MXPA97003385A - Human cell-lines - Google Patents

Abstract

La invención se relaciona con un método para producir líneas celulares humanas y células y líneas celulares producidas por tal método. El método comprende el uso de células precursoras o sin diferenciar, tratadas con un agente de inmortalización, el cual es susceptibles a las condiciones ambientales para proporcionar la activación/desactivación selectiva del agente de inmortalización y para la activación selectiva de la diferenciación.

Description

"i HUMAN CELL LINES DESCRIPTION OF THE INVENTION The invention relates to a method for producing human cell lines; and cells and cell lines when they are produced by such a method. It is widely recognized that it would be advantageous to have cell models in vi tro, simulating the conditions in vivo. Ideally, cell models should be able to propagate in the culture, express specialized tissue functions and allow fundamental biological problems to be solved by simple manipulation of culture conditions. Therefore it is not surprising discover that researchers have spent many years trying to perfect cell models in vi tro and in doing so, they have discovered that normal differentiated cells do not generally proliferate in culture and often end up expressing their specialized function. In effect until 1965 Leonard Hayflick reported that when human lung fibroblasts are observed in tissue culture, the number of divisions these cells can support is limited. Similar observations have been made for a wide variety of tissue types and indeed It has been found that each type of tissue or cell undergoes a characteristic number of divisions before cellular senescence or apoptosis. To overcome what would appear to be cell death related to age or senescence, the researchers investigated aberrant tumor cell lines that are capable of growing in a culture well beyond the normal level of growth found for a normal cell of the same type. tissue, that is, the cells are immortalized. Advantageously, these immortalized cells can retain the ability to express tissue-specific functions. Therefore, it would seem that immortalized cells can be favorable tools for in vi tro investigations. Indeed, historically the generation of cell lines was found in the observation that tumor cells do not exhibit apoptosis. Thus the primary cell lines were obtained only as tumor cells or spontaneously immortalized variants of cells, which grow easily in tissue culture. Subsequently, the discovery that certain viral oncogenes have the ability to confer indefinite growth for several types of normal cells, leads to the rapid generation of non-human cell lines by transfection of these immortalized genes directly into desired normal cell types in vi tro . The genes • Immortalizers can be introduced into cells by a variety of strategies, such as transfection and retroviral mediated gene insertions. In this way the use of immortalized genes has facilitated the delivery of a wide variety of non-human cell lines from different tissues. In the past fifteen years it has been possible to produce non-human cell lines that retain the functions differentiated by the transformation of normal cells with chemical carcinogens (1), oncogenes (3) and tumor viruses (4, 5). Workers have also attempted to produce human cell lines that retain differentiated functions using oncogenes (2) and tumor viruses (6). However, although it is possible to produce human cell lines that have some differentiated functions, these human cell lines do not go beyond a few replications before apoptosis or senescence. Therefore, it is believed that such cell lines are of little value for in vi tro investigations. In view of the considerable success experienced in producing non-human cell lines it is confusing and frustrating that until now, it has not been possible to use the same techniques to produce human cell lines successfully, for the term cell line lines are successfully understood. immortalized, which retain their specific characteristics of the fabric. It will be apparent that in the absence of immortalization and specific tissue characteristics the generated cell lines can not be used as reliable in in vitro cell models. It is interesting to note that the production of immortalized murine cell lines can be provided using any of the foregoing techniques, while it is not possible to provide immortalized human cell lines. The difference, in part, may be related to the expected expectation of the organism from which the cells are derived. For example, the life expectancy of a mouse is approximately 2 years, while the life expectancy of a human is approximately 70-80 years and therefore, it is possible that due to this significant difference in the expectation of life , there may be more severe regulation of the replication of human cells and this severe regulation may, in part, be responsible for the overall profound lack of success in the production of differentiated human cell lines. The invention is based on a surprising discovery that it was found that contrary to expectations, it is possible to produce an immortalized human cell line, which expresses the specific functions of the tissue, when the method of the invention is practiced, which method involves the use of immature cells, without differentiating or precursors. Although such cells have been used before to study differentiation - previously no one had realized that such cells can be used routinely to provide immortalized human cell lines expressing the tissue-specific functions observed in the mature differentiated phenotype. Therefore, it is important to note that although undifferentiated cells have been used to provide cell lines for the purpose of studying the process of differentiation, where one would expect to start with an undifferentiated cell, if one wants to study the process that leads to differentiation, nobody has thought to use the cells without differentiating as a source to provide a cell line, when you want to simply study the differentiated cell. On the contrary, it is usual to take a differentiated cell and then immortalize the differentiated cell for the purpose of producing a human cell line. Therefore, it is interesting to note that the method of the invention goes against conventional teaching. It is also of interest to note that when the undifferentiated cells are used to produce human cell lines for the purpose of studying the process of differentiation and when a controllable immortalization agent has been used, such as the T antigen of the large SV40 tumor, the method has always involved activating and deactivating the immortalization agent at preselected intervals along the differentiation path, such that these predetermined intervals differentiation products can be identified, for the purpose of establishing markers for trajectory mapping of differentiation. On the contrary, the method of the invention relates the use of an undifferentiated cell, which is allowed to progress continuously towards terminal differentiation for the purpose of investigating the differentiated cell therefore once again, it can be seen that the method of invention goes against conventional teaching. Therefore it can be seen that there is a need to provide immortalized human cell lines, which can be used as cell models in vi tro and therefore it is an object of the invention to provide a method that produces such cell lines; and cells and cell lines when they are produced by such a method. According to a first aspect of the invention, therefore, a method for producing human cell lines is provided, the method comprises: a) immortalizing an undifferentiated or human precursor cell of a given tissue type using an immortalization agent, which includes or has associated with it a control means, whereby the activation of the control means end the immortalization and allow the digestion of the cell without differentiating or precursor, b) culturing the immortalized cell to produce a homogeneous population of human cells, c) activating the control means to end the immortalization and activate the differentiation, and d) allow the differentiation of the cells to produce completely differentiated cells of the given tissue type. It can be seen from the foregoing that the method is characterized by the use of undifferentiated cells or precursors to produce a desired, fully differentiated human cell line. It is believed that the choice of undifferentiated cells or precursors will determine the nature of the cell line. In this way, for example, an osteoblast cell line will be provided by the use of bone marrow stromal cells; an osteoclast cell line will be provided by the use of precursors or hematopoietic derived osteoclasts; a heart cell line will be produced by the use of myocardial precursor cells; a kidney cell line will be provided by the use of kidney cell precursor cells; a muscle cell line will be provided by the use of muscle precursor cells; a skin cell line will be provided by the use of epithelial precursor cells; a line of liver cells will be provided by precursor cells of the hepatocytes; a lung cell line will be provided by precursor cells of lung cells; and T lymphocytes & B will be provided by the use of lymphocyte progenitor cells. Therefore, it can be seen from the previous examples, that the nature of a given cell line can be determined to have with respect to the type of differentiated cells or precursors used in the method of the invention. Surprisingly it has been found that the use of undifferentiated cells or precursors in the method of the invention provides an immortalized human cell line, which retains the functional characteristics associated with the type of cell from which the cell line is derived. Therefore we have been able extraordinarily to provide the human cell lines to be used as cell models in vi tro. The cell lines are immortal and reliable. In a preferred embodiment of the invention, immortalization is achieved using conventional transfection techniques and preferably the immortalizing agent is an immortalizing gene which is an oncogene, even more preferably, the immortalizing agent is a viral oncogene which can be integrated Stably in the genome of the host cells. Ideally, the immortalizing agent is a construct, preferably a retroviral construct, which includes an oncogene whose oncogene can be virally derived or a human derived oncogene. Any known oncogene can be used such as myc, ras, src, etc. Alternatively, immortalization may be affected using physical or chemical means. For example, immortalization may be affected by exposure of cells to radiation or chemicals which are known to promote cell division beyond the normal level found when a cell is not exposed to two physical or chemical media. In a preferred embodiment of the invention, the control means is sensitive to environmental conditions such as temperature, pH or ionic concentrations. In yet another preferred variety of the invention, the immortalizing agent and the control means are integrated, i.e. the immortalizing agent is itself controllable. In this way, the immortalizing agent and the control means may consist, for example, of a single entity such as a temperature sensitive oncogene. Alternatively, the immortalization agent and the control means may be two independent entities but in any case, ideally the activation / deactivation of the control means ideally has a direct effect such as in one embodiment, a reciprocal effect on the immortalization agent. For example, when the control means is activated the immortalization agent is deactivated. On the contrary, when the control means is deactivated the immortalization agent is activated. Ideally, control can be achieved with respect to the culture under ambient conditions, for example the preferred embodiment of the invention, the immortalization agent is temperature sensitive and the control is represented by a temperature sensitive activator, such that approximately , above or below a given first temperature, the immortalization agent is activated to immortalize the selected cell type, but at, approximately, or above a second temperature, the immortalization agent is deactivated and in this case the immortalization ends and the differentiation is allowed to proceed to provide a homogeneous population of cells of a given cell type. Preferably, the immortalizing agent is the SV40 T antigen, which is permissive, i.e., the viral gene is expressed in an active form at 33 ° C and not permissive, i.e. the viral gene is expressed in an inactive form at 39 ° C, in this way cells immortalized using this agent are sensitive to temperature for differentiation. Only cells when transformed using the SV40 T antigen differentiated at the non-permissive temperature and the survival crisis of a condition which is typically followed by apoptosis. In the survival crisis the cells were immortalized. It is believed that the immortalization characteristic is due to the use of the undifferentiated or precursor cells in the method of the invention. In the preferred embodiment that allows differentiation of cells, it comprises culturing the cells in the presence of a differentiating agent, for example to produce osteoblasts, the differentiating agent is Vitamin D3, ideally in the presence of Vitamin K. In a Preferred alternative modality that allows the differentiation of the cells, comprises cultivating the cells in the presence of a differentiating agent, for example to produce osteoblasts the differentiating agent is dexamethasone. In a preferred embodiment that allows differentiation of cells, it comprises culturing the cells in the presence of a differentiating agent, for example to produce adipocytes, the differentiating agent is rabbit serum or an extract thereof.

