MXPA97003493A - In vitro induction of dopaminergi cells - Google Patents

In vitro induction of dopaminergi cells

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Publication number
MXPA97003493A
MXPA97003493A MXPA/A/1997/003493A MX9703493A MXPA97003493A MX PA97003493 A MXPA97003493 A MX PA97003493A MX 9703493 A MX9703493 A MX 9703493A MX PA97003493 A MXPA97003493 A MX PA97003493A
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cells
dopaminergic
nerve
growth factor
tissue
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MXPA/A/1997/003493A
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Spanish (es)
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MX9703493A (en
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Weiss Samuel
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Neurospheres Holdings Ltd
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Priority claimed from US08/482,079 external-priority patent/US5981165A/en
Application filed by Neurospheres Holdings Ltd filed Critical Neurospheres Holdings Ltd
Publication of MX9703493A publication Critical patent/MX9703493A/en
Publication of MXPA97003493A publication Critical patent/MXPA97003493A/en

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Abstract

The expression of hydroxylase in nerve cells in vitro can be included by contacting nerve cells with a culture medium comprising at least one member of the fibroblast growth factor family in combination with a conditioning medium or molecules of the beta family of the transformation growth factor. The method induces nerve cells contained from normally non-dopaminergic nerve tissue, for example the striatum and the cortex to express tyrosine hydroxylase. Cells can be used to treat neurological conditions in patients that require dopaminergic cells

Description

Induction in Vi tro of Dopaminergic Cells Priority Requests This application is a continuation of U.S. Patent Application Number 08 / 482,079, filed June 7, 1995, which is a continuation of U.S. Patent Application Number 08 / 339,090, filed on 14 BACKGROUND OF THE INVENTION It has been established that phenotypic expression and survival of differentiating nerve cells and the survival and metabolism of established nerve cells is driven by a variety of extracellular signals. The neighboring cells and processes surrounding the nerve cells play an important role in the regulation of cell differentiation, metabolic function and survival, largely through the release of growth factors and other regulatory factors. Many neurological diseases, including Parkinson's disease, are the result of the degeneration of specific groups of nerve cells. In the case of the evil of Parkinson, the degeneration of a group of dopamine-containing cells, which connect the ventral segment and the substantia nigra (located in the ventral portion of the mesencephalon, or middle brain) with the striatum, have been implicated in the etiology of the condition.
In order to understand the factors that result, or could prevent, the degeneration of these dopaminergic pathways, we have extensively studied tissue obtained from the mesencephalon region. The embryonic neurons that contain dopamine, derived from the mesencephalic tissue, are difficult to grow since the dopaminergic neurons do not survive well in culture. However, these crops show better survival or modified biochemical activity, or both, when grown in a conditioned medium or when treated with growth factors. Embryonic tissue taken from the mesencephalon has been cultured in conditioned culture media (CM) derived from the glial B49 cell line of the rat, the glial cell line of the R 33 nerve retina and the JS Sch annoma cell line [Engle, J., et al., J. Neurosci. Res., 30: 359-371, (1991)]. In all three cases, CM significantly increased the survival of cultured dopaminergic neurons. The improved survival of the dopaminergic neurons was not due to the proliferation of dopaminergic neurons but was attributed to the effects of the CMs on the existing gual cells derived from the mesencephalic culture and the resulting interactions between the gual cells and the dopaminergic neurons, more than to a direct effect on dopaminergic neurons. Cultivation of mesencephalic embryonic tissue in MCs prepared with mesencephalic astrocytes, or culturing the tissue together in a layer of astrocytes derived from the mesencephalon, rescue dopaminergic neurons from death induced by serum deprivation. Astrocytes or CMs prepared from astrocytes taken from the striatum and the cerebral cortex had significantly weaker protective effects [Takashi a et al., J. Neurosci. , 14 (8): 4769-4779, (1994)]. In one report, CM derived from cortical astrocytes had no effect on the survival or proliferation of dopaminergic cells but altered the biochemistry of this cell population, resulting in a small increase in their dopamine uptake. [Gaul and Lübbert, Proc. R. Soc. Lond. B, 249: 57-63, (1992)]. It is believed that growth factors, many of which are present in CMs of nervous tissue, are responsible for regulating the survival and metabolism of the dopaminergic neuron, either directly, or through its effects on adjacent cells. It has been reported that growth factors that have a direct effect on the survival of the dopaminergic neuron include interleukin-6 (IL-6) [Hama et al., Neurosci. , 40 (2): 445-452 (1991)], the neurotrophic factor derived from the brain (BDNF) [Hyman et al., Nature, 350: 230-232, (1991)], the basic fibroblast growth factor (FGF-2, formally referred to as bFGF) [Dal Toso et al., J. Neurosci. , 8 (3): 773-745 (1988); Ferrari et al., Dev. Biol. 133: 140-147 (1989); and the neurotrophic factor derived from the glial cell line [GDNF], secreted by the rat B49 cell line [Lin, L.G., et al., Science, 260: 1130-1132 (1993)], all of which specifically improve the survival of dopaminergic neurons in cultures dissociated from the rat or mouse embryonic mesencephalon without increasing the number of neurons or gual cells. GDNF dramatically increases the morphological differentiation of dopaminergic neurons, resulting in greater external growth of neurites and larger cell body size. It has been reported that some growth factors, for example nerve growth factor (NFG) (Hatanaka and Tsuki (1986), Dev. Brains Res. 30: 47-56), platelet derived growth factor (PDGF) and Interleukin 1 (IL-1) (Engele &Bohn (1991), Neurosci 11 (10): 3070-3078); Mayer, E. (1993) Dev. Brain Res. 72: 253-258) and nerve growth factor (NGF) (Engle &Bohn, ibid) support the survival of the dopaminergic cell in embryonic mesencephalon tissue through a mechanism mediated by the glial cell. In vivo studies indicate that damage caused by mechanical or chemical damage to the dopaminergic pathways between the mesencephalon and striatum can be significantly reduced by treatment with epidermal growth factor (EGF) [Pazzoli et al., Movement Disorders 6 (4) 281-287, (1991)] and BDNF (Hyman et al., Supra). In vitro treatment with cyclic AMP, but not FGF-2 or NGF, increased the survival of mesencephalic dopaminergic neurons cultured in response to the chemically induced degeneration produced by l-methyl-4-phenylpyridinium.
