurological evaluations were performed at several post-operative intervals, as well as PET scans of these can be used to determine the metabolic activity of the implanted cells. These and similar procedures can be used for any implantation of NRP cells for any of the purposes indicated in this invention. The success of the procedure is determined through invasive analysis with, for example, nuclear magnetic resonance image scanners, and / or through functional recovery analysis according to methods well known in the art.

EXAMPLE 21 Use of Genetically Engineered NRP Cells 15 and / or their Derivatives for Transplantation In this example, NRP cells were genetically modified ex vivo prior to introduction to or near disease regions to express gene products that will make the cells transplanted resistant to destruction in vivo and / or to Expressing gene products that provide trophic support to host cells and / or to express gene products that limit destructive processes occurring in the host. Genetic modification is performed through any of the techniques known to those skilled in the art, including but not limited to transfection of calcium phosphate, transfection of DEAE-dextran, Ar ^ M ^^^ att ^^^^^^^^^^^^^^^^ j ^^^^^^^^^^^^^^^^^^, transfection of polybrene, electroporation, lipofection, virus infection, and the like. The gene products that could make the cells resistant to destruction in vivo and / or to express gene products that provide trophic support to the host cells and / or to express gene products that limit destructive processes occurring in the host include but are not limited to insulin-like growth factor-1, decay acceleration factor, catalase, superoxide dismutase, members of the neurotrophin family, glial-derived neurotrophic factor, ciliary neurotrophic factor 10, leukemia inhibitory factor, ligand fas, cytokines that inhibit inflammatory processes, receptor fragments that inhibit inflammatory processes, antibodies that inhibit inflammatory processes, and so on.

EXAMPLE 22 Use of NRP Cells and / or their Derivatives for the Classification of Potential and Neurologically Therapeutic Compositions NRP cells or their derivatives or mixtures thereof cultivated in vitro can be exposed to compositions of interest at varying doses, and the response of cells It was verified during several periods. The induction of expression of new levels or increased levels of proteins such as enzymes, receptors, and other cell surface or neurotransmitter molecules, 25 amino acids, neuropeptides and biogenic amines can be analyzed rf? jMj. ^ * »^^« = j > * ^ * ^ - »> - ^ »^^ - - - > ^^ s «^ - ^^ * ^ dti & ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ with any of the techniques known in the art which can identify the alteration of the level of said molecules, including protein assays, enzymatic assays, receptor binding assays, linked enzyme-linked immunosorbent assays, electrophoretic analysis, liquid chromatography analysis high performance, Western stains, and radioimmune assays. Nucleic acid analysis, such as Northern hybridization, can be used to examine the levels of mRNA that codes for those molecules, or for the enzymes that synthesize those molecules. The cells can also be used to classify compounds capable of promoting the division of NRP cells and / or their derivatives by determining the ability of the compounds to cause increases in the number of NRP cells or to promote DNA synthesis, as measured by, for example, example, incorporation of bromodeoxyuridine or titrated thymidine. Cells can also be used to classify compounds that promote the survival of NRP cells and / or their derivatives by applying the compounds to cells in conditions where they could be expected to die (eg, exposure to neurotoxic agents, withdrawal of all trophic factors) and examine cell survival using any of the techniques well known to those skilled in the art. Cells can also be used to classify compounds that specifically inhibit binding to particular receptors, looking at the ability of such compounds blocking to block the response produced by the union of the ^^ hk ^^^^^^^^^^^^^^^^? g ^^^^^^^^ j ^^^ agonist to said receptors. The cells can also be used to classify compounds capable of activating particular receptors using ligand binding assays well known to those skilled in the art, or by looking at said physiological alterations as associated with receptor activation, such as fluxes in calcium levels, or other alterations well known to those skilled in the art. Alternatively, the cells treated with these pharmaceutical compositions can be transplanted to an animal and its survival, the ability to form neurons and to Expressing any of the functions of these cell types can be analyzed through any of the methods available in the art.