In yet another preferred embodiment of the invention, the human cell line also includes a safety feature, which allows selective inability or destruction of the cell line. This safety feature is of advantage where the cell line is to be used for the purpose of transplantation or in any other way, whether it is permanent or temporary, attached to, or administered to, or stored in an individual. The safety feature allows the cell line to be selectively disabled and by this means make it less dangerous, or destroyed in cases where the cell line is thought likely or shown to have the potential to become tumorigenic in vivo, or it is thought to be in any way dangerous to an individual. Preferably the security feature comprises a gene whose products act either directly or indirectly to dismantle or destroy the cell line. For example, the gene can be a gene which, in the presence of certain agents, such as for example antiviral agents, produces a cytotoxic product. An example of such a gene would be the gene that codes for viral thymidine kinase (vTK). This gene anxiously converts the prescribed antiviral drugs to cytotoxic intermediates. Another example of a gene which can be used as a safety feature is the cytosine deaminase (CD) gene. The product of this gene makes the cells vulnerable to the effects of 5-fluorocytosine and results in cell death. In a preferred embodiment of the invention, the security feature is expressed together with the immortalization oncogene. This arrangement is preferred, because it means that the immortalization gene is unlikely to be expressed in the absence of the security feature and vice versa. The co-pending original application GB 94 22236.1, teaches how a vector can be produced which is provided for the co-expression of the security feature, which could be linked with the immortalization oncogene. In a preferred embodiment of the invention, the safety feature gene is positioned downstream of the immortalization oncogene and ideally followed 3 'for example, to a poliovirus derived from the internal ribosomal entry site sequence (IRES). This arrangement ensures that the promoter / enhancer element (s) controlling the transcription of the immortalization oncogene also controls the transcription of the security feature. It will be apparent to those skilled in the art that other provisions may be provided to allow the co-expression of the immortalization oncogene and the security feature and it is not intended that the above example should be considered in a form which limits the scope of protection provided by the request. According to another aspect of the invention, a method for the production of osteoblast cells comprising exposing the Cell Lines is provided.