(MPP +) [Hyman et al., Supra; Hartikka and collaborators, J. Neurosci. Res. , 32: 190-201, (1992)]. Although the use of the CMs of astrocytes stimulated by FGF-2 improves the absorption of dopamine, the use of these CMs did not have a protective effect when the neurons selected chemically using MPP + (Gaul and Lübbert, supra). Many of the cells obtained from embryonic tissue that normally give rise to dopaminergic neurons (ie, normally dopaminergic tissue), for example the mesencephalon and the olfactory bulb, will eventually differentiate into dopaminergic neurons under primary culture conditions. However, a larger number of cells, in tissue obtained from areas of the brain normally dopaminergic, can be induced to differentiate dopaminergic cells by co-cultivation with feeder cell layers derived from nerve tissue. The dopaminergic neurons of the olfactory bulb show an increase in the number of five times when the embryonic olfactory bulb neurons are cultured together with epithelial olfactory neurons. It is believed that a soluble factor, the gene-related calcitonin peptide (CGRP), which is present in epithelial cells, is responsible for the induction of additional dopaminergic neurons in the olfactory bulb [Denis-Donini, Nature, 339: 701- 703, (1989)]. The co-culture of neurostriatal tissue and rat substantia blacke from one to three weeks in glial cell feeder layers obtained from the region of the substantia nigra induces the expression of dopaminergic cells as indicated by the immunoreactivity of tyrosine hydroxylase (TH + ) in the tissue grown in both areas. However, when the same tissues were cultured together with normal cells of the neurostriated tissue, only dopaminergic cells were observed in the tissue of the substantia nigra but not in the neurostriated tissue [Beyer et al., Neurosci Lett. , 128: 1-3, (1991)]. The mechanism underlying the occurrence of TH-IR in neurostriatal tissue (an area that does not contain dopaminergic cells in adults) was not determined. However, it may have been due to the induction of TH + cells in the cell feeder layer of the substance in response to the presence of striated tissue; to the induction of the dopaminergic properties of striated cells; or to the promotion of the survival of dopaminergic cells in the striated tissue, it is reported that said cells occur transiently during development in the striated tissue (Tashiro et al. (1989), Neurosci Lett., 97: 6-10) and in the cortex. (Satoh and Suzuki (1990), Dev. Brain Res. 53: 1-5). Small numbers of TH + cells (140 TH + cells / cm 2) have been induced in rat embryonic cortex tissue using a combination of BDNF and dopamine, in a culture medium containing 10% fetal calf serum. Less TH + cells were seen when BDNF, or dopamine were used alone (Zhou et al. (1994), Dev. Brain Res. 81: 318-324). A few cells of mouse embryonic striatal tissue can be induced to express TH + by being coated with FGF-1 and an improved result can be obtained using a combination of FGF-1 and an unidentified fraction > 10 kD obtained from muscle tissue (Du et al. (1994) J. Neurosci 14 (12): 7688-7694).
The degeneration of the dopaminergic neurons of the substantia nigra that characterizes Parkinson's disease is usually treated using pharmacological interventions to increase the natural supply of endopamine increasingly inferior to striated tissue. However, there are problems related to drug treatment, for example the emergence of drug tolerance and possible side effects. Nerve implants, using embryonic black substance tissue, have shown some potential to relieve experimentally induced Parkinsonism in rodents and primates and some human Parkinson's patients. However, implant survival is poor and only limited amounts of embryonic dopamine tissue are available. On average, 4 to 10 fresh human embryos are required to obtain enough dopaminergic neurons for a single human transplant (Winder et al., N Engl J Med 327: 1556-1563 (1992)). The preferred treatment would involve the prevention of, or a reduction in the amount of degeneration that occurs. Once the damage has occurred, it would be preferable to replace the lost cells by implanting new dopaminergic neurons using cells derived from nerve cells that have proliferated in culture preferably from a non-tumor cell line, or from cells that have not been intentionally immortalized in order to induce proliferation and, more preferably, would be derived from a patient's own nerve tissue. Alternatively, a less invasive treatment would be to participate in the in vivo manipulation of the patient's own population of nerve cells in order to replace the function of damaged dopaminergic neurons. The prior art suggests that cultures of dopaminergic cells can be obtained through the use of glial feeder layers, or the application of certain growth factors or conditioned media to mesencephalon tissue and other dopaminergic tissues. These treatments can induce differentiation, increase survival, or increase the metabolism of cells of normally dopaminergic tissue that has been cultured in vi tro. However, culture methods for inducing cells from other non-dopaminergic regions to differentiate into dopaminergic cells are limited. It has been demonstrated that a feeder layer of cells from regions such as the substantia nigra and the olfactory bulb (areas that normally contain a relatively high population of dopaminergic cells) can be used to induce the appearance of dopaminergic cells in certain tissues of the central nervous system. embryonic (CNS), there is no evidence that non-dopaminergic nerve tissue cells could be used as feeder layers in the tissue culture designed to induce the appearance of dopamine in tissues, for example striated tissue, that do not normally contain dopamine. certain purposes, especially transplantation and certain drug testing procedures, it would be advantageous to use fully defined culture conditions to induce differentiation of dopaminergic cells.It would be particularly advantageous if the cells were obtained from both dopaminergic and non-dopaminergic nerve tissue sources, au thus maximizing the number of dopaminergic cells that could be generated from a single embryo. There is a need in the art for a reliable method to induce nerve cells, derived from all regions of the brain, from the tissue obtained from animals of all ages, to differentiate into dopaminergic cells in the presence or absence of a feeder layer substrate. . In particular, it would be advantageous to induce the expression of dopamine in cells derived from regions that do not normally contain dopaminergic cell bodies, for example striated tissue, but which require dopamine to function normally.
