WO2012164137A1 - Stem cells and neural crest cells derived from olfactory ensheathing glia, and uses thereof - Google Patents

Abstract

The invention relates to the production of stem cells and neural crest cells (neuron, glia and others) from the olfactory ensheathing glia (OEG, also called olfactory ensheathing cells or OEC), to the cells thus produced and to the uses thereof. More specifically, the invention relates to the stem cells and the precursors of the neural crest (or others) that are derived from the dedifferentiation of the olfactory ensheathing glia, that can be differentiated and give rise to different cell types, generally from the neural crest, but not necessarily. The invention also relates to the use of stem cells and the different cell lines obtained from the olfactory ensheathing glia, and the products derived from all the cell types produced, for treating illnesses; to cell therapy; to the search for, and design of, new medications; and to cellular, molecular, genomic and pharmacogenomic studies of human pathologies.

Description

 MOTHER CELLS AND NEURAL STYLE CELLS DERIVED FROM THE OLFATORY ENVELOPE GLIA, AND ITS APPLICATIONS

SECTOR OF THE TECHNIQUE

The invention falls within the technical sector of medical therapies, more specifically in the use of cells and their derivatives in the treatment of diseases and in diagnosis. STATE OF THE PREVIOUS TECHNIQUE

Cell transplants constitute a promising therapeutic strategy to repair and / or replace tissues and cells damaged by various skin, cardiac, metabolic, genetic, muscular, etc. pathologies, as well as the nervous system (degenerative, ischemic, trauma diseases, demyelinating, among others) (Revised in (Bjorklund, et al., 2000; Jain, 2009; Reier, 2004)). When the cells do not come from the patient himself, but are obtained from animals (xeno-transplant) or other individuals (alo-transplant), the host's immune system does not recognize the cells as their own and the patient must receive immunosuppressive medication to avoid implant rejection. However, if the donor of cells and the recipient thereof are the same individual (autologous therapy) the transplanted organs, tissues or cells are compatible with the host's immune system, and such therapy is not needed to avoid rejection. By not requiring the use of lifelong immunosuppressants in recipient patients, complications and side effects of this type of medication are avoided and the problem of finding a compatible donor is also eliminated. The performance of an autologous therapy also offers the advantage that the grafted cells or tissue integrate better than when the origin is from another individual or another species. All these advantages have favored that in the field of cell therapy the development of treatments using cells that allow the three options is preferred: auto-, alo- and xeno-transplantation. This is also the preference in the case of the generation of organs, tissues and cells from stem cells since, in this way, the patient himself solves the problem of getting a compatible donor, the risk of implant rejection is avoided. , and their cells could also be used in other immunologically compatible individuals. In addition, adult stem cells allow the availability of cellular phenotypes relevant to human pathology that otherwise could not be obtained from an adult individual.

 In the case of pathologies of the nervous system, the choice of the type of cell to be transplanted has depended on the type of lesion to be treated and the intrinsic reparative capacity that the cells of choice presented. In the case of axotomy pathologies, in which there is an interruption of nerve fibers without neuronal death, the purpose of transplanting cells is to achieve the regeneration of the injured axons and that they reestablish the appropriate connections with their targets so that they recover lost functions as a result of aggression. Cells that, spontaneously or after manipulation, produce survival factors and / or axon growth promoters (Ruff, et al., 201 1; Sahni, et al., 201 1; Tetziaff, et al. , 201 1). In the case of degenerative pathologies, the object of cell transplants is twofold. On the one hand, replace the cells that have degenerated, either neurons or glia, with others that can replace their function. On the other hand, transplants should promote the survival of host and transplanted cells, and rescue those cells that have not yet degenerated.

A limitation of cellular regenerative therapy in the case of nervous system pathologies is that neurons cannot be obtained from the patient. It is not advisable to perform biopsies of the brain, cerebellum, brain stem, spinal cord or peripheral nervous system to obtain neurons because this would generate additional damage to the patient. In the case of the central nervous system, obtaining glia is also limited for the same reason. In addition, neurons (peripheral and central) and oligodendroglia extracted from the adult nervous system have very low survival and little or no ability to divide, so it would be difficult to obtain a sufficient number of cells for transplantation. To alleviate this problem, stem cells from sources outside the central nervous system are being used to, from them, try to generate the different types of neural lineage.