Human according to the invention to a differentiation agent. Preferably the differentiating agent is Vitamin D3, ideally in the presence of Vitamin K. Preferably the differentiating agent is dexamethasone. According to another aspect of the invention, there is provided a method for the production of adipocytes from Human Cell Lines according to the invention, which comprises using a differentiation agent. Preferably, the differentiating agent is rabbit serum or an extract thereof. According to yet another aspect of the invention, there is provided a method for identifying an agent responsible for stimulating differentiation to produce adipocytes, the method comprising exposing the Human Cell Line according to the invention to the agent and observing any of the characteristics of the Differentiated phenotype.

Preferably the agent is produced by extraction of rabbit serum using a separation technique, for example, ionic separation, chromatography, protein precipitation, etc. According to another aspect of the invention, the use of rabbit serum to produce adipocytes is provided either from a cell line, preferably a bone marrow cell line, or at least one precursor cell, preferably a precursor cell from a cell line. bone marrow. According to another aspect of the invention, a composition is provided, which contains an agent that affects the differentiation process. Preferably the composition is a pharmaceutical composition. Preferably the agent stimulates the differentiation process. Alternatively, the agent blocks or prevents differentiation, the differentiation of which results in the supply of adipocytes. The bone marrow of osteoporotic patients often contains tissue grade, which is often mentioned as fatty bone marrow. This observation, together with the recognition that the same stromal cells of human bone marrow can be derived from adipocytes and osteoblasts allows us to propose that osteoporosis can be a consequence of disturbances' in the differentiation of bone marrow stromal cells to the lineage of bone marrow. osteoblasts, in favor of the formation of adipocytes. The information provided here, therefore, gives the address for the man skilled in the art, to seek this process of differentiation for the supply of pharmaceutical agents, capable of controlling it. For example: the separation agents from the separation (ionically, CLAP, alternative chromatography, protein precipitation and separation, etc.) of normal rabbit serum can be used to determine the agent or agents that induce differentiation for the lineage of adipocytes. These agents can be used to recognize the path of the intracellular transduction signal involved in the differentiation process and the subsequent development of agents to effect this path. It is contemplated that this information may also lead to the identification of agents to control obesity. According to another aspect of the invention, cells or cell lines produced according to the method of the invention are provided. Accordingly, at least one homogeneous population of immortalized human cells provided with means for terminating immortalization is provided, such that a homogeneous population of differentiated human cells is provided.

According to yet another aspect of the invention, there is provided the use of immature, undifferentiated cells or precursors to terminally produce differentiated human cell lines that express the specific functions of the tissue. The invention will now be described by way of example only, with reference to the human bone cell line and with reference to the following figures in which.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effects of temperature and Dexamethasone (5xl0 ~ 7M) on the activity of alkaline phosphatase in bone marrow human stromal clone 7 cells. Table 1 shows the effects of temperature and Dexamethasone on the expression of mRNA in an immortalized clone of human bone marrow stromal cells. Figure 2 shows the staining of a human bone marrow stromal cell clone, immortalized, untreated at 33 ° C - x 100. Figure 3 shows staining of human bone marrow stromal cell clone immortalized at 39 ° C treated with dexamethasone 5 x 10"7M at 39 ° C.

Figure 4 shows the staining of an immortalized human bone marrow stromal cell clone and the population mixed with and without Dex 5 x 10 ~ 7 M at 39 ° C. Figure 5 shows the staining of a human marrow stromal cell clone, immortalized with Dex 5 x 10 ~ 7 M at 39 ° C. Figure 6 is a bar graph which shows the effects of several agonists on the cAMP levels of clone 7 cells. The cells are penetrated with 1 mM IBMX for 5 minutes and then treated with the appropriate dose of the agonist during 20 minutes, cAMP levels are quantified using an EIA, and the amounts of CAMP for each treatment are compared with the control (only IBMX) and are expressed as a percentage. Figure 7 is a bar chart which shows the synthesis of Osteocalcin from the cells of clone 7 over a period of 4 days. The cells are treated with various concentrations of l ^ dlOH ^ D-g in the presence of vitamin K for 4 days. Then the media is removed and the amounts of osteocalcin are measured using a R.I.A. and osteocalcin levels normalized to ng of osteocalcin per 10,000 cells. Figures 8, 9, 10 and 11 are photographs which represent the oil-red-0 staining of clone 7 of bone marrow stromal cell line, immortalized.