Recently, it has been shown that multipotent nerve germ cells, obtained from embryonic and adult tissue, can be proliferated in vitro to generate large numbers of germline progenies, which, under the right conditions, can differentiate into neurons and glia (applications PCT number WO 93/01275, WO 94/16718, WO 94/10292, and WO 94/09119). It would be advantageous to generate dopaminergic cells of the proliferated progeny of multipotent nerve germ cells, derived from any area of the CNS. Accordingly, it is an object of this invention to provide a method for inducing large numbers of nerve cells obtained from the normally non-dopaminergic tissue to differentiate them, in vi tro, in dopaminergic cells in order to provide a reliable source of dopaminergic cells for various applications, such as, transplants to patients with dopamine deficiencies and for drug testing procedures. It is a further object of the invention to provide a method for inducing proliferated, undifferentiated progeny of multipotent nerve germ cells, derived from any area of the CNS that is known to contain said cells, to differentiate them into dopaminergic cells, in order to provide unlimited amounts. of dopaminergic cells for transplantation, drug testing and other purposes. Additionally, it is an object of the invention to provide tissue culture methods that use fully defined culture conditions, and therefore, do not require the presence of a cell feeder layer, conditioned medium, or serum, to increase the number of dopaminergic cells obtained from a single embryonic brain. Such cells would have use in specific applications for example transplants to patients with dopamine deficiencies and for certain drug testing procedures. These and other objects and features of the invention will be apparent to those skilled in the art from the following detailed description and the appended claims. It is not believed that any of the above references disclose the present invention as claimed and is not assumed as a prior art. References are offered in order to provide background information. Summary of the Invention A method for inducing the expression of tyrosine hydroxylase in nerve cells in vivo is presented. The method comprises contacting nerve cells with a culture medium comprising at least one member of the fibroblast growth factor family and at least one additive selected from a group consisting of conditioned medium and molecules of the beta family. of transformation growth factor. The method induces nerve cells obtained from normally non-dopaminergic nerve tissue, eg, striated tissue and cortex, to express tyrosine hydroxylase. A method for treating a neurological disease in a patient requiring dopaminergic cells is also presented. The method comprises contacting nerve cells with a culture medium comprising at least one member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family to produce differentiating dopaminergic cells. and differentiated and transplant the dopaminergic cells to the patient. Description of the Drawings FIGURE 1. Divisioning subependymal cells, which were in the subventricular zone of an adult mouse, were labeled with repeated injections of BrdU for a period of 24 hours. Within 30 minutes after the last injection, the mice were sacrificed and their brains removed. The subentricular zones were removed and the dissociated cells were cultured on poly-L-ornithine coated coating strips using the complete medium with or without conditioned media derived from the rat glial cell line B49 and FGF-2 (20 ng / ml) . After three days of plaque culture, the cells were fixed and processed for dual-brand indirect immunocytochemistry for TH (Eugene Tech, polyclonic 1: 1000) and BrdU (Amersham, monoclonal, 1: 100). Cells labeled with BrdU (dividing subependymal cells) that were also immunoreactive to TH were seen only in the experiments in which conditioning medium and growth factor were used. (A) A single BrdU immunoreactive cell (arrow) is TH immunoreactive (B), arrow) suggests that adult proliferating subependymal cells can be induced to express TH in the presence of conditioned medium and growth factor. FIGURE 2. Single-generated, non-dissociated, six-day-old neurospheres were grown in glass-coated strips coated with poly-L-ornithine in a DMEM / F12 hormone-mixing medium with conditioned media derived from the line of rat glomerular cell B49 and FGF-2 (20 ng / ml). Immunocytochemical analysis 24 hours later revealed the presence of TH + cells. (A) Phase contrast micrograph of a neurosphere 24 hours after plaque culture. (B) The same sphere of A, processed for TH + immunohistochemistry, reveals the presence of at least one TH + cell, with nerve morphology. FIGURE 3. Single, non-dissociated, second-pass neurospheres of six days were labeled with BrdU and plated in a confluent bed of astrocytes derived from striated tissue. 24 hours after plating, cells were processed for dual-label indirect immunocytochemistry for TH (Eugene Tech, polyclonic 1: 1000) and BrdU (Amersham, monoclonal, 1: 100). (A) A BrdU immunoreactive cell (arrow) is (B) TH immunoreactive (arrow). (C) TH immunoreactive cells (arrow) are also immunoreactive to MAP-2 (D), demonstrating other nerve characteristics, in addition to morphology. Detailed Description of the Invention Induction In Vitro of Dopaminergic Cells Derived from Primary Cells Obtained from Nerve Tissues Normally Not Dopaminergic The term "dopaminergic nerve tissue" refers to the tissue of regions of the CNS that are known, in the mature state, to contain significant amounts of dopaminergic cell bodies. Dopaminergic cells are nerve cells whose presence in nervous tissue is determined by the presence of tyrosine hydroxylated (TH) or the presence of dopamine decarboxylase or the absence of dopamine betahydroxylase, or all of these things, within the cells, techniques of polymerase hip reaction, or antibodies directed against dopamine. Tyrosine hydroxylase is the enzyme that limits the coefficient of the biochemical pathway with leads to the production of dopamine and is commonly used in the art as a marker of dopaminergic neurons. The dopaminergic nervous tissue is found in regions of the retina, the olfactory bulb, the hypothalamus or, the dorsal motor nucleus, the solitary fascicle of the nucleus, the gray matter that surrounds the silvio canal, the ventral segment and the substantia nigra. The term "normally non-dopaminergic nerve tissue", as used herein, refers to the tissue of the developed CNS regions that are not dopaminergic nerve tissue. Using the methods presented herein, primary cells derived from dissociated nerve tissue are induced to express tyrosine hydroxylase. The term "primary nerve cell" refers to a cell obtained from nervous tissue that has not been passed in vi tro (to a secondary culture). Nerve cell cultures are prepared by removing tissue from an animal using a sterile procedure, dissociating the tissue to generate a suspension of primary cells, and placing the cells in any known medium to support the survival of the cells. The primary cells are exposed to a culture medium composed of growth factors that induce the production of dopamine in cells obtained from normally non-dopaminergic nerve tissue. The term "growth factor" refers to a biological factor (i.e., a functional biologically active substance in the cells of the CNS) for example a protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, or other substance that have a growth, proliferative, differentiating or trophic effect on nerve cells, either individually, or in combination with other factors. A growth factor that induces dopamine