MXPA97007195A - Celu culture method - Google Patents

Abstract

A method for selectively culturing a pre-selected subpopulation of cells from a heterogeneous cell population in vitro, comprising the steps of: (a) introducing a selectable marker (eg, a positive and / or negative selectable marker) into the heterogeneous cell population, where the marker is subject to differential activity / expression in the preselected sub-population, and (b) selectively culturing the pre-selected sub-population on the bases of differential activity / expression with respect to the selectable marker.

Description

"CELLULAR CULTURE METHOD" The present invention relates to cell culture methods and, in particular, to cell culture methods for the production of an essentially homogeneous population of cells (e.g., neuronal cells) in vitro. The invention also relates to neural cells (e.g., human neural cells) that have a selectable marker introduced into them (e.g., a positive and / or negative selectable marker). The central nervous system is currently subject to intense research, but its enormous complexity at the cellular level has been opposed to a complete understanding of its function. Although increasingly selective methods have been developed for specific irradiation of their ex vivo neural cell populations (such as immunostaining, in situ hybridization, histochemistry, etc.) the separation and purification of these subpopulations as living cells presents serious difficulties. Several approaches have been adapted to address this problem. For example, techniques such as differential centrifugation have been used for enrichment for specific neural cell phenotypes. Other methods have been based on the use of specific substrates of the fluorescent cell type to irradiate cell subplopulations, or the use of fluorescent tracer dye materials injected into a specific termination area of a given set of neurons (Schaffner et al. J. Neuroscience 7: 3088). In each case, the separation of irradiated cells from non-irradiated cells is by fluorescence activated cell sorting (FACS). Another method is based on the identification of markers bound to the extracellular membrane specific for a specific cell type (Urakami &Chiu (1990) J. Nuroscience 10: 620). Here, the separation in this example is achieved by panning the population of cells mixed in a layer of the adherent antibody to produce complexes of the cell antibody from which the cells of interest can be subsequently dissociated for further study. According to another known method, the progeny cells giving rise to specific cell populations are immortalized by oncogenic transduction or by subculture of spontaneous cell overgrowth. Possibly with the exception of the immortalization technique of the precursor cell, most of these methods have not acquired extensive application. This is partially due to the enormous heterogeneity of phenotypes within the central nervous system, in such a way that the markers and separation techniques used have to be extremely specific to avoid cross-reaction with undesired cell types. Secondly, the degree of purification required to obtain homogenity is often two or more orders of magnitude: this enrichment is too large for FACS or panning, the result being a degree of contamination. Third, the availability of extracellular markers sufficiently abundant but nevertheless, suitably specific that are also common for groups of the neural cell better characterized (e.g., inertgic dopa cells or glutamatergic cells) is limited. Likewise, most of these enrichment techniques require relatively immature neural cells to prevent their destruction during the procedure. Finally, these considerations are even more restrictive for human neural cells due to the difficulties in obtaining appropriate amounts of fresh human brain tissue. The present invention provides a method for selectively culturing a preselected sub-population of cells from a heterogeneous population in vitro, comprising the steps of: (a) introducing a selectable marker (e.g., a positive and / or negative selectable marker) ) in the heterologous cell population whose marker is subject to differential expression / activity in the preselected sub-population; and (b) selectively culturing the preselected sub-population on the basis of the expression / differential activity therein of the selectable marker. The preselected sub-population of cells can be an essentially homogeneous population of cells of a specific cell type or cell class. For example, the preselected sub-population may be a specific class of neural cells. In the case of neural cells, the preselected population can be selected on the basis of the characteristics of the transmitter (e.g., neurons containing dopamine or acetylcholine can be selectively cultured according to the method of the invention.) The selectable marker will not need introduced into each cell constituting the heterogeneous cell population: for most purposes, it is sufficient if the significant proportion of the cells receives the selectable marker, however, preferably, the selectable marker (s) is introduced into a a large proportion (for example, essentially in its entirety) of the heterogeneous cell population As explained herein, the method of the invention finds specific application in the selective culture of specific classes of essentially normal neural cells. The method of the present invention is of general application and can be used for