MXPA97003386A - Crops neura - Google Patents

Abstract

The invention relates to a method for producing nerve cell lines to a homogeneous population of cells having preselected biochemical / functional characteristics. In addition, the invention relates to the provision of a homogeneous population of cells, which can be selectively made to undergo apostosis. Finally, the invention relates to nerve cell lines provided by the method of the invention.

Description

NEURAL CROPS DESCRIPTION OF THE INVENTION This invention relates to a method for producing neural cultures and particularly, but not exclusively, to neural cell lines; and to the cells and cell lines produced by such a method. The invention also relates to neural cell lines of humans and animals, and particularly, but not exclusively to nerve cell lines. Nerve cells are highly differentiated cells that comprise a cell body and procedures, the latter subdivided into dendrites and axons. Nerve cells vary considerably in shape and size in different parts of the body. For example, the granule cells of the cerebellum have a diameter of 5 micrometers, while the large motor cells of the anterior horn of the spinal cord are up to 120 micrometers in diameter. In addition, the axons of nerve cells vary from approximately thousandths of a micrometer in length to 1 meter in length. In addition to this variation in shape and size, nerve cells also vary in the nature of the receptors expressed on their cell surface and the nature of the secreted neurotransmitters for the purpose of effecting the transmissions of nerve cells. This difference in biochemistry can be used for classification purposes. In this way, in simpler terms, nerve cells can be classified as, for example, adrenergic, cholinergic, serotonergic, dopaminergic, and so on, according to the nature of their neurotransmitters. This biochemical mode of classification can be further subdivided in order to identify a total scale of nerve cells secreting different neuropeptides that are brought to function as neurotransmitters or neuromodulators such as the neuropeptides beta endorphin, met-enkephalin, somatostatin, luteinizing hormone - hormone of release, thyrotropin releasing hormone, substance P, neurotensin, angiotensin 1, angiotensin 2, vasoactive intestinal peptide, neuropeptide Y, peptide related to the calcitonin gene, etc. or alternatively, the amines or amino acids, adrenaline, noradrenaline, octopamine, serotonin, histamine, gamma aminobutyric acid and taurine. The aforementioned list is not intended to be exhaustive but rather serves to illustrate the nature of the biochemical diversity of nerve cells. It is widely recognized that it could be immensely advantageous if it were possible to ideally provide a homogeneous population of nerve cells in culture and thus provide, for example, a homogeneous population of nerve cells either from a given location in the central nervous system, or alternatively a population Homogeneous nerve cells that exhibit either predetermined morphological characteristics or biochemical characteristics. For example, it could be highly advantageous if it were possible to provide a homogenous population of nerve cells, which are characterized by the transmitter secreted in response to activation or alternatively the receptor occupied in response to activation. With the population of nerve cells it may be possible for research biologists to take significant advantages in understanding the nervous system and for industrial biologists to manufacture and test drugs, agents or entities that affect the functioning of a given population of nerve cells with a view to develop therapeutically active agents. Furthermore, if it were possible to provide a homogenous population of nerve cells, it might be possible to provide nerve cells of a given classification for the purpose of transplantation. This could be particularly appropriate in cases where degeneration or damage of nerve cells has occurred. For example, it is well known that Parkinson's Disease is related to the degeneration of nerve cells and a corresponding lack of dopamine secretion by nerve cells. In this way, if it were possible to provide a homogeneous population of nerve cells that secrete dopamine then it would be possible to transplant such nerve cells and in this way mitigate or alleviate or even reverse the symptoms of Parkinson's Disease. Similarly, other forms of dementia that are characterized by a progressive degeneration of nerve cells could be treated in a similar way. Similarly, acute destruction of nerve tissue can be treated through nerve cell implants comprising a homogeneous population of nerve cells and / or implantation of a selected combination of nerve cells from different homogeneous populations. However, the above-mentioned as nerve cell diversity and also the post-mitotic nature of nerve cells tends to impose severe restrictions on the number of cells that can be obtained in vi tro for research and / or transplantation using conventional cell culture techniques. For this reason, attempts have been made to provide nerve cell cultures by culturing primary oral tissue or by fusing primary cells with tumor cells. However, the tumor cells are irreversibly transformed and have a history defined by the disease. Its use as cell models is therefore highly questionable and also because of the potential tumorigenicity of such tissue, they can not be used for the purpose of transplants. Attempts to provide homogenous populations of nerve cells have also been made using carcinogen-induced transformation both in vi tro and in vivo and also by spontaneous transformation, i.e., by growth of cells from primary cultures without any deliberate gene population. However, it has been found that another restriction in the provision of homogenous populations of nerve cells refers to the fact that most neural tumors are human gliobastornas and thus have nothing to do with the uncontrolled division of functional nerve cells. . Other workers have transfected neural cells with oncogenes in order to establish neural cell lines. Some workers have shown that it is possible to induce oncogenes in primary neural cells and obtain cell lines, however, these cell lines are not nerve cell lines. They do not function as nerve cells nor are they homogeneous populations of definable nerve cells (1).