production will bind to a receptor in a cell and induce the cell to start expressing, or increase its expression of messenger RNA (mRNA) for dopamine precursor molecules and enzymes involved in production of dopamine. A preferred growth factor is a member of the fibroblast growth factor family (for example FGF-1 or FGF-2), or equivalent growth factors that are capable of binding to the FGF receptors of the cells. Growth factors FGF and equivalents can be used alone or in combination with other growth factors. Growth factors are generally added at concentrations of about 1 to 100 ng / ml, usually from about 5 ng / ml to 60 ng / ml. An optimal concentration is in the range of FGF, with 20 ng / ml being the most preferred. When a member of the FGF family, heparin sulfate, is used, a glycosaininoglycan molecule can be added which facilitates binding of the FGF to its receptors to culture the medium at a concentration of 0.2 μg / ml - 20 μg / ml, preferably at concentration of 0.4 μg / ml - 4 μg / ml. The most preferred concentration is about 2 μg / ml. In a preferred embodiment, the culture medium comprises FGF in combination with a member of the transforming growth factor beta family (TGFβ). The TGFß family includes myelin basic proteins (BMP-2, BMP-4, BMP-5, BMP-6, BMP-7), activins A &; B, decapentaplégico (dpp), 60A, OP-2, dorsalina, GDFs (1, 3, and 9), nodal, MIS, Inhibin a, transforming growth beta factors (TGF-ßl, TGF-ß2, TGF-ß3 , TGF-ß5), and glial-derived neurotrophic factor (GDNF) (see Atrisano et al., (1994) J. Biochemica et Biophysica Acta vol 1222: 71-80). If TH + cells are to be used for transplantation purposes or certain drug testing procedures, it is preferable to use a fully defined culture medium that has the necessary nutrients and hormones to facilitate the survival of the cells. By "completely defined", it means that all the components of the medium are known. Numerous defined culture media are available. A preferred medium, referred to herein as "Complete Medium", comprises a 1: 1 mixture of Dulbeco's Modified Eagle Medium and F12 nutrient (GIBCO) plus 0.6% glucose, 2mM glutamine, 3mM sodium bicarbonate, 5mM buffer HEPES and a defined hormone mixture and salt mixture (Sigma, 10% by volume) including 25 μg / ml insulin, 100 μg / ml transferrin, 20 μM progesterone, 50 μM putrescine, and 30nM of selenium chloride. The culture medium can comprise conditioned media (CM), which are culture media that have been exposed to living cells and therefore contain substances, for example growth factors, that have been released by the cells. However, the addition of the CMs becomes undefined to the culture medium, and is therefore not preferred when the cells are to be used for transplantation purposes and certain drug testing procedures. CM can be obtained from any tissue that can be cultured and that induces the expression of dopamine in cells derived from normally non-dopaminergic nerve tissue or that attenuates the effects of a growth factor that induces dopamine, or both. Any amount of CM can be used (0-100%). In general, the culture medium will comprise approximately 25 to 100% CM. Preferred CMs are derived from the same cells.
Particularly preferred are the CMs derived from the glial B49 cell line of rat [Schubert et al., Nature, 249: 224, (1974)] and CMs derived from astrocytes. The primary cell cultures are plated, preferably at a density in the range of about 102 to 10 7 cells / ml, more preferably at a density of about 10 6 cells / ml. The cells can then be cultured in any suitable container, for example a tissue culture flask, beaker or petri dish. The container may or may not have a surface to which the cells can adhere. In cases where it is desirable for the cells to adhere, it is usually necessary to treat them with a substance that provides an ionically charged surface for example poly-D-lysine, poly-L-ornithine, Matrigel, laminin, fibronectin, and other known surfaces that induce cell adhesion. The cells are able to adhere to certain plastics. However, when using glass substrates, it may be desirable to treat their surface. When adhesion is not desired, glass substrates or certain untreated plastic tissue culture substrates can be used. A culture vessel treated with poly-L-ornithine provides a particularly suitable surface to which the cells can adhere. Alternatively, the cells can be grown together in a bed of feeder layer of any type or combination of cell type as opposed to a treated substrate. A feeder bed for other nerve cells, for example neurons, astrocytes or oligodendrocytes derived from any region of the CNS, is preferred for a joint culture. The cultures are kept close to the physiological conditions as possible. The pH should be between pH 6 and 8. Preferably approximately between pH 7.2 and 7.6, more preferably at a pH of about 7.4. Cells should be maintained at a temperature close to physiological levels, between 30 and 40 ° C, more preferably between about 32 to 38 ° C and more preferably between about 37.5 ° C. Cells should be maintained at approximately 5% C02, 95 % 02, and a humidity of 100%. However, the conditions of the crop may vary. For example, the addition of 1% fetal bovine serum (FBS) results in an increase in the number of TH + neurons detected after the cultures have been grown together in a glial cell feeder bed for 24 hours and also increases the amount of TH + cells detected when the cells are grown in an undefined culture medium in the absence of a feeder layer. A preferred embodiment is to cultivate primary cells in a culture medium containing the CMs derived from the glial cell line of rat B49 and FGF-2 or a combination of EGF and FGF-2. A preferred embodiment for cells grown in a defined medium for transplantation and other purposes is to cultivate the primary tissue directly on a substrate coated surface, for example coating strips coated with poly-L-ornithine, in a defined culture medium (e.g. Complete Medium) and a combination of FGF-2 with activin or BMP-2. The induction of dopaminergic cells is determined using any method capable of measuring the presence of dopamine, for example immunocytochemistry using antibodies directed against adopamine, or by measuring the biochemical activity of the dopaminergic cells by measuring the absorption of dopamine. The presence of precursor molecules that participate in the synthesis of adopamine can be measured. For example, immunocytochemical analysis to detect the presence of tyrosine hydroxylase, or assays, for example polymerase chain reaction and hybridization techniques in itself that detect mRNA for the enzymes involved in the synthesis of dopamine are useful tools for measuring the presence of dopaminergic cells. The identification of dopaminergic neurons is achieved using morphological analysis of neurons or by dual or triple labeling to show the presence of dopamine or dopamine precursor in addition to immunoreactivity for neuron-specific enolase (NSE), tau neurofilament proteins -1 and MAP-2 or for neu-N (a nervous nuclear antigen) or ß-tubulin and / or bromodeoxyuridine (BrdU), or all of these, which mark cells that are actively dividing. In Vitro Induction of Dopaminergic Cells Derived from the Progeny of Proliferated Multipotent Nervous Cells in Vitro from Nervous Tissue of Embryonic and Adult Mammals. Multipotent nerve end-cells have been reported and their potential use described [Reynolds and Weiss, Science, 255: 1707 (1992); Reynolds et al., J Neurosci. , 12: 4565 (1992); Reynolds and Weiss, Restorative Neurology and Neuroscience, 4: 208 (1992); Reynolds and Weiss, Neural Cell Death and Repair, ed Cuello (1993)].