culti selectively select other sub-populations of cells. It is known in the art that mammalian neural cells can be translated into a heterologous genetic material. There are many methods for transducing eukaryotic cells and other cells, but the characteristics of neural cells are such that natural methods of transfection are currently the most useful (Miller (1992) Nature 357: 455). Therefore, transduction with a virally packaged genetic material, for example, is not only more efficient, but results in lower neural cell mortality during the actual process than does, for example, the precipitation of calcium phosphate, electroporation, microprojectile bombing or microinjection. Transduction of mammalian neural cells from the central nervous system both in vivo (Culver et al. (1992) Sciece 256: 155) and in vitro (Stringer &Foster (1994) Brain Res 79: 267) has already been described. The genetic material that can be introduced into living cells can include both positive and negative selectable markers. A positive selectable marker is one that allows the survival of the transduced cell under conditions that would kill cells that do not express the selectable phenotype. A negative selectable marker confers sensitivity to cells that express it in such a way that they are destroyed under conditions that are relatively harmless to other cells. A wide variety of selectable markers are available. Genes that are extensively applied as positive selectable markers include the bacterial neomycin phosphotransferase (neo; Colbere-Gerapin et al. (1981) J. Mol. Biol. 150: 1), hygromycin phosphotransferase (hph; Santerre et al. (1984)). Gene 30: 147) and phosphoribosyl transferase from xanthinguanin (ftp; Mulligan &Berg (1981) Proc. Nati. Acad. Sci. USA 78: 2072). The herpes simplex virus type 1 thymidime kinase (HSV-1 TK, Wigler et al. (1977) Cell 11: 2233), adenine phosphoribosyl transferase (APRT: Wigler et al. (1979)) are also used as positive selection markers. Proc. Nati, Acad. Sci. USA 76: 1373) and hypoxanthine phosphoribosyl transferase (HPRT; Jolly et al. (193; 83) Proc. Nati, Acad. Sci. USA 80: 477). These latter markers should be used in cells that have a specific mutant genotype (for example, one that leads to a deficiency in the gene product on which the selection is based). Preferred negative selectable markers include gene coding products involved in programmed cell death (apoptosis), for example, the gene for p53. These negative selectable markers can be activated by inducing the expression of the gene in question (for example, by using a promoter that responds to tetracycline, as described below). The use of products that encode the genes involved in apoptosis have the advantage that the transient expression (in many cases of 30 minutes or less) of the gene may be sufficient to force the cell to die, allowing a reliable negative selection and very strict Some of the aforementioned genes also confer negative as well as positive selectable phenotypes. They include the genes HSV-1, APRT, APRT and gpt. These genes encode enzymes that can catalyze the conversion of certain nucleoside or purine analogs into cytotoxic intermediates. For example, ganciclovir nucleoside analogue (GCV) is a good substrate for the thymidine kinase HSV-1, but it is a deficient substrate for the natural thymidine kinase that is found in the mammalian cells. Consequently, GCV can be used for efficient negative selection of cells expressing the HSV-1 TK gene (St. Clair et al. (1987) Antimicrob Agents Chemotherap, 31: 844). The xantinguanin phosphoribosyl transfersas can be used for both positive and negative selection that was expressed in wild-type cells (Besnard et al. (1987) Mol.Cell. Bio. 7: 4139). Cytosine deaminase can also be used as a selective selection marker by converting harmless 5-fluorocytosine, cytotoxic 5-fluorouracil (Polak & amp;; Scholer (1976) Chemotherapy (Basel) 21: 113). Selectable markers are usually used in both prokaryotic and eukaryotic genetic engineering to allow recovery of the mixed population of cells that have undergone a rare genetic change. For example, they can be used in physical association with another gene encoding a product of interest to select cells that have adopted the other gene together with the selectable marker. As an example, the neo gene has been used to monitor genetically modified cells that are taken from patient samples after gene therapy has been carried out. It has also been proposed to use negative selectable markers as a safety device in gene therapy (Lupton et al. (1991) Mol Cell Cell Biol. 11: 3374). Many gene therapies involve the removal of somatic cells from the patient, the introduction into them of a therapeutic gene (the expression of which restores a biochemical lesion) followed by the introduction of the cells into the patient. Since the reintroduced genetically modified cells can finally prove to be detrimental to the patient's health (for example, if they are shown to be immunologically incompatible or malignant), a negative selectable marker can be introduced together with the therapeutic gene to allow (if necessary) ) the subsequent selective elimination of genetically modified cells. A number of vectors carrying positive or negative selectable markers have been produced and are readily available to those skilled in the art (for review, see Miller (1992) Nature 357: 455). Others can be easily assembled using normal gene cloning techniques.