Transfection techniques used in the past have involved the use of retroviruses due to the ability of such viruses to stably integrate into the host cell genome. In addition, transfection has been performed using a temperature sensitive mutant of simian DNA virus 40 (SV40). An SV40 gene encodes a large tumor antigen (T), which is required for the initiation and maintenance of the transformation. The integration of viral genes into host cell genomes requires that the host cell undergo at least one round of DNA synthesis. Therefore, it is presented that when the integration of a viral gene into a host cell is required, the target cells are limited to mitotic neural cells. Therefore, transfection techniques have been taken in such cells. Although it has been possible to produce cell lines, that is, it has been possible to immortalize the transfected cells, it has not been possible to produce immortalized cells with the required degree of differentiation, which would make such cells useful tools for further investigation, study or use. . This could be because immortalization prevents the terminal differentiation of nerve cells. Actually, typically the cells that go into crisis and apostosis. For example, when immortalization of neural cells occurs using SV40 T, a homogenous population of cells can be cultured, however, at an unacceptable temperature of 39 ° C the expression of the active viral protein ceases and the cells enter the differentiation. However, the differentiation does not continue until the end, the cells go into crisis and apostosis occurs. Furthermore, it has been widely known that it is extremely difficult to provide a culture of differential neural or nerve cells either for use in transplants and / or for use in the testing of drugs, agents or entities, which affect the functioning of a given population of cells. nerve cells with a view to developing therapeutically active agents. It is difficult to provide the culture of nerve cells, especially when it comes to providing, enormously, a homogeneous population of nerve cells or a heterogeneous population of nerve cells including a relatively small number of phenotypes, since, among other things, it is very difficult to provide the differentiation of such nerve cells. Typically, it is difficult to provide differentiation of primary nerve cells in culture. Therefore, an object of the invention is to provide a method for producing nerve cell lines, which represent homogeneous populations of nerve cells that are not only functional but also their character can be reliably defined. In other words, it is an object of the invention to provide a method for providing a stable nerve cell line, which is subject to its phenotype. For example, using the invention it is possible to provide a homogeneous population of functional serotonin cells or of acetylcholine cells or adrenaline cells, etc. It is a further object of the invention to provide a non-mitotic cell line whose non-mitotic characteristics persist even in the presence of factors and / or conditions that could normally promote mitosis. Moreover, an object of the present invention is to provide a cell line, which survives at low densities. It is also an object of the invention to provide a method for producing nerve cell lines, which can be selectively made to enter into apostosis so that the apostosis procedure can be studied with a view to gaining a greater understanding of the procedure and also with a view to to manufacture drugs, agents or entities that affect apostosis through engineering. A further object of the present invention is to provide a population of nerve cells, homogeneous or otherwise that are fully differentiated.

The method of the invention is based on a surprising observation. Using conventional transfection techniques, selected neuronal cells can be immortalized. However, as with many other workers, until the presentation of the invention, they were unable to provide fully functional differential nerve cells. However, when the method was modified to produce cell lines, it was found that it was possible to induce total differentiation of the nerve cells when they were exposed, following the transfection of immortalization, to predetermined conditions. These conditions involved exposing the cells to an environment from which they come and particularly, but not exclusively, to the mitotic environment from which they come or to conditions that resemble the environment from which they come and thus provide an artificial imitation of the environment where they come from. The observation could also produce an in vitro culture of nerve cells, which have not been immortalized. In this case, the primary nervous tissue was first replicated by exposure to a replicating agent (8 and 9) and then led to differentiation by exposing the cell culture to the aforementioned environment from which such primary tissue comes or to the conditions that resemble such an environment.

By the term, the environment from which they are derived means a region of the central nervous system, and more preferably a region of the central nervous system in, adjacent to, or functionally related to the natural location in the central nervous system of the cultured cells. If a mitotic environment is accepted, therefore, a region of the central nervous system is accepted, which is mitotically active and most preferably a region of the central nervous system in, adjacent or functionally related to the natural location in the central nervous system. of the cultured cells. It would appear that having cells exposed to the environment from which they are derived means that the cells in that environment secrete agents, such as, for example, cytokines, growth factors, transmitters, etc. or perhaps such cells comprise surface-based factors. of removable cells, which can produce a differentiation response. Furthermore, it has been found that it is possible to use tissue and cells of different species in order to work with the invention. For example, it is possible to cultivate human nerve cells and expose such human nerve cells to such an environment or such an artificial environment, which is derived from the central nervous system of a rat. Conversely, it is possible to cultivate rat nerve cells and exposing such nerve cells to an environment or an artificial environment, which is derived from human. Therefore, it could be that the agents that produce neuronal differentiation of the invention are agents that can produce effects of transverse species. That is, these agents are biologically active in at least both rat and human systems and, therefore, are likely to be of the same structure or of a similar structure. It has been found that by modifying the method in such a way that the transfected cells or cultured cells are exposed to the conditions of the original environment at least from which the first cell of culture originates, they can produce differentiation. The nature of the factors involved in this stage is unclear. In addition, when using transfected cells it is preferred to employ a method that includes the provision of a switch, which allows to control immortalization and apostosis. Using this method, it has been found that cultured nerve cells do not spontaneously suffer apostosis, a frustrating feature of previously cultured neural cell lines, but rather can be selectively controlled if the cell lines remain immortalized or enter into apostosis.