The - Figures 8 and 9 samples the cells after 3 days of treatment with 10% rabbit serum at 39 ° C at an amplification of xlOO. Figure 10 shows cells after 3 days of treatment with 10% rabbit serum at 39 ° C at an amplification of x200. Figure 11 shows the cells after 3 days of treatment with 10% fetal bovine serum at 39 ° C at an amplification of x 100. Figure 12 is a bar diagram showing the effects of Dexamethasone on the activity of the alkaline phosphatase in three clones of human fetal cells immortalized at 39 ° C for 7 days. Figure 13 shows a femur derived from a human fetus 7-9 weeks gestation and approximately 600 μm in length. Figure 14 shows immunohistochemical analyzes of the cells in a culture of a human cortical precursor immortalized with the SV40T oncogene sensitive to temperature.

Immortalization of Bone Marrow Stromal Cells The human bone trabecula is immersed in medium comprising EMEM + 10% fetal calf serum, plus L-glutamine, plus lx penicillin / streptomycin (all from Sigma Chemicals) and shaken for release the population of bone marrow cells. After 24 hours, to allow the population of stromal cells to adhere to the surface of the tissue culture flask (Costar, UK Ltd), the medium was replenished to eliminate the population of non-adherent cells. The population of adherent cells was then transfected with a temperature-sensitive mutant of the large tumor antigen (T) derived from simian virus-40 using retroviral transduction. Any standard method of transfection of this sequence (together with binding to an appropriate promoter to activate its expression, for example the LTR promoter) would suffice, such as precipitation of the DNA with calcium phosphate, electroporation or microinjection, but transduction is chosen retroviral because of its simplicity of use.

Cultivation of Immortalized Cells to produce a Population of Homogeneous Cells In summary, amphotropically packed retroviral particles comprising this construct and a geneticin resistance marker, G418 (kindly donated by Dr.-M. O'Hare, Institute of Cancer Research, Royal Marsden Hospital, Lincoln's Inn, Sutton, Surrey and also _ Professor P. Gallimore, The University of Birmingham, UK) is added to the medium together with polybrene (Sigma Chemicals) to a final concentration of 0.8 mg / ml. The viral titer was adjusted to give a low transduction efficiency of 0.0002% which produces an average of 20 colonies of cells immortalized per flask, each colony derived from a single cell. Two hours after the addition of the virus, the culture medium is replaced with fresh medium. The cultures are maintained at 33 ° C, the permissive temperature for the active form of the SV40-T oncogene product. Five days after transduction, geneticin is added to the medium (0.4 mg / ml) for another 10 days to eradicate cells which have not been incorporated into the retroviral vector.

Differentiation of the cells _ _ Between 14-20 days after transduction, the individual colonies of replication cells were identifiable. The clones were selected on the basis of being well separated from other colonies in replication, the cells in each colony numbering between 100-1000 cells. These are harvested by ring cloning and expanded to near confluence in 2 75 cm flasks (Costar, UK Ltd.) which equivalent to approximately 22 divisions of a single cell before freezing the aliquot deposits. The cell samples generated in this way are grown in the well culture for one year and have some clones which have experienced more than 60 divisions (10-8 cells). The samples were also used to characterize the cell and determine that they possess the ability to differentiate into cells similar to mature osteoblasts. The differentiation is carried out by exposing the cells to the non-permissive temperature of the oncogene (39 ° C) and a differentiating agent such as dexamethasone or Vitamin D-1.

Provision of human cell lines that include a selectively controllable safety feature Another preferred embodiment of the invention is the preparation of homogeneous populations of cells by retroviral transduction, but they also incorporate a safety feature which allows cells to be selectively destroyed if It needs to be done This would be seen as an advantage, when such cells are used for transplantation in patients to alleviate the symptoms of for example, neurodegenerative disorders, osteoporosis or osteoarthritis, for example. The safety feature would allow the transplant to be selectively destroyed, for example, from situations where the transplanted material may become tumorigenic in vivo. The ways in which this can be done are numerous and well known to man with skill in art. For example, the addition of the viral thymidine kinase (vTK) gene, under the control of an appropriate motor, for the transduced ts-SV40-T cells, would mean that the cells express SV40-T, it could also express the vTK gene. This gene avidly converts drugs "Written antivirals such as ganciclovir or acyclovir, in cytotoxic intermediates, which destroy the cells in which it is expressed." Such a suicide gene would be a particular advantage to eradicate the graft, if necessary.Another example of such a safety activator molecular is the cytosine deaminase (CD) gene.The cells which express the CD become sensitive to 5-fluorocytosine and die in their presence, while the cells that do not express the CD gene remain without effect. preferred invention should not be seen as limited to vTK and CD as negative selection markers since the man skilled in the art, I could easily replace these with alternatives. Another aspect of the preferred invention, whereby a security mechanism is placed within the cells to serve as a negative selection component, would be to have the security component expressed together with the immortalization oncogene. This would be particularly preferred since it would mean that the immortalization gene is unlikely to be expressed in the absence of the negative selection security mechanisms and vice versa. The man skilled in the technique of constructing the vector would be very capable of making such a construction and the vector refers to one of four other patent applications, GB 94 22236.1 in this regard. For example, the negative selection gene (eg, CD or vTK) can be placed downstream of the immortalization gene and then to but 3 'for example, a poliovirus derived from the internal ribosomal entry site sequence (IRES). In this way the same promoter / enhancer elements that control the transcription of the immortalization gene would also control the transcription of the security element. This is because they could be transcribed as a complete unit, including the IRES sequence, which can be placed between them. The IRES sequence allows translation of the sequences downstream of it, which codes for a protein separated from the 5 'sequence of it. The ability to provide such a vector, once given the idea, is within the range of experience of man with skill in art.