The method of the invention may include the above induction of duplication in mixed populations of embryonic neural cells, using supplements in the culture medium such as epidermal growth factor or fibroblast growth factor, or by prior transfection with immortalization oncogenes to allow this duplication. Alternatively, non-expansion cell cultures can be used. Preferably, at an early stage after plaque culture, cells are transduced with a positive selectable marker and a negative selectable marker, both operably linked with an expression element. The expression element may be specific for a particular region of the central nervous system, a specific type of neural cell, or a specific sub-population of neurons. After the cultures have reached the required level of expansion, the cells can allow themselves, at least partially, to differentiate. The appropriate drug that can be applied in such a way that the non-transduced cells and those transduced cells without the specific active expression element are eliminated, while the cells transduced with the active element (which leads to the expression of the selectable markers) will be resistant. in downstream). By way of example only, the expression elements for use in the invention can be selected from: promoters and / or enhancers that are specifically active in: (i) dopaminergic, serotonergic, GABAergic, cholinergic or peptidergic neurons, or sub-populations of the same; (ii) Sch ann cells, oligodendrocytes, astrocytes, microglia and sub-populations thereof; (iii) stages of specific embryogenesis and (iv) other specific non-neural tissues. Particularly preferred for use in the present invention are the promoters responsive to tetracycline described in e.g., Furth et al., (1994) PNAS USA (Volume 91, pages 9302-9306). These promoters can be used as an element in a regulatory system that responds to tetracycline to effect temporal and / or spatial control of gene expression in vivo. Alternatively, they may be selected from promoters and / or enhancers that direct gene transcription for: (i) specific neurotransmitter receptors; (ii) ion channels; (iii) receivers involved in the disconnection of the ion channel; (iv) cytokines, growth factors and hormones; and (v) any substance that is produced and secreted specifically in a paracrine, autocrine or endocrine fashion. For examples, see Table 1. The invention provides a method for culturing human cells and other mammalian cells (e.g., neural cells) and selecting for a sub-population of cells based on the genetic material contained therein. , producing homogeneous cultures of a single cell type. These cultures can be delivered to a variety of uses including basic, electrophysiological, neurochemical and developmental experiments. In addition, purified neural cell populations will be useful in more clinically applied studies, such as an assessment of the feasibility of transplantation to alleviate the symptoms of a degenerative disease of the central nervous system and find application in different forms of therapy, prophylaxis and diagnosis. These diseases include: (i) Parkinson's disease or parkinsonism, the preselected sub-population of cells being dopamionergic neurons of the "substantia nigra"; (ii) Huntington's disease, the preselected sub-population of cells being neural cells of the stratum; (iii) Alzheimer's disease, the sub-population is preselected from cells that are neurons containing acetylcholine, serotonin and / or noradrenaline with neo and paleocortical; or (vi) multiple sclerosis, the preselected sub-population of cells that are oligodendrocytes of the brain.

TABLE I Promoter Type of tissue / cell Application Reference Tyrosine Catecholaminergic Alzheimer Stacho ick and others (1994) Hydroxylase Parkinson's neurons Mol Brain Res 22,309-19 TSH receptor Thyroid cells Hypothyroidism Ikuyama & Nakata (1994) Jap J Clin Med 52,962-8 BSF1 GABAergic neurons Epilepsy Motejlek and others (1994) J Biol Chem 269,15265-73 Human dopamine Neurons of Alzheimer's Hoyle et al. (1994) J ß-hydroxylase noradrenaline Nurosci 14,2455 -63 Thyroglobulin Thyroid cells Hypothyroidism Pigeon and others (1994) Biochem J 298,537-41 2-Neuroglycan cell receptor Diseases Ding et al. (1994) Molecular serotonin in neurodegenerative projection areas Brain Res 20,181-91 neonatal serotonergic CD4 expressing CD4 receptor AIDS Nakayama et al. (1993) T-lymphocytes Int. Imunol 5,187-24 Transplantation of Li Neurons Disease and Others (1993) Neuro-acetyl choline acetylcholine Motoneuron from chem Res 18,271-5 Human Alzheimer Example 1: Preparation of a homogeneous culture of human dopaminergic neurons The thymidine kinase (tk) gene of Herpes simplex virus (HSV) and the neo gene are functionally linked to a promoter that is active only in neurons containing dopamine, eg, those that control the expression of tyrosine hydroxylase. (see eg, as described by Harrington and others (1987) Nucí Acids, Res. 15: 2363). The construct is then cloned into the appropriate cloning site of a retroviral vector and used to transfect an amphotropic retroviral packaging cell line (e.g.,? -crip (for review see Molecular Virology: A Practical Approach (Editors AJ Davison &RM Elliott) IRL Press, 1993). The tissue is dried from embryonic human ventral mesencephalon (approximately 5 to 8 weeks gestation) and grown in the dissociated culture. The topaminergic precursor cells are induced to duplicate by application of a fibroblast growth factor (Mayer et al. (1993) Neuroscience 56: 389) epidermal growth factor (Reynolds &Weiss (1992) Science 2545: 1707) or by transduction oncogene (Stringer et al. (1994) Brain Res. 79: 267). Shortly after plating, the cultured cells are transduced with retrovirally packaged selectable markers, and the cultures are allowed to expand. When sufficient numbers of cells are produced, the cultures are incubated under conditions that lead to cessation of neuronal division. The cultures are then treated with geneticin to remove the non-translucent cells as well as the translucent cells that do not express the tirokin hydroxylase, but leaving neurons containing tyrosine hydroxylase transduced.