Embryonic stem cells are being used since, because they are pluripotential, they can give rise to neurons and glia (Kim, et al., 2009). For example, they have been used in animals and in stem cell transplants, both embryonic and adult, and oligodendrocyte precursors to promote myelination of naked axons (reviewed in (Mirón, et al., 201 1)). In experimental models of Parkinson's disease, and in some cases in people, stem cells, dopamine neuroblasts, and other dopamine-producing cells (carotid body cells, genetically modified, etc.) have been transplanted (Reviewed in (Fricker-Gates , et al., 201 1)). Transplants of stem cells and their derivatives of neural lineage are also being used to repair other degenerative, traumatic and ischemic diseases of the nervous system (Rodríguez, et al., 201 1; Uccelli, et al., 201 1). Several groups are choosing to pre-differentiate the stem cells before being transplanted, towards certain lineages of the neural lineage, such as oligodendroglia, for therapeutic purposes (Sharp, et al., 201 1). However, the use of embryonic stem cells has important disadvantages. It is difficult to control these cells in vitro without inducing their differentiation towards unwanted and non-neural cell types. In addition, in order to obtain a specific phenotype, which is needed for a pathology of the nervous system, embryonic stem cells must undergo several differentiation processes generally through treatment with compounds Chemicals or genetic manipulation. In addition, the use of embryonic stem cells has some controversy regarding the ethical problems derived from using embryos to obtain them. Another drawback is that the cells come from another individual (allotransplant) and therefore are generally rejected by the host. Although this problem could be remedied by performing a "therapeutic cloning", this technique has not been sufficiently developed in humans and there are also ethical conditions as this technique is prohibited in some countries. The safety of embryonic stem cells is also not clear since, being poorly differentiated cells, they have a high risk of generating teratocarcinomas. Even if they have been differentiated before use, they can undergo a process of de-differentiation.

 Adult stem cells from bone marrow have also been used in order to treat pathologies of the nervous system autologously (reviewed in (Reekmans, et al., 201 1; Sahni, et al., 201 1; Wright, et al. , 201 1)). However, these adult stem cells, which have their origin outside the nervous system, seem to have restricted their ability to generate neural lineages, and there is controversy regarding their ability to generate neurons (reviewed (Wright, et al., 201 1)) . In addition, because they are multipotential cells, their safety for patients is also not clear since it has also been described that they could generate tumors (Stagg, 2008; Worthley, et al., 2009; Zhu, et al., 2006) They are also beginning to use adult stem cells from the olfactory mucosa of the nasal cavity. These cells are also multipotential and undifferentiated, so their safety is not clear. But, in addition, the olfactory mucosa stem cells do not generate all neural phenotypes since they do not differentiate to neurons and only glia can be obtained from them (Wetzig, et al., 201 1).

There seems to be, therefore, a need to have a source of neurons and glia of the nervous system, accessible, that restrictively generate only cellular types of the nervous system (to avoid tumor risk derived from cellular multipotentiality), and that is located both in the peripheral and central nervous system to be able to generate cells from both.

The olfactory envelope glia (OEG) or olfactory envelope cells (OEC) constitute a unique cell type that allows the regeneration of axons in the olfactory system of mammals throughout their lives (Ramon-Cueto, et al., 1995). These cells involve the olfactory axons throughout their journey in the peripheral nervous system (SNP) of the olfactory epithelium and the lamina propria, and within the central nervous system (CNS) of the olfactory bulb, which constitutes, the latter, somewhat exceptional ( Blanes, 1898; Doucette, 1984; Golgi, 1875; Raisman, 1985). When these cells are isolated from the olfactory mucosa or the olfactory bulb and are transplanted into the injured central nervous system they promote the regeneration of axons and their reconnection and as a consequence the functional recovery of the paraplegic individual (reviewed in (Mackay-Sim, et al. , 201 1; Ramon-Cueto, et al., 201 1)). The transplanted OEG also promotes remyelination of axons when they have lost their myelin sheath, as occurs in multiple sclerosis (reviewed in (Sasaki, et al., 201 1)). This innate plasticity of the olfactory envelope glia is what has served as the basis for the present invention, which provides a source of neural stem cells, neurons and glia from these adult and differentiated glial cells of the mammalian nervous system . The first time that OEG cultures were obtained was in 1992 (Ramón-Cueto et al., 1992; Pixley 1992) and since then there are more than 200 scientific publications that, using different culture methods, obtain these same cells well from olfactory bulb (central nervous system, CNS) or nasal cavity (peripheral nervous system, SNP), either of the mucosa or of the lamina propria (Ramón-Cueto et al., 1993; Barnett et al., 1993; Chuah et al., 1993; Alexander et al., 2002; Au et al., 2003; reviewed in Higginson et al., 201 1). The first time the use of OEG to promote neuronal regeneration and repair of the nervous system was described in the year 1993 in an in vitro study (Ramón-Cueto et al., 1993) and one year later in an in vivo study (Ramón-Cueto et al., 1994; Ramón-Cueto et al., 1995; L¡ et al ., 1998; Ramón-Cueto et al. 2000; Navarro et al., 1999). Until December 2010 there are more than 60 scientific publications on the use of the OEG to repair the lesions and pathology of the central and peripheral nervous system (reviewed in Ramón-Cueto et al., 201 1). Some patent documents, such as WO2000AU01327 or WO01 / 30982, emerged years later on methodologies published in scientific journals, in which the culture of OEG from the lamina propria is described, or WO2007 / 069927 in which it is used for the cultivation of OEG olfactory bulb a previously published method.