Experiments to show the functional characteristics of human bone marrow stromal cells _ _ The characterization of the clones that have been produced using this method and which have been cultivated during a year in culture, has shown that the cells retain the expected characteristics of an osteoblast precursor bond and under stimulation to differentiate, potentiate the osteoblast phenotype. mature.

The differentiated bone marrow cells which are responsible for the production of bone are known as osteoblasts. A recognized indicator of osteoblast activity is a measure of the activity of alkaline phosphatase - an active enzyme in the production of bone or hydroxyapatite minerals. In Figure 1 it can be seen that bone marrow stromal cells immortalized at the permissive temperature of 33 ° C exhibit an alkaline phosphatase activity of approximately 0.3p-NP / mg protein / RH. Exposure of these immortalized cells at the permissive temperature of 33 ° C to dexamethasone - a differentiating factor results only in a slight increase in alkaline phosphatase activity, ie of approximately 0.5p-NP / mg protein / HR . In contrast, the same cells immortalized at a non-permissive temperature of 39 ° C in the absence of any differentiating agent showed an increased amount of alkaline phosphatase activity. In effect, the alkaline phosphatase activity increases to approximately 0.9p-NP / mg protein / RH. Therefore, it would seem that the non-permissive temperature of 39 ° C differentiation is carried out and this enzyme activity characteristic of the differentiated cell type increases. Another increase in this activity is observed when the immortalized bone marrow stromal cells are cultured at the non-permissive temperature of 39 ° C and further exposed to dexamethasone - a differentiating agent. Under these conditions the alkaline phosphatase activity increases to approximately 2.5p-NP / mg protein / RH. This activity is approximately 5 times higher than in cells exposed to the dexamethasone differentiation agent at the permissive temperature of 33 ° C. These data show that the cells immortalized according to the method of the invention, can be selectively made to differentiate in the non-permissive temperature of 39 ° C and the differentiation results in the formation of cells that have functional characteristics similar to those of the osteoblasts, in other words, the differentiation results in the production of ssteoblasts. Similarly, at the non-permissive temperature of the oncogene (39 ° C) and in the presence of differentiating agents such as dexamethasone or Vitamin D3, they substantially regulate their alkaline phosphatase activity (Figure 1) . The data in Table 1 similarly show that the bone marrow stromal cells immortalized according to the method of the invention can be made "to differentiate into functional osteoblasts." The table shows the expression of the mRNA in an immortalized clone of marrow stromal cells. Human bone markers were investigated to identify agents typically characteristic of an immortalized condition.These agents are shown on the far left side of Table 1. At the permissive temperature of 33 ° C the human bone marrow stromal cells immortalized at 33 ° C, all the agents except interleukin 3 and interleukin 4 were expressed. The use of dexamethasone at this temperature showed the same pattern of agent expression, however there was a slight reduction in interleukin 1 alpha and interleukin 1 beta and the expression of GM-CSF and a slight increase in the expression of TNF alpha. For a non-permissive temperature of 39 ° C, there was a marked difference in the pattern of mRNA agent expression. As in the previous, there was no expression of interleukin 3 or interleukin 4. However,In addition, there was also no expression of interleukin 1 alpha, interleukin 1 beta, interleukin 8, GM-CSF and TNF alpha previously expressed. However, interleukin 6 and collagen 1 are still expressed at this temperature. At the non-permissive temperature of 39 ° C dexamethasone increased the expression of interleukin 6 and interleukin 8, but reduced the expression of collagen 1. If the 2 previous patterns of expression, that is the expression of the immortalized cell and the expression of the differentiated cell, are compared with the expression of osteoblast-like cells, it can be seen that these cells grow at the non-permissive temperature of 39 ° C that those cells exposed to conditions of differentiation, present a pattern of expression almost identical to that of the cells similar to osteoblasts. The only difference is that osteoblast-like cells express interleukin 8, whereas differentiated human bone marrow stromal cells only express this agent in the presence of dexamethasone. The above data show that immortalized human bone marrow stromal cells can be made to differentiate at 39 ° C and when they are differentiated, they present a range of characteristics, by means of protein expression, almost identical to that of cells similar to osteoblasts. Therefore, these data suggest that the immortalized human cell line can be made to differentiate to produce the osteoblasts. In summary, cytokine studies and growth factor expression show cells in the undifferentiated state express IL-lalfa and IL-lbeta along with IL-6, IL-8, GM-CSF and TNFalpha, as well as collagen of the type I matrix protein. When treated with dexamethasone or Vitamin D3 for a period of up to 7 days, the expression of IL-lalfa, beta is lost along with GM-CSF, TNFalpha and type I collagen. In addition to the expression of IL-6 and IL- 8 is maintained in the presence of both Vitamin D3 and dexamethasone. In addition, no expression of IL-3 or IL-4 is "observed at any stage." Interestingly, the profile of the growth factor / cytokine that has been identified in these cells after treatment with the differentiation agents, reflects the observed profile of the cells similar to human osteoblasts differentiated in the primary culture (Table 1) In addition, we have found that if the clones of transduced cells are left at the non-permissive temperature of the oncogene in the presence of the differentiation agents, "for example (Vitamin D3 or Dexamethasone) and 10 mM B-glycerol phosphate, the cultures that express the osteocalcin protein and mineralize after 20 days (Figures 2, 4, 4 and 5). Figure 2 shows immortalized human bone marrow stromal cells, undifferentiated at 33 ° C. It can be seen that there is no evidence of mineralization or osteocalcin protein in this figure. In contrast, Figure 3 shows human bone marrow stromal cells, immortalized when exposed to differentiation agents at the non-permissive oncogene temperature of 39 ° C. The mineralization is clearly evident. This feature represents the differentiated cell and thus indicates that the immortalized human bone marrow stromal cell clones of the invention can be prepared to differentiate completely to express the mature phenotype. Figure 4 again shows the staining of an immortalized human bone marrow stromal cell clone and a mixed population of cells' both immortalized in the presence and absence of dexamethasone at 39 ° C. It can be seen that relatively little differentiation occurs at 39 ° C in the absence of a differentiating agent. However, in the presence of a differentiation agent a significant increase in the differentiation that occurs and in this way the mineralization is observed. Furthermore, it can be seen that the phenotypic characteristics are observed both in the mixed population and in an individual clone. Figure 5 shows the staining of a single clone under conditions of differentiation, ie at 39 ° C and in the presence of the dexamethasone differentiation agent. The data in Figures 2, 3, 4 and 5 indicate that cells can be made to differentiate completely in such a way that they express the phenotype of a mature osteoblast. Figure 6 shows further evidence that bone marrow stromal cells, immortalized and then differentiated according to the invention, produce phenotypically similar cells to osteoblasts. Figure 6 shows the cyclic AMP response of differentiated cells for two agonists, namely prostaglandin E2 (PGE2) and parathyroid hormone (PTH). The data indicate a clear cyclic AMP response for agonists, which is typical of cell responses of the osteoblast lineage. Yet another evidence of the immortalized bone marrow stromal cells, after undergoing differentiation form progenitor osteoblast cells was demonstrated by Figure 7. Figure 7 shows the effect of Vitamin D, in the presence of Vitamin K, on the expression of the osteocalcin protein in human bone marrow stromal cells immortalized as determined by radioimmunoassay (Nchols Institute). In the absence of Vitamin D3 there is no expression of osteocalcin (see "control"), the marker of osteoblast differentiation. The bone cell differentiator Vitamin D3, however, induces a dose-dependent expression of osteocalcin. Therefore, it can be seen from these data, together with the mineralization data (Figures 2 to 5) and also the alkaline phosphatase data (Figure 1), that the differentiated cells are osteoblast progenitor cells.