EXAMPLE 23 In this example, the cells were harvested from rat spinal cords E13.5, and the E-NCAM immunoreactive neuronal restricted precursor cells were harvested by immunological panning according to The procedure of Example 3. These cells were then labeled with a cell tracer and transplanted to different cortical regions using a glass microelectrode. The animals were sacrificed after 3.5, 10 or 21 days, and the brain was sectioned according to the well-known methods in the art. It was shown that said transplanted cells ^ Aig ^^^ gjí ^^ fe They survive and differentiate three times.

EXAMPLE 24 In this example, the cells were harvested, isolated and plated in a 35 mm dish as described in Example 3. The cells were then incubated with a retroviral construct containing a low green fluorescent protein (GFP) reporter gene. a cytomegalovirus (CMV) promoter. The cells were allowed to recover for 8 hours and then analyzed for GFP expression. GFP expression was detected after 24 hours of infection, and the expression of GFP persisted for up to 2 weeks, at which time the experiment was concluded. These results show that ectopic genes can be expressed in NRPs under a heterologous promoter, and that infected cells continue to stably express the ectopic protein for several weeks. twenty LIST OF SEQUENCES < 10 > Rao, Mahendra, S. Mayer-Proschel, Margot Kalyani, Anjali J. < 120 > Neural Precursors of Restricted Lineage < 130 > T5530, CIP < 140 > < 141 > 1998-07-02 < 150 > US 08 / 909,435 < 151 > 1997-07-04 < 160 > 14 < 170 > WordPerfect 8.0 < 210 > 1 < 211 > 21 < 212 > DNA < 213 > Rattus norvegious < 400 > 1 gcacatactc agacgaagcc to 21 < 210 > 2 < 211 > 24 < 212 > DNA < 213 > Rattus norvegious < 400 > 2 agcagccaag arggagcaat agac 24 < 230 > 3 < 211 > 23 < 212 > DNA < 213 > Rattus norvegious < 400 > 3 ctgaatactg gctgaatgac atg 23 < 210 > 4 < 211 > 23 < 212 > DNA < 213 > Rattus norvegious < 400 > 4 aaattaatga caactccaa gac 23 < 210 > 5 < 211 > 20 < 212 > DNA < 213 > Rattus norvegious < 400 > 5 gcagcatagg cttcagcaag 20 < 210 > 6 < 211 > 19 < 212 > DNA < 213 > Rattus norvegious < 400 > 6 gtagcaggtc cgcaaggtg 19 < 210 > 7 < 211 > 21 < 212 > DNA < 213 > Rattus norvegious < 400 > 7 gaatctttc tcctggtggt g 21 < 210 > 8 < 211 > 21 < 212 > DNA < 213 > Rattus norvegious < 400 > 8 gatcaaagc cccgtacaca g 21 < 210 > 9 < 211 > 18 < 212 > DNA < 213 > Rattus norvegious < 400 > 9 gcagaatccc acctgcag 18 < 210 > 10 < 211 > 19 < 212 > DNA < 213 > Rattus norvegious < 400 > 10 gttgctggca tcgaaagag 19 < 210 > 11 < 211 > 24 < 212 > DNA < 213 > Rattus norvegious < 400 > 11 gcacagacat ggttgggata ctga 24 < 210 > 12 < 211 > 20 < 212 > DNA < 213 > Rattus norvegious < 400 > 12? O gcagggctgt tctggagtcg 20 < 210 > 13 < 211 > 20 < 212 > DNA < 213 > Rattus norvegious 15 < 400 > 13 ccaccgtgtt cttcgacatc 20 < 210 > 14 < 211 > 19 < 212 > DNA 20 < 213 > Rattus norvegious < 400 > 14 ggtccagcat ttgccatgg 19

Claims (59)

1. - A pure population, isolated from neuron precursor cells restricted to the mammalian CNS.