Stem cells can be obtained using different compositions of the culture medium. For example, WO 2010/056075 describes a method for obtaining and culturing stem cells that includes ICAM 5, Activin A, decorin, IGFBP7, glypican 3, among other compounds. In WO201 1/062013 neural stem cells and precursors of the neuronal lineage are obtained using an inductor containing a compound represented by a general formula. A different method for also obtaining neural stem cells is presented in WO201 1059920. US2008 / 107633 describes a standard definition culture medium, a carbohydrate source, a buffer, a source of hormones, and LIF for generating neuronal stem cells, to stimulate its proliferation and the generation of neuronal and glial cells. US2005 / 0245436 describes a different composition of the culture medium, based on the use of pheromones and luteinizing hormone, to obtain neural stem cells that can be converted into neurons and glial cells. WO 2009/002559 adds LIF and type I collagenase to the culture medium to obtain the neural stem cells, and withdraws LIF for the differentiation of these neural stem cells into neurons and glial cells. Other examples of patent documents that use different culture media to obtain neural stem cells are the following: WO 2008/109320, WO2010 / 052904, WO2010147803. Therefore, one skilled in the art knows that different culture conditions and media can be used to obtain stem cells, neural stem cells and precursors of the neural lineage.

Patent documents also emerged that use adult differentiated OEG cultures to obtain OEG cell lines by genetic modification (WO02 / 088337 and WO05 / 012513) or that use differentiated OEG in combination with a pharmacological compound (AU2003 / 249092) . However, there is currently a need to obtain neural stem cells from differentiated OEG.

There are patent documents that isolate existing neural stem cells in the olfactory mucosa and propagate them in culture (WO 2003/064601; WO 2007/02061 1; WO 2007/02061 1; US 2007/0141035; CN101591642; WO 2010/051531 ; WO 2010/077294). These patent documents directly isolate the stem cells and precursors already existing in the tissue, and all use the nasal mucosa or nasal epithelium as the starting tissue and not the olfactory bulb. None of these documents. These patent documents directly isolate the stem cells and precursors already existing in the tissue, and all use the nasal mucosa or nasal epithelium as the starting tissue and not the olfactory bulb. None of these documents obtain neural stem cells from OEG, or from differentiated cells isolated from these tissues, or from cells that are present in both the SNP and the CNS, which are substantial differences of our invention. Stem cells that are only present in the mucosa, and are already undifferentiated cells, are cell types other than OEG, a cell type already differentiated and that is present both in the mucosa and in the olfactory bulb. Therefore the starting product is different and so is the final product. In addition, mucosal stem cells are found only in the peripheral nervous system (SNP) while differentiated OEG is found in both the SNP and the system. central nervous (CNS). All this gives an advantage to the present invention because the neural stem cells object of this invention are obtained from a controlled, already differentiated cell type, which is not multipotential and, in addition, is found both in the peripheral nervous system (SNP) as in the central nervous system (CNS).

Therefore, there is currently a need to obtain neural stem cells obtained from a controlled, already differentiated cell type that is not multipotential and that is also found in both the peripheral nervous system (SNP) and the central nervous system ( SNC).

DESCRIPTION OF THE INVENTION It is desirable to have a source of neurons and glia, from differentiated cells of the nervous system, from cells of the neural line in which there is no multipotentiality (to avoid the risk of tumor formation), which It can be obtained from adults to guarantee an autologous therapy, and that the source of these differentiated cells is located both in the peripheral and central nervous system, in order to offer the possibility of obtaining the neurons and glia of both systems.

We have designed a method and have generated stem cells and cells of the neural line (neurons, glia, others) from the olfactory envelope gland, by de-differentiating these cells and their subsequent differentiation into other cell types. The present invention allows, for the first time, to obtain cells of the neural lineage (neurons, glia, others) from adult differentiated cells also of the neural lineage that are found both in the peripheral nervous system and in the central nervous system, for the disease therapy, for diagnosis and for the evaluation of the biological activity of various agents. The nervous structures that are used for the generation of The cells object of the invention are accessible structures whose elimination or biopsy does not cause negative sequelae. This invention also offers all the advantages of autologous therapies: the risk of rejection by the host is avoided, the use of immunosuppressants is not required for life in recipient patients, which avoids the complications and side effects of this type of medication, it also eliminates the problem of finding a compatible donor and the ethical conditions of using embryos. A first aspect of the invention consists in the generation of neural stem cells and neural progenitors derived from dedifferentiated olfactory envelope glia. The olfactory enveloping glia is preferably obtained from the olfactory bulb (central nervous system), as it is of special relevance for pathologies of the central nervous system (CNS) because both the origin and derived cells will be from the CNS, without this excluding their obtaining of the olfactory mucosa and lamina propria (peripheral nervous system). That is, the envelope glia to generate neural stem cells and progenitors of the neural lineage can be obtained from both the olfactory mucosa (including the lamina propria) and the olfactory bulb. Both the olfactory bulb and the olfactory mucosa are surgically accessible nerve structures and whose biopsy or extraction causes minimal risks to patients.