In addition, it has been found that cell lines when differentiated, are subjected to their phenotype and thus retain their phenotypic characteristics even when the environment from which they are derived, is removed and / or exposed to factors such as serum Fetal bovine In addition, it has also been found that cell lines do not exhibit mitosis, again, even under conditions that could promote mitosis, and, what is more, cell lines are able to survive at low densities. According to a first aspect of the invention there is thus provided a method for producing large populations of neural cells, the method involves: a) improving the replication of a first undifferentiated neural cell, or neural cell precursor cell or stem cell precursor, b) exposing such replicated neural cells to either an environment from which the first neural cell originates or to an environment that resembles such an environment; and c) allowing the differentiation of such cells to produce fully differentiated active neural cells. It is evident, from the foregoing, that by using the method of the invention, a neural cell precursor cell can be cultured and / or immortalized and thus produce a homogeneous population of cells. However, successful differentiation is carried out by exposing the cells to any environment from which the first nerve cell comes or alternatively to an environment that resembles that environment. In this way, it is possible to produce a homogeneous population of fully differentiated active neural cells. In a first embodiment of the invention, the environment from which the first nerve cell originates is any region of the central nervous system, however, more preferably, such an environment is an environment in, adjacent, or functionally related to the natural location in the central nervous system from which the cultured cells are derived. The term, an environment that resembles such an environment, is also constructed accordingly. More preferably, such an environment is a mitotic environment, that is, it comprises cells that undergo mitosis. It would seem that in this case the agent or agents that produce the differentiation process is capable of being released or expressed and somewhat affecting the differentiation by the cells within the mitotic cell environment. Preferably, such nerve cells and tissue from the natural or artificial environment are derived from a single species. However, alternatively such nerve cells and such tissue can be derived from different species. For example, nerve cells can be derived from fetal human tissues, while the environment and very specifically the tissue of such an environment can be derived from another animal species such as rats, mice, monkeys, and so on. In a preferred embodiment of the invention, immortalization is achieved using conventional transfection techniques and preferably transfection involves the incorporation into the genome of the cell of an oncogene, such an oncogene favors the establishment of cell division beyond the normal level found when a cell is not transduced with an oncogene, in other words the oncogene immortalizes the cell. Alternatively, immortalization can be effected using physical or chemical means. For example, immortalization can be effected by exposing the cell to radiation or chemicals (2), which are known to promote the division of cells beyond the normal level found when a cell is not exposed to physical and chemical means. Ideally, transfection is performed using a virally derived oncogene such as mic, src, ras, SV40T or a retroviral construct that includes any of the aforementioned oncogenes and / or any human oncogene. A retroviral construct is favored due to its ability to stably integrate the host cell genome.