Figures 8, 9, 10 and 11 show a series of photographs and provide evidence that the immortalized osteoblast precursor cells can, under alternating conditions, also differentiate to become adipocytes. This is important because: it underscores the fact that it truly has precursor cells; and is "" adapted to the central dogma, since it was thought that bone marrow stromal cells are also derived from adipocytes - hence early bone marrow precursors may also have the ability to go either to a bone marrow pathway. differentiation of bone or an adipocyte depending on the nature of the stimulation. Figures 8, 9 and 10 clearly show that rabbit serum, or at least one agent in rabbit serum, activates a differentiation process which produces adipocyte cells as shown by red staining. Figure 11 shows that in the absence of rabbit serum red staining does not occur, indicating that there is no adipocyte formation. This result provides further evidence that the early precursor cells of the bone marrow may have the ability to differentiate to produce adipocytes or osteoblasts. The photographs depict the Aceiterojo-O stain (a fat cell marker) of clone 7 of the immortalized bone marrow stromal cell line. This occurs after three days of treatment with the medium containing normal rabbit serum (but not fetal bovine serum) which shows that the population of bone marrow stromal cells, which in the presence of Vitamin D3 and / or dexamethasone becomes to an osteoblast differentiation path, it is able to become an adipocyte when cultivated under appropriate conditions. This confirms the precursor state of the immortalized cells. In addition to the previous staining, changes in specific gene expressions have also been demonstrated. First, the expression of lipoprotein lipase (LPL), an early marker known for adipocytes, can be identified within a few days of treatment with rabbit serum. Second, the expression of type I collagen, a marker of the osteogenic lineage, disappears after a few days of treatment with normal rabbit serum. The expression of LPL and the disappearance of type I collagen, therefore provide another support for the bipotential nature of the human bone marrow stromal cells that have been generated. In this way it is believed that they are using genuine precursor cells to label human cell lines. Figure 12 depicts the levels of alkaline phosphatase in a series of three stromal cell lines of human bone marrow that have been derived from fetal bone. None have alkaline phosphatase activity at baseline (control) levels but when stimulated with dexamethasone for 7 days, two of the three clones (clones 10 and 14) can be induced to express alkaline phosphatase activity. The key point is that the absence of dexamethasone, no alkaline phosphatase activity is detectable. In this way, these precursors have not been selected based on the expression of alkaline phosphatase. Both clones 10 and 14 will be mineralized after 14-20 days in culture in the presence of dexamethasone and phosphate. The remaining clone, clone 2 can not be induced to express alkaline phosphatase activity by treatment with dexamethasone and will be mineralized in the culture. Figure 13 shows a femur derived from a human fetus 7-9 weeks gestation and approximately 600 μm in length. The femur has been cultured for a period of 14 days to allow expansion of replicating cell populations, which can be seen flowing from all regions of the femur. This was before the immortalization of the cells by the transduction of the retroviral temperature sensitive oncogene. Therefore the figure shows the nature of the population of the cells that were used to produce some of the cell lines of the invention.

Longevity of the Human Bone Marrow Stromal Cell Line - - Using the method of the invention, it was surprisingly found that the human cell line successfully differentiates to produce functional cells, which cells avoid the crisis and thus the apoptosis. Therefore the cell line continues to survive and the current human bone marrow stromal cell line has gone through 40 divisions over a period of one year producing 10- ^ cells. The cell line continues to survive and in addition the cells of the line continue to show functional characteristics typical of the tissue type of the differentiated cell. In this way it has been able to produce an immortalized human cell line, which comprises differentiated cells that retain their functional characteristics.