A second aspect of the invention consists in obtaining in vitro different cell types from the neural lineage (neurons, glia, others), without these cell types being exclusive of others, originating from the stem cells and the precursors derived from the surrounding glia. -differentiated.

A third aspect of the invention is the use of all these cells derived from the olfactory envelope glia, to generate others by their genetic modification or manipulation of any kind. A fourth aspect of the invention consists in the use of any of the cells derived from the olfactory envelope glia mentioned previously, and / or the products or molecules derived therefrom, in the treatment of pathologies or diseases.

A fifth aspect of the invention consists in the use of any of the cells derived from the olfactory envelope glia mentioned above, and / or the products or molecules derived therefrom, in the preparation of pharmacological compounds or medicaments that can be used for regeneration. of organs and tissues or for the treatment of diseases and pathologies. Another aspect of this invention is these compounds or medicaments mentioned. A sixth aspect of the invention consists in the use of any of the cells derived from the olfactory envelope glia mentioned previously, and / or the products or molecules derived therefrom, for the evaluation of the biological activity of different agents (compounds, cells, gene expression products, molecules, drugs, or others) both in vitro and in vivo.

A seventh aspect of the invention consists in the use of any of the cells derived from the olfactory envelope glia mentioned previously, and / or the products or molecules derived therefrom, in the design or conduct of diagnostic tests.

The present invention relates to stem cells and neural lineage cells all derived from olfactory envelope glia, and their use. Stem cells and neural types (neurons and glia) obtained from the adult enveloping glia can be used for the treatment of various pathologies of the nervous system. These cells and / or their derived molecules may be used either alone or in combination with others. therapeutic strategies of various kinds (transplants of other cells, gene therapy, administration of compounds, etc). They can be used in the treatment of various diseases such as degenerative, ischemic, traumatic, genetic, autoimmune, demyelinating diseases, spinal cord injuries and degenerations, brain, optic nerve, peripheral nerves, Parkinson's disease, Huntington's disease , alterations of the retina, multiple sclerosis, amyotrophic lateral sclerosis, pathologies of the cerebellum, bulbaric spinal ataxias, without these excluding other diseases or pathologies for which the invention is also useful.

The present invention relates to cells consisting of stem cells and precursors obtained from olfactory glia or OEG or GEO (also called olfactory envelope cells or OEC).

The present invention relates to cells comprising various cell lineages (neurons, glia or others) generated from olfactory envelope glia (OEG) and / or derived from stem cells obtained from olfactory envelope glia.

The present invention relates to the use of stem cells and the different cell types (neurons, glia, others) generated from the olfactory glia, or any molecule or molecules that these cells produce, for the treatment of diseases and lesions of the nervous system of mammals, including humans, as well as to treat any other pathology in which these cells may be useful.

The present invention relates to the combination of stem cell transplants and / or the different cell types (neurons, glia, others) generated from the olfactory envelope glia, with another or other therapeutic strategy / s for the treatment of diseases and injuries of the nervous system of mammals, including humans, as well as to treat any other pathology in which these combinations may be useful. The present invention relates to the new cell lines or cell types that are obtained after genetic or other manipulation of the stem cells and the different cell types (neurons, glia, others) generated from the olfactory envelope glia or derivatives her. The present invention relates to the new molecules produced by the stem cells and / or the different cell types (neurons, glia, others) generated from the olfactory envelope glia or derivatives thereof, which have biological activity, as well as the molecules known in which a new biological effect or activity is described. Also the use of these substances as pharmacological targets or for the development of new products for diagnostic, therapeutic or other purposes.

The present invention relates to drugs or drugs that are designed to enhance the effect of the new or known molecules identified, produced by the stem cells and / or the different cell types (neurons, glia, others) generated from the glia olfactory envelope or derivatives thereof. The use of these drugs, alone or in combination with stem cell transplants and / or with the different cell types (neurons, glia, others) generated from the olfactory envelope glia, and also the use of these drugs in combination with any molecule or molecules that these cells produce, all for the therapy of diseases and injuries of the nervous system and also for the therapy of those pathologies in which they may be useful.

The present invention relates to the use of stem cells and the different cell types (neurons, glia, others) generated from the olfactory envelope glia or derivatives thereof, or the use of any molecule or molecules that these cells produce, to perform in vitro tests that allow testing of new compounds, and for the search and development of new therapeutic agents.

The present invention relates to the use of stem cells and the different cell types (neurons, glia, others) generated from the olfactory envelope glia or derivatives thereof, to conduct genomic studies of pathologies, and also pharmacogenomics, where The activity of a compound that is used in the nervous system could be related to the gene structure of the individual.