In a first embodiment of the invention, the immortalization agent includes or has been associated with the same control means, whereby the activation of the control means ends with immortalization and causes the cell to enter into apostosis. It is preferred that the immortalization of the cell with an immortalizing agent ideally take place during the last division before the migration of the proliferative zone and the onset of terminal differentiation. This is due to the probability of producing a cell line that has only one set of functional characteristics increased. Immortalization before this preferred time can be taken, but the probability that the precursor cells adopt several different phenotypes after differentiation is increased. In a preferred embodiment of the invention, the control means respond to the culture or environmental conditions such as temperature, pH or ionic concentrations. For example, in a preferred embodiment, the immortalizing agent is temperature sensitive and the control is thus represented by a temperature sensitive switch so that approximately or below a given first temperature, the immortalizing agent is activated with in order to immortalize the selected type of nerve, but in, approximately or above a second temperature, the immortalization agent is deactivated and in this case the immortalization ends and the apostosis continues. The immortalization agent and the control means may comprise, for example, a single entity such as a temperature sensitive oncogene. Alternatively, the immortalization agent and the control means can be two independent entities, but in any case, ideally the activation / deactivation of the control means has a reciprocal effect on the immortalization agent. For example, when the control means are activated, the immortalization agent is deactivated. Conversely, when the control means are deactivated, the immortalization agent is activated. This ability of the control means to deactivate the immortalization agent is a means to terminate the immortalization so that apostosis can occur. By exposing the cells to the original environment, it may involve the transplantation of such homogeneous population of cells back to the central nervous system, or more preferably, a location in the central nervous system in, adjacent or functionally related to the original environment of the first cell or alternatively, and more preferably, simply extracting a population of cells from the central nervous system or from the original environment and placing such a population extracted very close to the homogenous population of cells. Ideally, such a chosen environment comprises mitotically active cells. In the case where the cell is exposed to a population extracted from cells then ideally such extracted cells are plated on a substrate and allowed to reach confluence either before being placed in contact with the homogenous population of cells or while they are in contact with the homogenous population of cells. Alternatively, such a population extracted from cells is developed to reach confluence and in the middle of such a population is added to the homogeneous population of cells in order to reach differentiation. Preferably, the homogenous population of cells are also exposed to one or more growth factors, such as fibroblast growth factor and / or epidermal growth factor. It will be evident from the foregoing that the nature of the homogeneous population of cells will be determined by the nature of the undifferentiated nerve cell or the nerve cell precursor cell. In this way, using the method of the invention it will be possible to produce cell lines of different nerve cells, whose function and properties will be determined by the nature of the undifferentiated nerve cells and the nerve cell precursor cells. In this way, the invention has a broad scale of application since the invention provides a method by which a wide scale of homogeneous populations of nerve cells in the culture can be developed. It is obviously significant for neurobiologists both from the point of view of research and from a technical point of view. Preferably, the immortalizing agent is, what is typically termed as, a soft oncogene such as a SV40 viral oncogene and more preferably, in the case where control means are preferred, the oncogene is the SV40 T antigen, the which is tolerant, that is, the active product of viral gene is expressed, at 33 ° C, and not tolerant, that is, the active product of viral gene is not expressed, at 39 ° C, in this way immortalized cells using this agent, are sensitive to temperature for apostosis. Only cells, when transformed using the SV40 T antigen and exposed to an environment, natural or artificial, that promotes differentiation, survive the crisis, a condition that is typically followed by apostosis. It can be seen that the environment also provides the release of substances or some effects cause the cells to survive the apostosis.

In a further embodiment of the invention, the cell line includes a security aspect that allows the incapacity or selective destruction of such a cell line. This security aspect is advantageous when the cell line is to be used for the purpose of transplantation or otherwise if it is permanent or temporary, attached to, administered to, or stored in an individual. This security aspect allows the cell line to be selectively incapacitated, and thus to render it harmless, or to destroy it, in cases where the cell line is likely to be created, or shown to have the potential to become tumorigenic in I live, or it is believed that it can be dangerous in any way to an individual. Co-pending patent application GB 9422236.1 teaches how a vector can be produced that provides for the coexpression of a security aspect in the form of a gene, which may or may not be linked to the immortalization oncogene. According to a further aspect of the invention, cells and / or cell lines produced according to the method of the invention are provided. Accordingly, there is provided at least one homogeneous population of immortalized cells, which can be made to differentiate completely, in order to provide a homogenous population of fully differentiated nerve cells; and / or alternatively there is provided at least one homogeneous population of immortalized cells provided with means for terminating immortalization and activating apostosis. According to a further aspect of the invention, cells and / or cell lines produced according to a method of the invention are provided which, when differentiated, retain their phenotypic characteristics and / or are non-mitotic and / or survive at low densities. One embodiment of the invention will now be described by way of example and for the purpose of showing only a specific reference to functional nerve cells that secrete serotonin. The following is illustrated through a table and a number of figures where; 1. Table 1 is a summary of experiments performed with the immortal nerve cell line of clone 1. 2. Figure 1 shows the current-voltage ratio at 30mM Ba of clone 1 cells, one to two weeks after the differentiation 3. Figure 2 shows the time course of the VDCC effects of the application of toxin to nerve cells of clone 1; and 4. Figure 3 shows the current-voltage relationship for clone 1 cells.

Immortalization of Cells Rat embryos of 12-13 days of gestation were dissected, and the probable raphe nucleus region comprising the rhombencephalon and ventral medulla and the medulla oblongata was removed. After dissociation through moderate trituration in the medium (Eagle's medium modified with Ham's F12 / Dulbecco's (50/50 v / v) supplemented with L-glutamine (2mM), penicillin: streptomycin (100 IU / ml: 10 μg / ml) and a modified supply solution [3,4] containing 5 ng / ml of basic fibroblast growth factor [5] (all supplied by Sig a), the cells were plated in tissue culture flasks of 162 cm2 coated with poly L-lysine / gelatin (Costar UK Ltd) at a density of 5 x 10 cells / ml, 20 ml per flask.After the cells adhered, the retroviral particles comprising a construct (tsA58) incorporated a temperature-sensitive form of the large tumor antigen of simian virus (ts) SV40-T and a resistance marker for geneticin, G418 '(denoted by Dr. P Jat, Ludwig Institute, Middlesex Hospital, London. deposit, details to be provided) [6], were added to the medium together with 0.8 μg / ml polybrene. Viral titration was adjusted to give a low transduction efficiency of 0.0002%, producing an average of 20 colonies per flask.