Additionally, the utility of these cells is described in published PCT applications numbers WO 93/01275, WO 94/16718, WO 94/10292, and WO 94/09119. As used herein, the term "nerve terminal cell" refers to an undifferentiated multipotent nerve terminal cell that can be induced to proliferate in vi tro the presence of a growth factor that induces proliferation, e.g. amfiregulin, the acid fibroblast growth factor (aFGF or FGF-1), the basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor alpha (TGFa), and the like. The germline nerve cell is capable of self-maintenance, which means that with each cell division, a daughter cell will also be a germ cell. The non-germinal cell line (ie, progenitor cells) of a single multipotent germinal nerve cell is capable of differentiating into neurons, astrocytes (type I and type II) and oligodendrocytes. Therefore, the germinal nerve cell is "multipotent" because its progeny have multiple differentiation routes. The term "nerve progenitor cell", as used herein, refers to an undifferentiated cell that is derived from a germline nerve cell, which is not itself a germ cell. A distinctive feature of a progenitor cell is that, unlike the germ cell, it has limited proliferative capacity and therefore does not exhibit self-maintenance. It is committed to a particular path of differentiation and, under the right conditions, will eventually differentiate into glia or neurons. The term "precursor cells", as used herein, refers to the progeny of nerve germ cells, and therefore includes both progenitor cells and daughter nerve germ cells. The CNS precursor cells derived from the germ cells can be cultured using the methods described in Example 3 below, and in the applied PCT application referred to above. In the embryo, tissue containing nerve germ cells can be obtained from any region of the CNS, including the striated tissue, the cortex, the septum, the thalamus, the ventral mesencephalon, and the spinal cord. However, in the adult, the nervous tissue is obtained preferably from the tissue that covers the various ventricles and passages of the CNS. For example, tissue can be obtained from the regions immediately surrounding the lateral ventricles (first and second) and the third ventricle of the forebrain, the material surrounding the brain, the fourth ventricle and the central canal. The germ cells responsive to the nerve tissue growth factor are cultured in a culture medium in the presence of at least one growth factor. The medium is preferably defined as serum free. Growth factors that can be used to induce proliferation, individually or in combination with other growth factors, include any growth factor that allows proliferating precursor cells, including any molecule that binds to a receptor on the surface of the cell to exert a trophic effect, or one that induces the growth of the cell. Such factors include dibasic acid fibroblast growth factors (FGF-1, and FGF-2), epidermal growth factor (EGF), an EGF-like ligand, enfiregulin, transforming growth factor alpha (TGFa), and the like. The cell is induced to divide giving rise to a cluster of undifferentiated cells that are not immunoreactive from the astrocyte tag, the glial fibrillary acidic protein (GFAP9, the nerve markers, the neurofilament (NF), the associated protein with the microtubule (MAP-2), and the neuron-specific enolase (NSE), or the oligodendrocyte markers, myelin basic protein (MBP) and galactocerebroside (GalC), however, the precursor cells within the cluster are immunoreactive to nestin, an intermediate filamentous protein found in cells of the CNS undifferentiated. The nestin marker was characterized by Lehndahl et al. [Cell, 60: 585-595 (1990)]. The mature phenotypes related to the types of nerve cells that can be differentiated from the progeny of the precursor cells are predominantly negative to the nestin phenotype. Given the continued presence of an itogen eg EGF, FGF or similar, the precursor cells that are found in the neurosphere continue to divide resulting in an increase in the size of the neurosphere and the number of undifferentiated cells [nestin (+), GFAP (-), NF (-), MAP-2 (-), NSE (-), MBP (-), GalC (-)]. In this stage, the cells are non-adherent and tend to form freely floating clusters characteristic of the neurospheres. However, the culture conditions can be varied such that although the precursor cells still express the nestin phenotype, they do not form the characteristic neurospheres. Differentiation of cells can be induced by any method known in the art that activates the cascade of biological events that lead to growth, including the release of inositol triphosphate and intracellular Ca 2+, the release of diacyl glycerol and the activation of protein kinase. C and other cell kinases, and the like. Treatment with phorbol esters, growth factors that induce differentiation, and other chemical signals may induce differentiation. The differentiation can also be induced by culturing the cells in plates on a fixed substrate eg flasks, plates or coating strips coated with an ionically charged surface eg poly-L-lysine and poly-L-ornithine and the like. Other substrates can be used to induce differentiation for example collagen, fibronectin, laminin, matrigel, and the like. Differentiation can also be induced by leaving the cells in suspension in the presence of a growth factor that induces proliferation, without reinitiation of proliferation (ie, without dissociating the neurospheres). A preferred method for inducing the differentiation of nerve germ cell progeny comprises culturing the cells on a fixed substrate in a growth factor-free growth medium that induces proliferation, after removal of growth factor-induced proliferation, cells adhere to the substrate (for example, plastic or glass treated with poly-ornithine), flatten, and begin to differentiate into neurons and gual cells. In this step the culture medium can contain serum, for example, 0.5-1.0% fetal bovine serum (FBS). However, for certain uses, if defined conditions are required, serum should not be used. Within 2-3 days, most or all of the germ cell progeny begin to lose nestin immunoreactivity and begin to express specific antigens for neurons, astrocytes, or oligodendrocytes as indicated by MAP-2, GFAP, and immunoreactivity. GalC, respectively, using immunocytochemistry techniques well known in the art. In summary, the germ cells of the CNS