The present invention relates to the use of stem cells and the different cell types (neurons, glia, others) generated from the olfactory glia or derivatives thereof, or use of any cell obtained by modification thereof (genetics). or others), or of any molecule or molecules that these cells produce, in obtaining tissues and organs. The present invention relates to the use of the stem cells and the different cell types (neurons, glia, others) generated from the olfactory envelope glia or derivatives thereof, or from any cell obtained by modification thereof (genetics or others), or the use of any molecule or molecules that these cells produce, in combination with other cells, compounds or pharmacological products for all purposes set forth in the previous points.

Other aspects of this invention are evident and are deduced from reading the description of the present invention.

Throughout the description and the claims the words "comprises", "consists" and their vain, are not intended to exclude other technical characteristics, additives, components or steps. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES

FIGURE 1. Figure 1A shows the appearance of an olfactory envelope glia culture before stem cells and precursors are obtained. Figure 1 B shows the aspect of olfactory envelope glia culture 3 days after the culture medium that produces its differentiation and the generation of stem cells and precursor cells has been administered. Figures 1 C and 1 D show the spheres formed of stem cells and precursors, which are obtained from olfactory envelope glia cultures after the culture medium that favors their differentiation has been administered.

FIGURE 2. Figure 2 shows the same sphere that contains stem cells and precursors but displayed differently. Figure 2A shows the sphere in clear field. Figure 2B shows that stem cells and precursors that are contained in the spheres are viable since they all contain the vital dye (CSFE). Figure 2C shows that the cells contained in the spheres are dividing, since they have captured the BrdU proliferation marker. FIGURE 3. Figure 3 shows the cell types contained in the spheres obtained from olfactory envelope glia after dedifferentiation, by means of marking for different molecules. This staining was performed after 8 days in vitro. Figures 3A and 3B show that in the same spheres there are precursors of neurons (β-111-tubulin positive cells) (A) and neural stem cells (nestin positive) (B). Figures 3C and 3D show that in the same spheres there are cells expressing p75, which is a marker of neuronal precursors (C) and glial cells (labeled with anti-GFAP) (D). FIGURE 4. Figure 4 shows the cell types obtained after differentiating the differentiated envelope glia (after differentiating the spheres). This staining was performed after keeping the cells 7 days in the differentiation culture medium. Figures 4A and 4B show that after differentiating the de-differentiated envelope glia, glial line cells that are not olfactory envelope glia (positive cells against GFAP and negative cells against p75) are obtained and olfactory envelope glia is also re-obtained ( GFAP and p75 positive). Compare panel A showing the staining against GFAP with the same field of the dyed culture against p75 of panel B. Figures 4C and 4D show that after differentiating the differentiated envelope glia, neurons (C) and glia (D) are obtained. ). Neurons are identified with β-ΙΙΙ-tubulin (Tuj) (C), and the glia with GFAP (D). The images are taken in a confocal microscope and show the same field of culture. Figure 4E shows the same field of culture of Figures C and D, in which we have analyzed in the confocal microscope if there is co-localization of both markers in the same cells. The non-existence of co-expression of β-ΙΙΙ tubulin and GFAP in the cells demonstrates that, after differentiating the de-differentiated envelope glia, neurons and also glia are obtained. Figure 4F shows that after differentiating the differentiated enveloping glia, oligodendroglial cells (positive versus 04) are obtained.

EXAMPLES

EXAMPLE 1. Obtaining cell spheres from olfactory envelope glia Adult OEG cultures are obtained from adult olfactory bulbs as previously described (Munoz-Quiles, et al., 2009; Ramon-Cueto, et al., 2000; Ramon-Cueto, et al., 1994; Rubio, et al., 2008). The OEG is maintained in sterile culture medium (DMEM, Dulbecco Modifier Eagle's Medium and F12) containing 2mM L glutamine, antibiotics and 10% fetal bovine serum. The serum that is used to supplement the medium can be human and thus be used from an autologous source. Once the cultures reach confluence, the cells are detached by the use of trypsin and seeded on untreated glass at low density (10,000 cells per cm ² ). The medium used to obtain stem cell spheres from OEG is composed of Neurobasal, B27 (2%), L-Glutamine (200mM), Human Epidermal Growth Factor (Hu EGF) (100pg / mL) and Factor of growth of beta fibroblasts (FGF β) (100pg / mL). The cultures are kept in an incubator with 5% CO2 at 37 ° C. Half of these cultures are changed every 2-3 days. After three days the first spheres begin to be seen and they increase in size by dividing the cells that compose them progressively. (Figure 1 ). By incorporating 5-bromo-2-deoxyuridin (BrdU), we have shown that the cells that form the spheres proliferate. To do this, we add 20 μΜ of BrdU to the crops on the third and seventh day after planting. The spheres are incubated for 24 hours with this compound and after this period they are washed with fresh medium. The cultures are fixed with 4% paraformaldehyde for 20 minutes and incubated with 0.07 M sodium hydroxide for 10 minutes. To detect the presence of BrdU in the cells, an anti-BrdU antibody and a fluorescent secondary antibody are used. (Figure 2). EXAMPLE 2. Immunocytochemical characterization of cells contained in the spheres Neurospheres are very dynamic cellular structures with different gradients of cell proliferation, survival, apoptosis and phagocytosis and contain progenitors of different cell types in different states of differentiation (Ahmed, 2009). For the characterization of the spheres, immunocytochemicals are performed using antibodies against Nestine, β-ΙΙΙ-tubulin (Tuj), acidic protein of the glial filaments (GFAP) and the low affinity receptor of nerve growth factor or p75. This last molecule has been identified in both neuroblasts and neural stem cells. Neural stem cells coexpress p75 and nestin, while neuronal line cells co-express p75 with Tuj.