2. The population according to claim 1, wherein said restricted neuron precursor cells are capable of self-renewal.

3. The population according to claim 1, wherein said restricted neuron precursor cells are capable of differentiation to neuronal cells of the CNS, but not to CNS glia cells.

4. The population according to claim 1, wherein said restricted neuron precursor cells express the embryonic neural cell adhesion molecule.

5. The population according to claim 4, wherein said neuron-restricted precursor cells do not express a ganglioside recognized by the antibody A2B5.

6. The population according to claim 4, wherein said restricted neuron precursor cells do not express nestin.

7. The population according to claim 1, wherein said restricted neuron precursor cells are selected from a mammalian embryo selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, swine, sheep. , lagomorphs and other rodents. ^ A ^ A? -A ^ ~ í '* A * 5"

8. - The population according to claim 1, wherein said cells are capable of differentiating neurons that are capable of releasing and responding to neurotransmitters.

9. The population according to claim 8, wherein said neurons demonstrate receptors for said neurotransmitters, and said cells are capable of expressing neurotransmitter synthesis enzymes.

10. The population according to claim 1, wherein said cells are capable of differentiating neurons, which can form functional synapses and / or develop electrical activity.

11. The population according to claim 1, wherein said cells are capable of stably expressing at least one material selected from the group consisting of growth factors for said cells, differentiation factors for said cells, maturation factors for said cells, and combinations of any of these.

12. A method for isolating a pure population of mammalian CNS-restricted neuron precursor cells, comprising the steps of: (a) isolating a population of stem cells from the mammalian multipotent CNS capable of generating both neurons and glia; (b) incubating the stem cells of the multipotent CNS in a medium configured to induce said cells to begin differentiation; ? »» .. (c) purifying from the differentiating cells a sub-population of cells expressing a selected antigen that defines neuron-restricted precursor cells; and (d) incubating the purified sub-population of cells in a medium configured to support their adherent growth.

13. The method according to claim 12, wherein said selected antigen defining restricted neuron precursor cells is an embryonic neural cell adhesion molecule.

14. The method according to claim 12, wherein said purification comprises a method selected from the group consisting of specific antibody capture, fluorescent activated cell sorting, and magnetic bead capture.

15. The method according to claim 14, wherein said method is specific antibody capture.

16. The method according to claim 12, wherein said multipotent CNS stem cells are neuroepithelial stem cells.

17. The method according to claim 16, wherein said isolation of a population of neuroepithelial stem cells of the CNS comprises: (a) removing a tissue from the CNS from a mammalian embryo at an embryonic development stage after of the closing of the neural tube, but before the differentiation of cells in the neural tube; (b) dissociating cells comprising the neural tube removed from the mammalian embryo; (c) plating the dissociated cells in an independent cell-feeder culture on a sub-stratum and in a medium configured to support the adherent growth of the neuroepithelial stem cells comprising effective amounts of the fibroblast growth factor and extract of chicken embryo; and (d) incubating the plated cells at a temperature and atmosphere that lead to the growth of the neuroepithelial stem cells.

18. The method according to claim 17, wherein said mammalian embryo is selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, swine, sheep, lagomorphs and the order of rodents. .

19. The method according to claim 17, wherein said sub-stratum is selected from the group consisting of fibronectin, vitronectin, laminin and RGF peptides.

20. The method according to claim 12, wherein said medium comprises effective amounts of the fibroblast and neurotrophin growth factor 3.

21. A method for isolating a pure population of neuron precursor cells restricted from the mammalian CNS. includes the steps of: (a) removing a tissue sample from the CNS from a mammalian embryo at a stage of embryonic development after neural tube closure, but before differentiation of glial and neuronal cells in the neural tube; (b) dissociating cells comprising the sample of the CNS tissue removed from the mammalian embryo; (c) purifying from the dissociated cells a sub-population that expresses a selected antigen that defines precursor neuron-restricted cells; (d) plating the purified sub-population of cells in a cell-feeder independent culture on a sub-stratum and in a medium configured to support the adherent growth of the neuron-restricted precursor cells; and (e) incubating the cells plated at a temperature and atmosphere that lead to the growth of the neuron-restricted precursor cells.