Example 2: Preparation of a homogenous culture of human oligodendrocytes.

The herpes simplex virus (HSV) thymidian kinase (tk) gene and the neo gene are operably linked in a promoter that is active only in oligodendrocytes, which controls the expression of galactocerebrosidase of oligodendrocyte-specific enzyme. The construct is packaged virally as in Example 1. A virus such as adenovirus could alternatively be used. The tissue of an embryonic or adult brain is dried and grown in a dissociated culture.

If necessary, duplication of cells is induced (for example, using cells from the HS2ts6 mice (Noble et al. (1991) WO 91/13150) and shortly after plating, the cells are transduced with gene coding for the selected and positive marker linked for example to the galactocerebrosidase promoter and to the negative selectable marker linked to a constitutively active promoter, such as cytomegalovirus The cell selection is obtained as in Example 1, yielding pure populations of oligodendrocytes.

Example 3: Preparation of a homogeneous culture of essentially normal human dorsal root ganglion cells expressing peptide related to the calcitonin gene.

The dorsal root ganglion (DRG) neurons can be grown in vivo using either embryonic, neonatal or adult tissue as a source or source material. The population of mixed cells will be grown for example, in a background layer of v.gr., neomycin-resisting non-neuronal cells prepared above to provide trophic support (Brenneman et al. (1987) J. Cell. Biol. 104: 1603 ). The DRG cells are transfected using adenoviral technology with a neo gene operably linked to the promoter for expression of peptide related to the calcitonin gene (CGRP).

After several days to allow the activation of the promoter and in this way the induction of the neo-expression, the DRG cells that do not actively express neo, will be destroyed by application of neomycin. The result will be a pure population of positive CGRP neurons differentiated from the human DRG, for in vitro use.

Example 4: Transduction of neural cells.

The primary cells of the human cortex, stratum, hypothalamus, mesencephalon, raphe complex, medulla oblongata and ventral horn of the fetal spine (at eight weeks gestation) were plated separately in gelatin / poly-L-lysine coated flasks. . The medium was a mixture of DMEM / Ham's F12 (1: 1) containing penicillin / streptomycin, L-glutamine (2mM) and a solution of modified material (Bottenstein and Sato, PNAS 76 (1979) 514-517; Romijn et al. , J. Neurophysiol, 40 (1981) 1132-1150) which contains the basic fibroblast growth factor (5ng / milliliter). Once the cells had adhered, the retroviral particles that comprise a building (tsA58) that incorporate a geneticin resistance marker (G418r) linked to an SV40T promoter, were added to the medium along with 0.8 microgram per liter of polybrene. After one hour, the culture medium was replaced by fresh medium. After five days, geneticin (0.4 microgram per milliliter) was added to the culture medium for an additional 10 days to eradicate the cone cells that had been incorporated into the retroviral vector. After 15 to 20 days, the clusters of the human neural precursor cells could be found from all the previously numbered areas that were capable of being duplicated in the medium containing FGF and which are also geneticin resistance. All exhibited a neuronal phenotype (for example, they were neuron-specific enolase-positive). Therefore, it is possible to transduce human neural cells stably from a variety of areas of the central nervous system, with gene constructs retrovirally packaged. Second, the construct may include a selection marker, such as geneticin resistance.

Claims (16)

R E I V I N D I C A C I O N E S:

1. A tissue graft implant comprising cells having inserted therein a positive selectable marker operably linked to a tissue or cell-specific expression element.