95-100% of the spheres obtained from olfactory envelope glia cultures contain cells of various types: neural stem cells and neuronal and glial progenitors. The presence of nestin and p75 positive cells indicates that the spheres contain neural stem cells. The presence of positive β-)-tubulin (Tuj) and positive cells against p75 indicates that the spheres contain neuronal precursors. The presence of some positive GFAP cells that are negative for the other markers indicates that there are glial line cells in the spheres. These glial cells are not olfactory envelope glia since GFAP and p75 do not colocalize in the same cells (compare Figure 2C and 2D) and the olfactory envelope glia expresses both molecules. (Figure 3).

EXAMPLE 3. Obtaining neurons and glia from spheres generated with olfactory envelope glia cultures

After eight days in culture with Hu-EGF and FGFβ, the spheres are transferred to new glass plates pre-treated with Poly-L-Lysine (25 pg / mL; 2h) and Laminin (10 pL / mL; 4h). The culture medium used for the differentiation of spheres in neurons and glia is Neurobasal, B27 (2%), L-Glutamine (200mM), without EGF or FGF. Said medium is changed every 2-3 days and the cultures are maintained at 37 ° C and 5% CO2. Three days after be sown, the cells have adhered to the substrate, differentiate and distribute forming colonies. After 7-8 days in culture, the cells are fixed with 4% paraformaldehyde and their phenotypes are characterized by immunocytochemistry using antibodies against GFAP, p75, β-lll-tubulin and 04.

After 8 days in the culture medium described, approximately 45% of the cells express β-lll-tubulin, which demonstrates a differentiation of the cells from the spheres to neurons. 69% cells express GFAP, demonstrating differentiation to glial line cells. GFAP is also a marker of precursors of certain types of neurons, so that the cells that express both, β-ΙΙΙ-tubulin and GFAP, are of neuronal lineage, while those that express only GFAP are of glial lineage. 56% of cells express 04, a marker of oligodendroglia precursors. The cells expressing only 04 demonstrate that the cells of the spheres have differentiated towards oligodendroglia. A small percentage of cells express 04 and also GFAP, which demonstrates their re-differentiation to olfactory envelope glia (Figure 4).

BIBLIOGRAPHY

Ahmed, S, 2009. The culture of neural stem cells. Journal of cellular biochemistry 106, 1-6.

- Alexander, CL, Fitzgerald, UF, Barnett, SC, 2002. Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype. Glia 37, 349-364.

 Au, E, Roskams, AJ 2003. Olfactory ensheathing cells of the lamina propria in vivo and in vitro. Glia 41, 224-236.

 Barnett, SC, Hutchins, AM, Noble, M 1993. They purified of olfactory nerve ensheathing cells from the olfactory bulb. Dev. Biol. 55, 337-350.

 Bjorklund, A, Lindvall, O, 2000. Cell replacement therapies for central nervous system disorders. Nature neuroscience 3, 537-544.

Blanes, T, 1898. On some doubtful points of the olfactory bulb structure. Rev Trim Micrograf 3, 99-127.

 Chuah, MI, Au, C, 1993. Cultures of ensheathing cells from neonatal rats olfactory bulbs. Brain Res. 601, 213-220.

- Doucette, JR, 1984. The glial cells in the nerve fiber layer of the rat olfactory bulb. Anat Rec 210, 385-391.

 Fricker-Gates, RA, Gates, MA, 2011. Stem cell-derived dopamine neurons for brain repair in Parkinson's disease. Regenerative medicine 5, 267-278.

- Golgi, C, 1875. Sulla fine anatomy of the bulbi olfatorii. Reggio- Emilia.

 Higginson, JR, Barnett, SC, 2011. The culture of olfactory ensheathing cells (OECs) -a distinct glial cell type. Exp. Neurol. 229, 2-9.