22. The method according to claim 21, wherein said selected antigen defining neuron-restricted precursor cells is an embryonic neural cell adhesion molecule.

23. The method according to claim 21, wherein said purification comprises a method selected from the group consisting of specific antibody capture, fluorescence activated cell sorting and magnetic bead capture.

24. - The method according to claim 23, wherein said method is specific antibody capture.

25. - The method according to claim 21, wherein said mammalian embryo is selected from the group consisting of human and non-human primates, equines, canines, felines, bovines, swine, sheep, lagomorphs, and of the order of rodents.

26.- A pure population of mammalian CNS-restricted neuron precursor cells isolated through the method of claim 12.

27.- A pure population of neuron precursor cells restricted from the mammalian CNS isolated through the method of claiming 21.

28.- A method for obtaining post-mitotic neurons comprising: (a) providing restricted neuron precursor cells and culturing the neuron-restricted precursor cells under proliferation conditions; and (b) changing the culture conditions of the neuron precursor cells restricted from proliferation conditions to a differentiation condition, thereby causing the neuron precursor cells restricted to differentiate into post-mitotic neurons.

29. The method according to claim 28, wherein said change of culture conditions comprises adding retinoic acid to the basal medium.

30. The method according to claim 28, wherein said change of culture conditions comprises removing a factor mitotic of the basal medium.

31. The method according to claim 30, wherein said mitotic factor is a fibroblast growth factor.

32. The method according to claim 28, wherein said change of culture conditions comprises adding a neuronal maturation factor to the basal medium.

33. The method according to claim 32, wherein said neuronal maturation factor is a member selected from the group consisting of sonic hedgehog, BMP-2, BMP-4, NT-3, NT-4, CNTF, LIF. , retinoic acid, brain-derived neurotrophic factor (BDNF), and combinations of any of the foregoing.

34. An isolated cellular composition comprising the neuron-restricted cells of the mammalian CNS of any of claims 1-7.

35.- A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 34 and a pharmaceutically acceptable carrier.

36. A method for treating a neuronal disorder in a mammal, comprising administering to said mammal a therapeutically effective amount of the composition of claim 34.

37. - A method for treating a neuronal disorder in a mammal, comprising administering to said mammal a therapeutically effective amount of the composition of claim 35.

38. - The method according to claim 34, wherein said composition is administered through a selected route of the group consisting of intramuscular administration, intraheal administration, intraperitoneal administration, intravenous administration and combinations of any of the foregoing.

The method according to claim 34, wherein said method also includes the administration of a member selected from the group consisting of differentiation factors, growth factors, cell maturation factors, and combinations of any of the foregoing. .

The method according to claim 39, wherein said differentiating factors are selected from the group consisting of retinoic acid, BMP-2, BMP-4 and combinations of any of the foregoing.

41. The composition according to claim 34, for use as a glia cell delivery vehicle of an agent selected from the group consisting of cell growth factors, cell maturation factors, cell differentiation agents and any combination of the above.

42. The composition according to claim 34, to be used as a delivery vehicle to supply trophic factors to neurons.

43.- A method for the treatment of neurodegenerative symptoms in a mammal comprising the steps of: (a) providing a pure population of neuronal restricted precursor cells; (b) genetically transforming said neuronal restricted precursor cells with a gene encoding a growth factor, neurotransmitter, neurotransmitter synthesis enzyme, neuropeptide synthesis enzyme, or substance that provides against free radical mediated damage, thus resulting in a transforeated population of glial restricted precursor cells expressing said growth factor, neurotransmitter, neurotransmitter synthesis enzyme, neuropeptide, neuropeptide synthesis enzyme, or substance that provides protection against damage mediated by free radical; and (c) administering an effective amount of said transformed population of neuronal restricted precursor cells to said mammal.