After 1 hour, the culture medium was replaced with fresh medium. The cultures were maintained at 33 ° C, the probable temperature for the active form of the product or oncogene of SV40-T. Five days after transduction, geneticin was added to the culture medium (0.4 mg / ml) for an additional 8 days to eradicate the cells that were not incorporated into the retroviral vector. Between days 14 and 20 after transduction, individual colonies of üfreplicació? "Clones" were selected based on being separated from other replication colonies, their circular shape and their morphology. The individual clones were harvested and expanded near the confluence in a 75 cm2 flask, i.e. approximately 23 divisions of an individual precursor, before the aliquots of cells were frozen. Also, the aliquots were placed in 12-well plates coated with poly-L-lysine / gelatin for analysis of potential differentiation characteristics. Alternatively, rat embryos were treated, as mentioned above, in order to provide cells in the tissue medium and these cells were then exposed to a replicating agent as described in references 8 and 9 before undergoing differentiation. as described later.

Cell Differentiation The cells were maintained in the constituents of the medium for replication above and at the probable temperature of 33 ° C, but the homogeneous population of nerve cells, now referred to as RAPHE CLONE 1 CELLS, were cultured at the bottom of a well with CELLS RAPHE primary (prepared as described above, but without the subsequent steps of transfection) as a non-confluent cell layer placed on a plate on PTFE inserts (Corning) Both immortalized and primary RAPHE cells were replicated until the primary RAPHE cells became confluent. At this point, the immortalized cells exhibited a much reduced replication rate. The primary RAPHE cells were removed, removing the insert and the immortalized cells began to exhibit a significant degree of differentiation. For example, 5-hydroxytryptamine was now synthesized without the requirement of precursor handling, ie, 5-hydroxytryptamine could now be demonstrated. The morphological differentiation was much more complex, since many tapering, branching dendrites were usually visualized. In addition, the cells developed several ion channels, including in particular calcium channels of type N. Very little or no apostosis was observed at the probable temperature, and the cells were refractory to the effects of serum that induce glial differentiation.

Cell Apostosis The above referred to the homogeneous population of clone 1 raphe cells can be made to enter apostosis using any of the following four methods. 1. The temperature was increased to an unlikely value (39 ° C) for 72 hours, in the presence or absence of a fibroblast growth factor or epidermal growth factor. Neural cells developed the ability to take 5-hydroxytryptophan (5HTP, the precursor of 5HT), the same 5HT, and to decarboxylate 5-hydroxytryptophan to 5 HT. No native 5HT was detected. The 5HT derivative of 5HTP was released, although the mechanism of such release is unknown. We demonstrated weak neurofilament and neuron-specific enolase-type immunoreactivity. The morphological differentiation was limited to the development of three or four neurites with a single branch. The cells also appeared to suffer extensive apostosis, so that after three days less than 10% remained. The remaining cells were probably neuronal. 2. The temperature was increased to an unlikely value (39 ° C) for 72 hours, in the presence of cyclic AMP plus fibroblast growth factor. Although the differentiation parameters described in the above are basically similar after the incubation of the cells with cyclic AMP, there was an increase in the degree of fiber development. The cells are probably neuronal. 3. The temperature was increased to an unlikely value (39 ° C) for 96 hours, in the presence of retinoic acid (10 μM) plus fibroblast growth factor. The survival of cells increased enormously, but the cells failed to develop the 5HT parameters described above. In addition, neuron-specific enolase staining was further reduced, whereas immunoreactivity of fibrillary acidic glial proteins was found in many but not all cells. They were taken on a flat morphology, and did not exhibit more fibrous extensions. The cells were probably equal. 4. The temperature was increased to an unlikely value (39 ° C) for 96 hours, in the presence of 5% fetal bovine serum plus 5% heat inactivated horse serum plus the fibroblast growth factor. The survival of cells was greater than after retinoic acid, and the cells lost the 5HT parameters described above. In addition, the specific enolase staining in the neuron was dramatically reduced, whereas the immunoreactivity of brightly acidic glial proteins was found in many cells. The cells took a flattened morphology, and did not exhibit more fibrous extensions. The cells were probably glial. It is believed that cells may undergo apostosis when they reach confluence.