have been isolated from a variety of embryonic and adult CNS regions including the striated tissue, the spinal cord, the brain stem and the hypothalamus. In each of these cases the germ cell of the isolated CNS shows self-maintenance and in the long run generates a large number of differentiated progene that includes neurons, astrocytes and oligodendrocytes. Therefore, germ cells are present in multiple regions of the CNS of the adult mammal and can be cultured, in vi tro, to obtain large numbers of undifferentiated nerve cells, whose differentiation can be regulated by the application of growth factors or other biological factors, or both Undifferentiated cells (either a suspension of intact cells or neurospheres) proliferated using these techniques can be cultured to generate dopaminergic cells using the same methods described above for the induction of dopaminergic neurons of the primary nervous tissue. Transplantation of Cultured Dopaminergic Cells Therapeutic compositions comprising purified populations of differentiated dopaminergic cells derived from the primary culture or from the proliferated progenitor progeny of nerve creminal cells can be prepared and can be administered to brain regions of an endopamine-deficient receptor. Alternatively, therapeutic compositions comprising differentiating cells that have been cultured in a culture medium that induces the formation of dopaminergic cells can be prepared. The composition is administered to the appropriate brain region, where the cells are implanted before finishing the differentiation process. After implantation, differentiation of dopaminergic cells can be completed in vivo. The composition may comprise purified cells, prepared using any suitable purification method. The composition may also comprise other types of nerve cells. Any suitable method can be used for the implantation of dopaminergic cells or precursor cells near the region of dopamine depletion. The methods presented in the United States Patent Number ,082,670 awarded to Gage et al. For injection of cell suspensions, for example fibroblasts, into the CNS can be used for the injection of differentiated dopaminergic cells prepared by the culture methods disclosed herein. Additional methods and approaches can be found in Neural Grafting in the Mammalian CNS, Bjoklund and Stenevi, eds., (1987). Xenoingertos or allografts, or both, may require the application of immunosuppressive techniques or the induction of tolerance of the host to improve the survival of the implanted cells. In some cases, it is possible to prepare differentiating or differentiated dopaminergic cells of the nervous system of the receptor itself (for example, the tissue removed during biopsy). In such instances, the dopaminergic cells can be generated in culture from the progeny of nerve terminal cells. Dissociated nerve tissue cells are cultured in the presence of a growth factor that induces proliferation, for example EGF or FGF. After adequate expansion of the amounts, the precursor cells are contacted with a growth factor or a combination of growth factors or a conditioned medium or combinations of conditioned media, or all of these, that induce the differentiation of dopaminergic cells. Preferably, the cells are proliferated and the expression of dopamine is induced using a defined culture medium. A composition comprising the differentiating or differentiated dopaminergic cells is administered to the appropriate region (to the appropriate regions) of the receptor brain. The composition may further comprise growth factors or other components that improve cell survival after implantation. Drug Testing Using Cultured Dopaminergic Cells The dopaminergic cells produced using the methods disclosed herein can also be used to test the effects of drugs and other compounds on dopaminergic cells. The test methods disclosed in pending joint applications may be used US Serial No. 08 / 311,099 and United States Serial No. 08 / 339,090. In general, the effect of drugs and other compounds on the ability of differentiating or differentiated cells to produce or metabolize dopamine or both processes would be measured. Example 1: Propagation of Primary Cultures The brains of E14 embryonic albino mice were placed in a phosphate-buffered saline (PBS) and dissected to obtain the striated tissue, the cerebral cortex and the mesencephalon. The nervous tissue was mechanically dissociated in a serum-free medium (Dulbecco's Modified Eagle Medium (DMEM) and nutrient F12 (GIBCO), using a Pasteur pipette burnished with fire. The cells were cultured in platelets at a density of 10 6 cells / ml in glass-coating strips coated with poly-L-ornithine (15 μg / ml; Sigma) in 24 Nuclon culture boxes in a volume of 0.5 ml / per glass. Complete Medium or were grown together in a glial feeder bed. Growth factors or conditioned media or 1% serum, or all of these elements, were added to the vessels as summarized in Example 6. Cells were incubated at 37 ° in a humidified atmosphere of 95% with air number / 5 % of Co2. Example 2: Induction of Tyrosine Hydroxylase Expression in Adult Subventricular Zone Derived Cells To label proliferating cells in the subependymal of the subventricular region of the brain, five injections of BrdU were applied to adult CDi mice (Sigma, 120 mg / kg) in sterile saline, administered at two-hour intervals [Morshead and van der Kooy, J. Neurosci. 1:49 (1992)]. Thirty minutes after the last injection of BrdU the animals were sacrificed. Striated tissue was removed, cut into 1 mm coronal sections and placed in artificial cerebrospinal fluid (aCSF, 124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 2mM CaCl2 26 mM NaHC03 and 10 mM D-glucose (pH 7.35, -280 mOsmol), aereated with 95% C02 at room temperature. After minutes in the CSF, the subventricular zones were microdissected, cut into small pieces and transferred to a centrifuge flask (Bélico Glass) with a magnetic stirrer, containing a low solution in Ca2-aCSF (124 mM NaCl, 5 mM KCL 3.2 mM MgCl2, 0.1 mM CaCl2 26 mM NaHCO3 and 10 MM D-glucose (pH 7.35. ~ 280 mOsmol), 1.33 mg / ml trypsin (9000 B7? EF (benzoyl-L-arginine ethyl ester) units / mg), 0.67 mg / ml of hyaluronidase (2000 units / mg) and 0.2 mg mi of cinuric acid This solution was aerated with 95% C02 / 5% 02 at 32 ° C at ° C. After 90 minutes, tissue sections were transferred to normal aCSF for 5 minutes and then placed in a DMEM / F12 