44. A classification method or compounds for neurological activity comprising the steps of: (a) providing a pure population of neuronal restricted precursor cells or derivatives thereof or mixtures thereof cultured in vitro; (b) exposing said cells or their derivatives or mixtures thereof to a selected compound at varying doses; and (c) verifying the reaction of said cells or their derivatives or mixtures thereof to said selected compound during selected periods.

45.- A method for the treatment of a disease neurological or neurodegenerative, which comprises administering to a mammal in need of such treatment an effective amount of neuronal restricted precursor cells or their derivatives or mixtures thereof.

46. The method according to claim 45, wherein said neuronal restricted precursor cells or their derivatives or their mixtures are caused to proliferate and differentiate in vitro before being administered.

47. The method according to claim 45, wherein said neuronal restricted precursor cells or their derivatives or their mixtures are caused to proliferate and differentiate in vitro before being administered, and then are caused to proliferate further and be differentiate in vivo after being administered.

48. The method according to claim 45, wherein said neuronal restricted precursor cells or their derivatives or their mixtures are caused to proliferate in vitro before being administered, and then they are made to differentiate in vivo before being administered. .

49. The method according to claim 45, wherein said neuronal restricted precursor cells or their derivatives or their mixtures are from a heterologous donor.

50.- The method according to claim 49, wherein said donor is a fetus.

51.- The method according to claim 49, wherein said donor is a young man.

52. The method according to claim 49, wherein said donor is an adult.

53. The method according to claim 45, wherein said neuronal restricted precursor cells or their derivatives or their mixtures are from an autologous donor.

54. The method according to claim 53, wherein said donor is a fetus.

55.- The method according to claim 53, wherein said donor is a young person.

56. The method according to claim 53, wherein said donor is an adult.

57. The method according to claim 45, wherein said derivatives thereof are obtained through differentiation of neuronal restricted precursor cells in vitro.

58. The method according to claim 45, wherein said derivatives thereof are obtained through genetic transduction of neuronal restricted precursor cells.

59. A method for isolating a pure population of neuron precursor cells restricted from the mammalian CNS, comprising the steps of: (a) providing a sample of embryonic stem cells of a mammal; (b) purify from mammalian embryonic stem cells a sub-population that expresses a selected antigen that defines restricted neuron precursor cells; (c) plating the purified sub-population of cells in a cell-feeder independent culture on a sub-stratum and in a medium configured to support the adherent growth of the neuron-restricted precursor cells; and (d) incubating the cells plated at a temperature and atmosphere that lead to the growth of the neuron-restricted precursor cells. SUMMARY A restricted, self-renewing stalk cell population has been identified to develop (embryonic day 13.5) spinal cords that can differentiate into multiple neuronal phenotypes, but can not be differentiated into glia phenotypes. This neuronal restricted precursor (NRP) expresses a highly polysialated or embryonic neural cell adhesion molecule (E-NCAM) and is morphologically distinct from neuroepithelial stem cells (NEP cells) and progenitors of spinal cord spinal glands on day 10.5 embryonic The NRP cells self-renew on multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin 3 (NT- #) and express a sub-group characteristic of neuronal epitopes. When grown in the presence of RA and in the absence of FGF, NRP cells differentiate into GABergic, glutaminergic immunoreactive neurons, and cholinergic. NRP cells can also be generated from multipotent NEP cells cultured from day 10.5 embryonic neural tubes. Clonal analysis shows that E-NCAM immunoreactive NRP cells arise from a NEP progenitor cell that generates other restricted CNS precursors. The E-NCAM immunoreactive cells derived from NEP undergo self-renewal in defined n medium and differentiate into multiple neuronal phenotypes in a mass and clonal culture. In this way, there is a direct linear relationship between A ^ A »s > multipotent NEP cells and the most restricted neuronal precursor cells present in vivo on day 13.5 embryonic in the spinal cord. Methods for treating neurological diseases are also described.