Differentiation Conditions Mesencephalic and medullary raphe neural cells of E12 - E13 of rat embryos were immortalized (El = conception day) using a temperature-sensitive oncogene as described above (Stringer et al., 1994). Under permissible conditions, ie in the presence of 5 ng / ml of fibroblast growth factor (FGF) (Sigma, product No. F3391) and at 33 ° C, the immortalized raphe precursor cells were replicated. In a clone (921203-6), which possessed all the characteristics of clone 921202-6 described in Stringer et al, (1994), changing the temperature to 39 ° C, but maintaining all the other conditions as before, caused the development of precursors of some characteristics of serotonergic neurons (5HT), such as neuron-specific enolase- (NSE) and neurofilament- (NF) immunoreactivity, a bright phase morphology with two or three bifurcation procedures, the ability to take serotonin via a vehicle of low affinity (V ^ = 36μM) and decarboxylar 5-hydroxytryptophan (5HTP) to serotonin. However, tryptophan hydroxylase activity was not demonstrated, and the cells failed to synthesize tryptophan serotonin. No calcium channels were demonstrated using patch fastening analysis. The development of raphe precursors of clone 921203-6 in the presence of primary cells dissected from the same ventromedial regions of the mesencephalon and medulla oblongata from which the clone was originally derived leads to improved differentiation of the clone, as long as a mitotic environment. To establish such conditions, the ventromedial mesencephalon and the medulla oblongata were dissected from E12-E13 rat embryos and plated into inserts coated with poly-L-lysine (PTFE membrane, 0.4μm pore size, Corning, product no. 25204-6), approximately one mesencephalon / medulla oblongata per insert. The primary cells were incubated exactly under the same replication conditions as those used to obtain replication in the immortalized precursors, ie, with 5 ng / ml of FGF, and at 33 ° C. After several days, the density of the primary cells reached confluence. At this time, the cells of clone raphe 921203-6 were placed in plates at low density on a 6-well plate (previously coated with gelatin and poly-L-lysine) and, after confirmation of their binding to the substrate, the Inserts containing primary cells were placed in the same wells, along with their conditioned medium. No direct contact between primary and clonal cells was possible; Diffusible factors in the common environment can have effects on both groups of cells, clonal and primary cells, but the effects on the trainer are undoubtedly direct. The incubation conditions were maintained exactly as before, that is, 33 ° C, with FGF. After 2-3 days, the immortalized precursors developed a highly differentiated morphology of two to three long procedures, branching, tapering (presumably dendrites) and a greater bright phase soma. Immunocytochemical analysis of clonal cells at this point demonstrated immunoreactivity of NSE-, NF- and serotonin, the latter case in the absence of loading with 5HT, 5HTP or tryptophan. Now, the calcium channels could be demonstrated, and also channels of type without P and without T, of type N were included. Once the inserts became confluent, both the primary and immortalized cells began to exhibit signs of tension and death. The serum failed to avoid this. However, removal of the insert and / or conditioned medium completely prevented cell stress and death. Despite the loss of medium conditions, the immortalized cells continued to exhibit all the parameters described above in the mature 5HT neurons. Using the midline region of the rat spinal cord E12-E13 capital, as the source of primary tissue, the differentiation of clonal cells is filled. The inclusion of fetal bovine serum and heat-inactivated horse serum (both 5%) in the culture medium had no apparent effect on the differentiated clonal serotonin neurons. In contrast, adding serum to the same cells that undergo rudimentary differentiation produced via the temperature change method that caused them to lose all their neural characteristics and adopt rather an astrocytic phenotype. Daily counts of the number of immortalized rafe neural precursors / differentiated serotonin neurons were made. Although all the cells continued to replicate during the first, second and third days, as soon as the onset of differentiation arrived, replication was morphologically apparent, which stopped even when the cells were still at 33 ° C. Removal of the insert after differentiation was induced and led to no increase in cell number; in addition, no mitotic antibody was evident. On the other hand, the removal of the insert before the differentiation began allowed the cells to continue dividing. In summary, returning to the raphe neural precursor cells immortalized in the mitotic environment, from which they originally originated, leads to a much more extensive differentiation than previously described methods can provide. The effect is directly on the same clonal precursor cells, and is mediated by either other cell types or cell-cell contact. In addition, such differentiation can take place in the presence of a continuous replication device, and quickly leads to a compromise with the chosen phenotype (e.g., a full-blown serotonin neuron), which is maintained even in the presence of factors that normally they will cause an alternative phenotype (for example, astrocytic) to be expressed. The removal of the conditioning factors does not cause the cells to change their phenotype now submitted. It is probably that the soluble factors present in the mitotic primary cell conditioned medium are responsible for inducing such differentiation, and may be related to the recently described N-terminal unfolding product of sonic urchin that is known to induce differentiation of the brain stem and spinal cord precursors to be made, respectively, dopaminergic neurons (Hynes et al, Neuron 15 (1995) 35-44 and cholinergic motoneurons (Roelink et al, Cell (1995) 445-455).