- Jain, KK, 2009. Cell therapy for CNS trauma. Molecular biotechnology 42, 367-376. Kim, SU, of Vellis, J, 2009. Stem cell-based cell therapy in neurological diseases: a review. Journal of neuroscience research 87, 2183-2200.

 Li, Y, Field, PM, Raisman, G, 1998. Regeneration of adult rat corticospinal axons induced by transplanted olfactory ensheathing cells. J. Neurosci. 18, 10514-10524.

 Mackay-Sim, A, St John, JA, 2011. Olfactory ensheathing cells from the nose: Clinical application in human spinal cord injuries. Exp Neurol 229, 174-180.

 Mirón, VE, Kuhlmann, T, Antel, JP, 2011. Cells of the oligodendroglial lineage, myelination, and remyelination. Biochimica et biophysica acta 1812, 184-193.

 Munoz-Quiles, C, Santos-Benito, FF, Llamusi, MB, Ramon-Cueto, A, 2009. Chronic spinal injury repair by olfactory bulb ensheathing glia and feasibility for autologous therapy. J Neuropathol Exp Neurol 68, 1294-1308.

 Navarro, X, Valero, A, Gudino, G, Flores, J, Rodríguez, FJ, Verdu, E, Pascual, R, Cuadras, J, Nieto-Sampedro, M, 1999.

Enshathing glia transplants promote dorsal root regeneration after spinal reflex restitution after multiple lumbar rhizotomy. Ann. Neurol 45, 207-215.

 Pixley, S 1992. The olfactory nerve contains two populations of glia identified both in vitro and in vivo. Glia 5, 269-284.

Raisman, G, 1985. Specialized neuroglial arrangement may explain the capacity of vomeronasal axons to reinnervate central neurons. Neuroscience 14, 237-254.

 Ramón-Cueto, A, Nieto-Sampedro, M 1992. Glial cells from the adult rat olfactory bulb, mmunocytochemical properties of puré cultures of ensheathing cells. Neurosci. 47, 213-220.

Ramón-Cueto, A, Pérez, J, Nieto-Sampedro, M, 1993. In vitro enfolding of olfactory neurites by p75NGF receptor positive ensheathing cells from adult rat olfactory bulb. Eur. J. Neurosci. 5, 1 172-1 180.

 Ramon-Cueto, A, Nieto-Sampedro, M, 1994. Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia transplants. Exp Neurol 127, 232-244.

Ramon-Cueto, A, Valverde, F, 1995. Olfactory bulb ensheathing glia: a unique cell type with axonal growth-promoting properties. Glia 14, 163-173.

 Ramon-Cueto, A, Lamb, MI, Santos-Benito, FF, Avila, J, 2000.

Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheathing glia. Neuron 25, 425-435.

 Ramon-Cueto, A, Munoz-Quiles, C, 2011. Clinical application of adult olfactory bulb ensheathing glia for nervous system repair. Exp Neurol 229, 181-194.

 Reekmans, K, Praet, J, Daans, J, Reumers, V, Pauwels, P, Van der Linden, A, Berneman, ZN, Ponsaerts, P, 2011. Current Challenges for the Advancement of Neural Stem Cell Biology and Transplantation Research. Stem cell reviews.

Reier, PJ, 2004. Cellular Transplantation Strategies for Spinal Cord Injury and Translational Neurobiology. Neurorx 1, 424-451.

Rodríguez, FD, Vecino, E, 2011. Stem cell plasticity, neuroprotection and regeneration in human eye diseases. Current stem cell research & therapy 6, 73-81.

 Rubio, MP, Munoz-Quiles, C, Ramon-Cueto, A, 2008. Adult olfactory bulbs from primates provide reliable ensheathing glia for cell therapy. Glia 56, 539-551.

 Ruff, CA, Wilcox, JT, Fehlings, MG, 2011. Cell-based transplantation strategies to promote plasticity following spinal cord injury. Exp. Neurol.

Sahni, V, Kessler, JA, 2011. Stem cell therapies for spinal cord injury. Nat Rev Neurol 6, 363-372. Sasaki, M, Lankford, KL, Radtke, C, Honmou, O, Kocsis, JD, 2011. Remyelination after olfactory ensheathing cell transplantation into diverse demyelinating environments. Exp Neurol 229, 88-98. Sharp, J, Frame, J, Siegenthaler, M, Nistor, G, Keirstead, HS, 2011. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants improve recovery after cervical spinal cord injury. Stem cells (Dayton, Ohio) 28, 152-163.

Stagg, J, 2008. Mesenchymal stem cells in cancer. Stem cell reviews 4, 1 19-124.

 Tetzlaff, W, Okon, EB, Karimi-Abdolrezaee, S, Hill, CE, Sparling, JS, Plemel, JR, Plunet, WT, Tsai, EC, Baptiste, D, Smithson, LJ, Kawaja, MD, Fehlings, MG, Kwon, BK, 2011. A

Systematic Review of Cellular Transplantation Therapies for Spinal Cord Injury. Journal of neurotrauma.