Chondroprogenitor Cell Lines __ As well as precursor cells are generated for the osteogenic lineage, which are capable of the diminished differentiation of an adipocyte pathway, chondroprogenitor cell lines were also produced which, in the presence of differentiation agents such as dexamethasone, will arrive to be hypertrophic chondrocytes. The cell lines have been generated by the transduction of the temperature-sensitive oncogene, retroviral as described above, using human fetal tissue as the material source (which must first obtain ethical approval). Some of the cell lines have the ability to mineralize in the culture in the absence of any of the factors, which induce differentiation and therefore, are already differentiated. In addition, although some cell lines do not present baseline levels of activity related to differentiated cell types. To allow this undifferentiated cell lines to potentiate the hypertrophic chondrocytes, the agent of aggregate differentiation, dexamethasone, to the culture medium of cell clones derived by the cloning of a single cell after immortalization. After several days of treatment with dexamethasone, it was shown that some of the clones could express type X collagen, a marker for hypertrophic chondrocytes. Type I collagen was not clearly expressed in these cells, showing that they were not of osteoprogenitor origin. The cells were also sensitive to 1.25 (OH) 2 3 and expressed high levels of alkaline phosphatase activity, two more markers of the hypertrophic chondrocyte-like phenotype. Also, when the cells are left in a monolayer culture for 10-14 days at 39 ° C, the non-permissive temperature of the oncogene and in the absence of added β-glycerophosphate, the cultures are mineralized. Again, these data clearly show the undifferentiated cells or true precursors are being used to provide human cell lines and that the origin of the precursor cells determines the phenotype of the differentiated cell line.

Neural Cell Lines In another embodiment of claim 1, the neural tissue for immortalization was dissected from human fetal material from 8-12 weeks of gestation; this is close to the optimal age for immortalization (via retroviral transduction) of forebrain cells such as striatal neurons and some cortical neurons, because they have not yet experienced their final replication in vivo. In this way they are still able to incorporate the retroviral oncogene into their genome and co-express it stably. Seven regions of 8-12 weeks of fetal CNS were dissected - cortex, striatum, hypothalamus, rostroventral mesencephalon, caudoventral mesencephalon, medullary encephalic, and dorsal and ventral spines of the spine. The dissociated cells from these regions were plated on a number of different substrates (gelatin / polylysine, fibronectin, uncoated plastic) and incubated in a defined medium.

(Stringer et al., 1994). The cells are transduced by the usual method (Stringer et al., 1994) with an amphotropic virus (PA317-CMV48T) (from P. Gallimore, University of Birmingham, UK) coding for the controllably expressed oncogene (ts-SV40T) bound to a geneticin resistance marker (G418). Since the neural precursors of the human CNS present a degree of replication potential intrinsically activated in the fibroblast growth factor (FGF) - the medium containing it, it is considered advantageous to allow both of the transduced and untransduced cells to expand in the same culture flasks in the same way. In this way, if other samples of fresh human material do not become available, it would be possible to simplify to retransfect existing cells to generate more clones. Therefore, once the mixed cells have reached confluence, the cultures were passed and a frozen ratio for possible subsequent use. The passed cells were treated with geneticin to eradicate the untransduced cells and after 10-12 days, small clones were apparent. The transduced cells, resistant only to G418 of each clone, are extracted for expansion. To achieve this, a method has been developed to activate the cells both to survive better and to replicate more rapidly during the early, critical expansion stages, by co-cultivating them with support cells maintained as feeder layers in well-cell inserts ( Corning). Once the clonal cells numbered around one to two hundred, they become self-supporting, with a markedly increased mitotic speed. At this stage, the inserts containing the support cells were no longer required and could be removed. Now several homogeneous clones have been isolated in this form, although they still have many hundreds of clones mixed, heterogeneous either frozen or continue to expand. The expanded clones of individual cells are continuously grown from their initial culture in May 1994. The differentiation of a clone from the human cortex has been analyzed in greater detail. The cells of this clone were plated on 24-well plates and expanded to the permissive oncogene temperature of 33 ° C for 2-3 days. The cells are then cultured at the non-permissive temperature of 39 ° C in the presence of a variety of agents and other cell types. These include nerve growth factor, ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor, glial cell-derived neurotrophic factor (GDNF), FGF, epidermal growth factor, platelet-derived growth factor, retinoic acid, and serums. different After 14 days under these conditions, the cells were fixed and immunohistochemically screened with a battery of cell-type specific antibodies such as neurofilament, neuron-specific enolase, glial fibrillary acidic protein, myelin-oligodendrocyte glycoprotein, nestin, vimentin and CDllb (labeled microglia). A neural phenotype was found that was morphologically and immunologically apparent after the precursor clone had been incubated in the presence of glial derived neurotrophic factors (see Figure 14). On the contrary, incubation with CNTF leads to a phenotype similar to the astrocyte. Interestingly, although the precursor cells are homogeneous, which have been expanded from a single cell, it appears that they can produce at least two different phenotypes under each set of conditions. This classification of multipotentiality is on the contrary to the multipotentiality observed with the raphe clones, where a set of conditions results in a homogeneous neuronal phenotype that is expressed (Stringer et al, 1994). Supposedly, the multiple phenotypes of the cortical clone reflect the initial stage of cortical development in which the precursors were isolated, when cells have a less restricted commitment to individual differentiation pathways. Nestin and vimentin were also identified from this clone. In Figure 14A the homogeneous precursor cells were incubated at the non-permissive temperature of the oncogene (39 ° C) in the presence of the glial-derived neurotrophic factor and allowed to differentiate. Some of the precursors (with arrows) developed a bright phase morphology and presented neuron-specific enolase immunoreactivity, a characteristic marker of neurons. Greater magnification is shown in Figure 14B. Other cells, however, adopted a different phenotype. The incubation with CNTF leads on the contrary to a phenotype similar to the astrocyte. The same precursors on the contrary were incubated with the ciliary neurotrophic factor (please see Figure 14C). Now they did not present any immunopositivity-NSE. However, the meshwork of immunoreactive fiber-GFAP (a marker for astrocytes) becomes prominent, most cells are positive. Interestingly, it has therefore been shown that undifferentiated cells, or precursors can be used to produce human cell lines with considerable success and that the nature of the differentiated phenotype of such cell lines is determined by the nature of the precursor cell, and in some cases, the nature of the differentiation agent to which it is exposed from the human cell line.