medium containing 0.7 mg / ml ovomucose (Sigma). The tissue was mechanically crushed with a Pasteur pipette widened to fire. The cells were centrifuged at 400 r.p.m. for 5 minutes and resuspended in the Complete Medium with or without conditioned medium from the rat glial cell line B49 (see example 5) and FGF-2 (20 ng / ml). They were plated on poly-L-ornithine-coated glass coater strips in 24-cup Nunclon tissue culture flasks and were incubated at 37 ° C, 100% humidity, aerated with 95% C0 / 5% 02 for three days. days. The cells were then fixed and processed for dual-labeled indirect immunocytochemistry for TH and BrdU, as outlined in Example 11. The presence of BrdU-labeled cells, which were also TH-IR, only in those cells treated with the CMs and FGF-2 (Figure 1), suggest that cells from the adult proliferating subventricular zone can be induced to express TH in the presence of CMs and growth factor. Example 3: Isolation and Propagation of Embryonic Germ Cells A. Mouse Germ Cells Embryonic CDX albino mice on day 14 (E14) (Charles River) were decapitated and the brain and striated tissue were removed using a sterile procedure. The tissue was mechanically dissociated with a burnished Pasteur pipette and placed in the Complete Medium. The cells were centrifuged at 800 r.p.m. for 5 minutes, floating residue was aspirated, and the cells were resuspended in the DMEM / F-12 medium for counting. Cells were resuspended in the Complete Medium with 16-20 ng / ml of EGF (purified from mouse submaxillary, Collaborative Research) or TGFa (human recombinant, Gibco), plated at 0.2 x 106 cells / ml in 75 cm2 tissue culture flasks (Croning) without previous treatment of substrate and housed in an incubator at 37 ° C, with 100% humidity, 95% air / 5% C02. The cells proliferated within the first 48 hours and at 3-4 days in vi tro (DIV), they formed neurospheres that separated from the substrate between 4-6 DIV. After 7 DIV, the neurospheres were removed, centrifuged at 400 r.p.m. for 2-5 minutes, and the pellet was mechanically dissociated into individual cells with a glass Pasteur pipette burnished in 2 milliliters of Complete Medium. 1 x 10 6 cells were further plated in a 75 cm 2 tissue culture flask with 20 ml of Complete Medium containing EGF. The proliferation of germ cells and the formation of new neurospheres were reinitiated. This procedure can be repeated every 6-8 days. B. Human Germinal Cells Fetal human anterior brain tissue was dissociated (week after conception 10.5), obtained following routine suction abortion procedures, with a burnished Pasteur pipette on fire and placed in the Complete Medium. The cells were centrifuged at 800 r.p.m. for 5 minutes, the floating solution was aspirated, the cells were resuspended in DMEM / F-12 medium for counting. Cells were resuspended in the Complete Medium with 20 ng / ml EGF (Chiron Corp.) and 10 ng / ml FGF-2 (R &D Systems), were cultured in platelets at approximately 1.5 x 106 cells / ml in culture flasks (Nunclon T175) without pretreatment of the substrate and housed in an incubator at 37 ° C, 100% humidity, 95% air / 5% C02. The cells proliferated after 5 days and by day 10 began to form neurospheres that were separated from the substrate from day 21. After 15 DIV, the neurospheres were removed and centrifuged at 1500 r.p.m. for 7 minutes. The granule was mechanically dissociated into individual cells by crushing 150 times in 2 ml of Complete Medium. The cells were counted and 1.5 x 106 cells / ml were reactivated in plates in each of the several culture flasks (Nunclon T175), with 25 ml of Complete Medium containing EGF and FGF-2, as above. The proliferation of germ cells and the formation of new neurospheres were reinitiated. This procedure can be repeated (that is, the passage of the cells can be done) every 2-3 weeks.
Example 4: Preparation of the Gaseous Feed Layers An astrocyte glial cell feeder layer was prepared from striated tissue obtained from postnatal mice (0-24 hours). The nervous tissue was dissected, separated and transferred to a 15 ml centrifuge tube that contained DMEM / F12 (1: 1) / 10% FBS. The tissue was dissociated by trituration with a glass pipette burnished on the fire and cultured on a platelet in Corning culture flasks containing 20 ml of DMEM / F12710% FBS at a density of 150,000 cells / ml. When the primary primary astrocyte cells reached confluence, the cells were dissociated (using trypsin EDTA) and plated again at 200,000 cells / cm 2 in poly-L-ornithine-coated glass-coating strips in culture flasks. 24 glasses After 3 to 4 days, the confluence was re-established. The cells obtained from the primary tissue culture (Example 1) or the second step neurospheres labeled with BrdU for 6 days (Example 3) were washed twice and resuspended in fresh medium. (free of serum, EGF or BrdU) before culturing the cells or neurospheres in the astrocyte feeder beds (Examples 6 and 7). Example 5: Preparation of the Conditioned Medium The conditioned medium (CM) was prepared from astrocytic cells or from a rat glial B49 cell line [Schubert et al., Nature, 249: 224, (1974)]. An astrocyte glial cell feeder layer was prepared (Example 4). The astrocytic layer was passed once before collecting the CM. The rat B49 glial cell line cells were cultured under the same conditions as the astrocytic cells. The cultures of the confluent astrocytes or the rat glial B49 cell line were rinsed once with PBS and twice with serum-free Complete Medium and covered in 20 ml of the same medium. The CM was collected 24, 48 and 72 hours after the incubation began and centrifuged at 1,000 to 2,000 r.p.m. to remove any cell or waste. The CM was stored at -80 ° C. Example 6: Induction of HT-IR in Cells Derived from Primary Culture Paradigm 1: Primary cultures derived from striatum tissue and cerebral cortex were prepared as outlined in Example 1. The Complete Medium (control), EGF (20 ng / ml, Chiron), recombinant FGF-2 (20 ng / ml, R &D Systems), and a combination of EGF plus FGF, astrocyte (ACM) or glial cell line B49 of conditioned medium ( BCM, see Example 5) were added individually or in combinations to individual vessels at the time of plate culture (time, t = 0). 1% FBS (Upstate Biotechnology Incorporated) was added to 50% of the vessels that did not receive the conditioned medium. Immunocytochemical analysis was performed for the detection of TH + cells (Example 5) 24 hours after plating.