 Uccelli, A, Mancardi, G, 2011. Stem cell transplantation in multiple sclerosis. Current opinion in neurology 23, 218-225.

Wetzig, A, Mackay-Sim, A, Murrell, W, 2011. Characterization of olfactory stem cells. Cell transplantation (in press).

Worthley, DL, Ruszkiewicz, A, Davies, R, Moore, S, Nivison- Smith, I, Bik To, L, Browett, P, Western, R, Durrant, S, So, J, Young, GP, Mullighan, CG , Bardy, PG, Michael, MZ, 2009. Human gastrointestinal neoplasia-associated myofibroblasts can develop from bone marrow-derived cells following allogeneic stem cell transplantation. Stem cells (Dayton, Ohio) 27, 1463-1468.

Wright, KT, El Masri, W, Osman, A, Chowdhury, J, Johnson, WE, 2011. Bone Marrow for the Treatment of Spinal Cord Injury: Mechanisms and Clinical Application. Stem cells (Dayton, Ohio) 29, 10.

 Zhu, W, Xu, W, Jiang, R, Qian, H, Chen, M, Hu, J, Cao, W, Han, C, Chen, Y, 2006. Mesenchymal stem cells derived from bone marrow favor tumor cell growth in alive. Experimental and molecular pathology 80, 267-274.

Claims

one . Neural stem cells and / or neural precursors derived from the olfactory envelope glia of an individual, characterized in that:  a) have the ability to form spheres when grown;  b) said spheres comprise viable cells;  c) said spheres comprise dividing cells;  d) said spheres comprise neural stem cells that express at least the Nestine marker. 2. Neural stem cells and / or neural precursors according to claim 1, characterized in that they form spheres comprising cells of the neuronal line that express at least the marker (3— III— tubulin and / or comprise cells of the glial line that express at least the GFAP marker. 3. Neural stem cells and / or neural precursors according to claims 1 and 2, characterized in that the olfactory envelope glia is located in a nervous structure of the central nervous system or peripheral nervous system of said individual. 4. Neural stem cells and / or neural precursors according to claim 3, characterized in that the nervous structure of the central nervous system is the olfactory bulb. 5. Neural stem cells and / or neural precursors according to claim 3, characterized in that the nervous structure of the peripheral nervous system is the olfactory mucosa or the lamina propria. 6. Neural stem cells and / or neural precursors according to claims 1 to 5, characterized by being obtained after a de-differentiation process comprising the following steps:  a) obtain a culture of adult differentiated cells of the olfactory envelope glia;  b) sowing said culture in a means of de-differentiation; and c) incubating said culture for a period of at least 3 days. 7. Neural stem cells and / or neural precursors according to claim 6, characterized in that the dedifferentiation culture medium comprises at least one growth factor. 8. Neural stem cells and / or neural precursors according to claim 7, characterized in that the dedifferentiation culture medium comprises at least one growth factor selected from the epidermal growth factor and / or the fibroblast growth factor. 9. Neural stem cells and / or neural precursors defined in claims 1 to 8, for use in the generation of at least one cell line through a differentiation process. 10. Neural stem cells and / or neural precursors, according to claim 9, for use in the generation of at least one neural cell line characterized by comprising cells of the neuronal line and / or the glial line. eleven . Neural stem cells and / or neural precursors according to claim 10, characterized in that at least one neural cell line comprises selected glial line cells between olfactory envelope glia cells and / or oligodendroglial cells. 12. Pharmaceutical composition comprising the neural stem cells and / or neural precursors defined in claims 1 to 8. 13. Neural stem cells and / or neural precursors defined in claims 1 to 8, and / or pharmaceutical composition defined in claim 12, for use in the therapeutic and / or diagnostic treatment of a pathology, lesion or disease. 14. Neural stem cells and / or neural precursors defined in claims 1 to 8, and / or pharmaceutical composition defined in claim 12, for use in the regeneration of organs and tissues. 15. Neural stem cells and / or neural precursors defined in claims 1 to 8, and / or pharmaceutical composition defined in claim 12, for use in the evaluation of the biological activity of various agents both in vitro and in vivo. 16. Neural stem cells and / or neural precursors defined in claims 1 to 8, and / or pharmaceutical composition defined in claim 12, for use in performing in vitro and in vivo tests for the search and development of new Agents 17. Neural stem cells and / or neural precursors defined in claims 1 to 8, and / or pharmaceutical composition defined in claim 12, for use in the design or conduct of diagnostic tests. 18. Neural stem cells and / or neural precursors defined in claims 1 to 8, and / or pharmaceutical composition defined in claim 12, for use in genomic and pharmacogenomic pathology studies, wherein the activity of a compound that Used in the nervous system is related to the gene structure of the individual.