Provision of Nervous Cell Lines including at least one selectively controllable safety factor Another preferred embodiment of the invention relates to the preparation of homogenous populations of cells through retroviral transduction, but also to the incorporation of a safety factor that allows the cells to be selectively deactivated and / or destroyed if necessary. This could be advantageous when such cell lines are used for transplantation in patients to alleviate the symptoms of neurodegenerative disorders. The safety feature could allow the transplant to be selectively destroyed in, for example, situations where the transplanted material can become tumorigenic in vivo and / or in situations where the transplanted material becomes otherwise harmful. The ways in which this can be done are numerous and well known to those skilled in the art. For example, the cell line can be transfected with a gene, which when activated acts, either directly or indirectly, to disable or destroy the transplant. Examples of such genes are well known to those skilled in the art and will not be described here in great detail. In a preferred embodiment of the invention, a security aspect can be coupled to the transformation oncogene, so that the coexpression of the two corresponding cell products occurs. This means that in cases where the oncogene can be activated too much, then the safety aspect could be overcome and in this way the damages associated with the tumorigenic nature of the oncogene. The coexpression can be obtained in a number of ways, for example, the safety gene can be placed downstream of the immortalization gene and then 3 'for example, a polyvirus derived from the sequence of the internal ribosomal entry site (IRES) . In this way, the same promoter / enhancer or enhancers that control the transcription of the immortalization gene could also control the transcription of the security aspect. This is because they are transcribed as a complete unit, including the IRES sequence which can be seated between them. The IRES sequence allows translation of downstream sequences which codes for a protein separated from the 3 'sequence thereof. The ability to provide such a vector, once the idea is presented, is within the range of any one skilled in the art.

Experiments that show the functional characteristics of differentiated nerve cell lines Functional ion channels Table 1 shows the functional activity of immortalized nerve cells of clone 1 under varying neurophysiological conditions. Twelve different cells were examined 2 or 4 weeks after differentiation to complete a completely differentiated nerve cell. Using the conventional arrangement holding techniques, the conductivity of ion channels within the nerve cells examined in either 5mM Ca or 30mM Ba was determined. With 5mM of Ca cells 2 and 7 showed a conductivity lower than 50pA. Cells 8 and 9 showed a conductivity greater than lOOpA. These results indicate that clone 1 included functional nerve cells. At 30mM of Ba, cells 8 and 9 showed functional ion channels having a conductivity greater than 200pA. Cells 11 and 12 also showed conductivity under these conditions. A weak signal less than 50pA was shown for cell 11 and a stronger signal greater than 200pA was shown for cell 12.

Exposure of clone 1 cells to known toxins that interfere with the conductivity of the calcium ion channel affected the conductivity of the functional nerve cells of clone 1. Specifically, a lmM w-CgTxGVIA, a known toxin that blocks blood channel N-type calcium, cell 8 was 100% affected. At a lower concentration of lOOnM, cell 9 was 70% affected. At lmM, cell 12 was also 70% affected. These results indicate that the factors that specifically affect the nerve cell conductivity affect the differentiated nerve cells in clone 1 and thus indicate that these differentiated nerve cells were fully functional nerve cells expressing phenotypic characteristics and more specifically nerve cells possessing calcium channels of type N. Using the toxin-Aga IVA, it is a toxin known because it blocks the calcium channels of type P, was less successful at a concentration of 50nM, cell 9 was insensitive. Referring now to Figure 1, current-voltage data are available for clone 1 cells. A scale of voltages between -85 and 50mv was applied to the cells of the invention. Simultaneously, the response of such cells was inspected by recording the current flow. The voltages above the rest potential produced a flow of current and thus opened the ion channels of the nerve cell. A depolarization potential was observed at approximately -50mv. This depolarization potential resulted in a generation of a potential action indicating that the cells are fully functional. The cells were inactivated at approximately lOmV. Figure 2 shows a dependent channel voltage driving course time and the effects of toxin application on this conductivity. Over a period of about 5 minutes, the application of w-CgTxGVIA resulted in a marked reduction in nerve cell conductivity. After a 10 minute interval, a second toxin was added and the current remained at approximately 70 pA. The current-voltage relationship is shown towards the inside of Figure 2, where it can be seen that the addition of -CgTxGVIA at a concentration of lOnM, significantly affected the conductivity of the nerve cell ion channels. The addition of w-AgalVA also affected nerve cell conductivity but much less markedly. Finally, Figure 3 shows the current-voltage relationship in the 30mM solution of Ba and Cl2 for clone 1 cells. At a depolarization potential of -'jOmV the nerve cell ion channels opened and a current of the order flowed. of -350pA. Also shown are cells that include fully functional voltage dependent potassium channels, which can be blocked using conventional physiological components (data not shown). The above data indicates that the nerve cell clones of the invention can be made to fully differentiate and thus exhibit phenotypic characteristics of a fully functional and thus totally differentiated nerve cell.