References 1. Stampfer MR, Bartley JC 1985. Induction of transformation and continuous cell-lines from normal mammary epithelial cells after exposure to benzo [a] pyrene. Proc Nati Acad Sci USA 82: 2394-2398. 2. Yoakum GH, Lechner JF, Gabrielson E, Korba BE, Malan-Shibley L, ill and JC, Valerio MG, Shamsuddin AM, Trump EF, Harris CC 1985. Transformation of human bronchial epithelial cells transfected by Harvcy'ras oncogene. Science 227: 1174-1179. 3. Amsterdam A. Zauberman A, Meir G, Pinhasi-Kimhi O, Suh BS, Oren M 1988. Contransformation of granulosa cells with simian virus 40 and Ha-RAS oncogene generates stable lines capable of inducing steroiogenesis. Proc Nati Acad Sci USA 85: 7582-7586. 4. Vitry F, Camier M, Czernichow P, Benda P, Cohen P, Tixier-Vidal A 1974. Establishment of a clone of mouse hypothalamic neurosecretory cells synthesising neurophysin and vasopressin. Proc Nati Acad Sci USA 71: 3575-3579.

. Isom HC, Tevethia J, Taylor JM 1980. Transformation of isolated mouse hepatocytes with simian virus 40. J Cell Biol 85: 651-659. 6. Rhim JS, Jay G, Arnstein P, Price FM, Sanford KK, Aaronson SA 1985. Neoplastic transformation of human epiermal keratinocytes by AD12-SV40 and Kirsten sarcoma virases. Science 227: 1250-1252. 7. Stringer B.M. J., et al., Raphne neural cell i mortalized with a temperature-sensitive oncogene, Developmental Brain Research 79: 267-274, 1974.

Claims (27)

1. A method for producing human cell lines, characterized in that it comprises: a) immortalizing an undifferentiated, human precursor cell of a given tissue type using an immortalizing agent, which includes or has a control means associated with it, what the activation of the control means ends the immortalization and allows the differentiation of the cell without differentiating or precursor, b) cultivating the immortalized cell to produce a homogenous population of human cells, c) activating the control means to complete the immortalization and activate differentiation; and d) allow the differentiation of the cells to produce completely differentiated cells of the given tissue type.

2. The method according to claim 1, characterized in that the immortalization agent is an immortalization gene.

3. The method according to claim 2, characterized in that the gene is a viral oncogene.

4. The method according to claim 1, 2 or 3, characterized in that the immortalizing agent is a construct.

5. The method in accordance with the claim 4, characterized in that the construct is a retroviral construct.

6. The method according to claims 1 to 5, characterized in that the control means is sensitive to environmental conditions.

7. The method according to any preceding claim, characterized in that the immortalization agent and the control means are integrated.

8. The method according to claim 7, characterized in that the integrated immortalization agent and the control means comprise a temperature sensitive entity.

9. The method according to claim 8, characterized in that the entity is an oncogene.

10. The method according to claim 8 or 9, characterized in that the immortalizing agent is the SV40T antigen.

11. The method in accordance with the claim 1, characterized in that the immortalization agent is a chemical medium.

12. The method according to claim 1, characterized in that the immortalization agent is a physical medium.

13. The method according to any preceding claim, characterized in that the process that allows the differentiation of the cells involved the exposure of the cells to a differentiation agent.

1 . The method according to claim 13, characterized in that the agent is Vitamin D3.

15. The method according to claim 13 or 14, characterized in that the agent is Vitamin K, either alone or in combination with Vitamin D3.

16. The method according to claim 13, characterized in that the agent is dexamethasone.

17. The method according to claim 13, characterized in that the agent is rabbit serum or an extract thereof.

18. The method according to any preceding claim, characterized in that the method further involves immortalization of an undifferentiated or precursor cell, human with an immortalizing agent and also a security means, which allows the selective incapacity and / or destruction of the line cell phone.

19. The method in accordance with the claim 18, characterized in that the method involves the transfection of the cell line with a gene, which in the presence of certain agents, produces an effect and / or cytotoxic product.

20. The method in accordance with the claim 19, characterized in that the gene is viral thymidine kinase.

21. The method according to claim 19, characterized in that the gene is cytosine deaminase.

22. The method according to claim 18, characterized in that the transcription of the immortalization agent also results in the transcription of the security means.

23. Cells or cell lines produced according to the method of the invention.

24. Cells or cell lines according to claim 23, characterized in that they comprise at least one homogenous population of immortalized cells provided with means for terminating immortalization, such that a homogeneous population of differentiated cells is provided.

25. Cells or cell lines according to claim 23 or 24, characterized in that they comprise at least one security means according to the invention.

26. Cells or cell lines according to claims 22 to 25, characterized in that the cell or cell lines are of human origin.

27. The use of immature, undifferentiated or precursor cells to produce terminally differentiated human cell lines, which express tissue-specific functions.