2. A process for producing an essentially pure sub-population of cells comprising the steps of: (a) providing a heterogeneous cell population in vitro; (b) transfecting, infecting (v.gr, with a retroviral vector) or transducing the heterogeneous cell population with a positive selectable marker operably linked to a tissue or cell-specific expression element to produce a heterogeneous or transfected population of cells transformed; (c) selectively culturing the cell sub-population of the heterogeneous cell population of step (b) on the basis of tissue-specific expression or cell of the positive selectable marker to produce an essentially pure sub-population of cells.

3. The tissue graft implant of claim 1, or the process of claim 2, wherein the cells further comprise a selectable negative marker (e.g., a gene coding product (s) involved in programmed cell death). or apoptosis, e.g., the p53 gene).

The invention according to claim 3, wherein the negative selectable marker is not subject to expression / differential activity (e.g., being constitutively expressed).

5. The invention of any of the preceding claims, wherein the cells are neural cells (e.g., neurons).

6. The invention according to claim 5, wherein the cells are human neural cells, thyroid cells, CD4 expressing the T-lymphocytes or cells of a specific non-neural tissue.

The invention according to claim 5 or claim 6, wherein the cells are neural cells that are selected from any of the dopaminergic, glutaminergic, catecholaminergic cells, GABAergic neurons, noradrenaline neurons, neuroglia cells of serotonergic projection areas , acetylcholine neurons or human dorsal root ganglion cells that express the peptide related to the calcitonin gene.

8. The invention of any of the preceding claims, wherein the tissue or cell-specific expression element is selected from: (A) promoters and / or enhancers that are specifically active in: (i) dopaminergic, serotonergic, GABAergic, neurons, cholinergic or peptidergic, and sub-populations thereof; (ii) Schwann cells, oligodendrocytes, astrocytes, microglia and sub-populations thereof; (iii) specific stages of embryogenesis; (iv) specific non-neural tissue; (v) the endocrine glands, lungs, muscles, gonads, intestines, skeletal tissue or part or parts thereof; (vi) epithelial, fibroblast, fat, mustocyte, mesenchymal or parenchymal cells; (vii) components of the blood system (e.g., T-lymphocytes, B-lymphocytes and macrophages), or (B) promoters / enhancers that direct gene transcription for: (i) neurotransmitter-specific receptors; (ii) ion channels; (iii) receivers involved in the disconnection of the ion channel; and (iv) cytokines, growth factors and hormones; (any substance that is specifically produced and secreted in a paracrine, autocrine, or endocrine manner.) 9.

The invention according to claim 8 (B), wherein the promoter is selected therefrom for tirocin hydroxylase, the TSH receptor. , BSF1, human dopamine β-hydroxylase, thyroglobulin, serotonin 2-receptor, CD4 receptor and human choline acetyl transferase 10.

The invention according to any of the preceding claims, wherein the positive selectable marker is selected from phosphotransferase. of neomycin, hygromycin phosphotransferase, xantinguanidine phosphoribosyl transferase, Herpes simplex virus type I thymidine kinase, adenosine phosphoribosyl transferase and hypoxanthine phosphoribosyl transferase.

The process of any of claims 2 to 10, wherein the population of heterogeneous cells is a primary cell culture, eg, a human neural primary (e.g., fetal) cell culture, for example comprising precursor / hemocytoblast neural cells.

12. Cells obtainable by the process of any of claims 2 to 11.

13. A tissue graft implant of any of claims 1 and 3 to 10 or cells of claim 12, for use in therapy, prophylaxis or diagnosis. .

14. The tissue graft implant of any of claims 1 and 3 to 10 or cells of claim 12 for the manufacture of a medicament or for use in therapy, prophylaxis, or diagnostics.

15. The invention according to claim 13 or claim 14, wherein the therapy is transplantation therapy.

16. The invention according to claim 15, wherein the transplantation therapy is for the treatment of: (i) Parkinson's disease and / or parkinsonism, the cells being dopaminergic neurons of the "subtantia nigra): (ii) korea of Huntington, being the cells, neural cells of the stratum; (iii) Alzheimer's disease, the cells being neurons that contain acetylcholine, serotonin and / or noradrenaline associated with paleo- and neocortex; (iv) lateral amitrópica sclerosis, being the motor neuron cells of the spinal cord; (v) multiple sclerosis, the cells being oligodendrocytes of the brain.