References 1. White L A. and Whittemore S R. Immortalisation of Raphe Neurons: an Approach to Neuronal Function in vi tro and in vivo, Journal of Chemical Neuroana tomy, Vol. 5: 327-330 (1992). 2. Stampfer MR, Bartley JC 1985. Induction of transformation and continous cells lines from normal mammary epithelial cells after exposure to benzo [a] pyrene. Proc Nati Acad Sci USA 32: 2394-2398. 3. Bottenstein, J E and Sato G H., Growth of a mouse neuroblastoma cell-line in serum-free supplemented medium, Proc. Na ti, Acad. Sci. , 76 (1979) 514-517. 4. Romijn H J., Mud M T., Habets, A.M.M.C. and olters P S., A quantitative electron microscopic study of synapse formation in dissociated fetal cerebral cortex in vitro, J Neurophysiol. , 40 (1981) 1132-1150. 5. Murphy M, Drago J and Bartlett P F. Fibroblast growth factor stimulating the proliferation and differentiation of neural precursor cells in vitro, J Neurosci Rea. , 25 (1990) 463-475. 6. Jat P S. and Sharp P A., Cell-lines established by temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the non-per issive temperature. Mol. Cell Biol, 9 (1989) 1672-1681. 7. Stringer BMJ, et al., Raphé neural cell immortalized wiht a temperature-sensitive oncogene, Developmental Brain Research 79: 267-274, 1974. 8. Reynolds BA and Weiff S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Sci. 255: 1707-1710, 1992. 9. Mayer E., Dunnett F. B., and Fawcett J. W. Mitogenic effect of basic fibroblast growth factor on embryonic mesencephalic domapinergic neurone precursors. Dev Brain Res 72: 253-258, 1993.

Claims (23)

1. A method for producing large populations of neural cells, characterized in that it comprises carrying out the following steps in the following order: a) improving the replication of a first undifferentiated neural cell or neural cell precursor cell, or precursor stem cell, b ) exposing such replicated neural cells to either a region, or part thereof, of the nervous system, or an extract thereof including homologues and analogues thereof, from which the neural cell originates; and c) allow the differentiation of the cells to produce fully differentiated active neural cells.

2. A method according to claim 1, characterized in that the environment from which the first nerve cell comes is any region of the central nervous system.

3. A method in accordance with the claim 2, characterized in that the environment is an environment in, adjacent, or functionally related to the neural location in the central nervous system from which the first undifferentiated nerve cell is derived.

4. A method according to claim 3, characterized in that the environment is a mitotic environment.

5. A method according to any of the preceding claims, characterized in that the nerve cells are exposed to a soluble extract of the environment.

6. A method according to any of the preceding claims, characterized in that the environment is of the same species as the first undifferentiated nerve cell.

7. A method according to claims 1 to 5, characterized in that the environment is of a different species from that of the first undifferentiated nerve cell.

8. A method according to any of the preceding claims, characterized in that replication is provided through the use of a replication agent such as a growth factor.

9. A method according to claims 1 to 7, characterized in that improved replication is provided through an immortalization agent.

10. A method according to claim 9, characterized in that the agent is an oncogene.

11. A method in accordance with the claim 10, characterized in that the oncogene includes, or has associated with it, control means.

12. A method in accordance with the claim 11, characterized in that the control means respond to culture or environmental conditions.

13. A method in accordance with the claim 12, characterized in that the control means respond to the temperature.

14. A method according to claim 13, characterized in that the oncogene is SV40T.

15. A method according to any of the preceding claims, characterized in that the method comprises an extract of cells of a region in, adjacent or functionally related to the original region from which the first undifferentiated nerve cell is derived.

16. A method according to claims 1 to 14, characterized in that the environments comprise a soluble extract taken from a population of cells physiologically located in a region in, adjacent or functionally related to the region from which the first nerve cell is derived not differentiated

17. A method according to any of the preceding claims, characterized in that the homogeneous population of cells is exposed to at least one growth factor.

18. A method according to any of the preceding claims, further characterized in that it includes transforming the first undifferentiated nerve cell with a safety-looking gene, which is either constitutive or can be activated selectively in order to be able, in any case , selectively disable or destroy the cell line.

19. The use of a nerve cell line, comprising a first undifferentiated nerve cell or nerve cell precursor that has been immortalized with an immortalization agent, which includes or has associated with it control means, so that the immortalization agent can be selectively activated / deactivated as a model to investigate apostosis, so that after culturing the immortalized nerve cell a homogeneous population of nerve cells can be provided before the confluence of control means that can be activated for remove the functional effect of the immortalization agent and lead the cell to apostosis.

20. Cell lines produced according to the method of claims 1-18.

21. A nerve cell line according to claims 1-18 subjected to a fully differentiated phenotype.

22. A non-mitotic nerve cell line according to claims 1-18.

23. A nerve cell line surviving at low densities according to claims 1-18.