The results are summarized in Table 1. The addition of 1% of FBS to CM-free vessels caused a three-fold increase in the amount of TH + cells recorded in the presence of growth factors. The combination of FGF-2 plus BCM produced the most profound results, which resulted in the generation of, on average, more than about 5,000 TH + cells / cm2.
TABLE 1 Average Number of TH-t Cells Generated per cm2 From Primary Cultures of Striated and Cortical Tissue Tissue E! STRIATED BARRIER free of FBS 1% of FBS free of FBS 1% of FBS control 4 2 12 29 EFG 10 * 18 14 * 25 * FGF 326 1128 72 295 EGF + FGF 350 1668 75 229 ACM 10 * - - - ACM + EGF 10 * - - - ACM + EGF 1468 - - - ACM + E + F 1645 - - - BCM 16. .
BCM + EGF 26 BCM + FGF 5397 BCM + E + F 4470 'Non-significant increase over control values. n = 4 Paradigm 2: Cells obtained from primary culture (Example 1) were washed twice and resuspended in fresh medium before co-culturing the cells in astrocyte feeder layers (Example 4) in the presence of Complete Medium (control) or Complete Medium more EGF, FGF-2 or a combination of EGF and FGF-2 (20 ng / ml of each growth factor), 1 μM of BrdU and 1% of FBS. Indirect immunocytochemistry (Example 11) was performed on the cells that had been cultured for 24 hours. A significant increase in TH-IR was detected striated tissue cells cultured together in the presence of FGF-2 or EGF plus FGF-2 when compared to cells grown only in the complete medium. A small but significant increase was observed in the presence of EGF alone. The primary cells derived from the cortex showed a similar EGF and FGF response alone (a significant increase over the control values) but the highest increase in TH-IR was seen using EGF and FGF-2 together. As indicated by BrdU uptake, few mesencephalon cells are mitotically active, even in the presence of growth factors. The results are summarized in Table II.
TABLE II Example 7: Induction In vi tro of TH expression using a Defined Culture Medium The primary cultures derived from the striated tissue and from the cerebral cortex were prepared as summarized in Example 1. They were added alone or in combination Complete Medium (control), FGF-2 (20 ng / ml; R & D systems), BMP-2 (50 ng / ml; Chiron Corp.) and activin (50 ng / ml; Chiron Corp.) (Medium Complete plus FGFA-2 and BMP-2 or Complete Medium plus FGF-2 and activin) to individual vessels at the time of plating. Immunocytochemical analysis was performed for the detection of TH + cells (Example 11) 24 hours after plating. The results are summarized in Table 3. The combination of FGF-2 plus activin or FGF-2 plus BMP-2 produced the most profound results, resulting in the generation of, on average, approximately 5,000 TH + neurons per cm2. In contrast, control, which comprised only the Complete, it produced an average of 2 TH + cells per cm2. Table 3: TH number + immunoreactive per cm2 Example 8: Induction of TH-IR in Progenies of Nervous Cell Derived Cell Precursor Cell Using Conditioned Paradigm 1: Primarily unique, non-dissociated generated neurospheres of 6 days (see Example 3) were grown on plates in coating strips. glass coated with poly-L-ornithine, in Complete Medium with or without CM derived from rat glial cell line B49 (Example 5) + 20 ng / ml FGF-2, and incubated at 37 ° C in 5% C02, 95 % air, 100% humidity. 24 hours after plaque culture, indirect immunocytochemistry for TH + (see Example 11) revealed the presence of TH + cells, with processes and nerve morphology, in the vessels containing CM and FGF-2 (FIGURE 2) but not in vessels that contained only the complete medium. Paradigm 2: Second pass, single, non-dissociated 6-day neurospheres (Example 3) were labeled with BrdU, washed twice and resuspended in fresh medium before co-cultivating the neurospheres in astrocyte feeder layers (Example 4) in the presence of Medium Complete or Complete Medium plus FGF-2 (20 ng / ml). Indirect immunocytochemistry was carried out (Example 11) on cells that had been cultured for 24 hours. TH-IR cells with nerve morphology that marked positive for MAP-2 in neurospheres cultured with FGF-2 were observed. Several of the cells TH + showed BrdU immunoreactivity (FIGURE 3). Example 9: Induction In vi tro of TH-IR in Progeny Derived from Nerve Terminal Cell Using Defined Medium Neuroespheres generated primarily single, not dissociated for 6 days (see Example 3) are grown on plates in glass strippers coated with poly-L-ornithine, in Complete Medium with a combination of 20 ng / ml FGF -2 and 20 ng / ml BMP-2 or a combination of 20 ng / ml of FGF-2 and 20 ng / ml of activin (Example 7), and incubated at 37 ° C in 5% C02, 95% air, and 100% humidity. The number of dopaminergic neurons is determined by TH immunoreactivity (Example 11). Example 10: Induction of TH-IR in Progenies Derived from Human Germinal Nervous Embryonic Cell Human germ cells were proliferated and passed 35 times to increase the number of cells, as described in Example 3B. Neurospheres of 12 days were obtained at the end of the pass. The neurospheres were washed, suspended and mechanically dissociated in Complete Medium. The neurospheres were grown in plates in glass strippers coated with poly-L-ornithine (15 μg / ml) (density: 0.3 million cells) in 24-cup Nunclon culture flasks either under direct control (Complete Medium) or conditions that induced TH (0.8 ml / well; 75% of CM derived from rat glial cell line B49 (Example 5) + 20 ng / ml of FGF-2). In addition, preparations of cells containing only CM (75%) or FGF-2 (20 ng / ml) were coated to determine their effects when used separately. The cells were incubated at 37 ° C in 5% C02, 95% air, 100% humidity. After DIV and 3 DIV the number of TH-IR cells was determined as indicated in Example 11. The results are shown in Table 4. Table 4: Number of TH + cells / cm2 Example 11: Immunocytochemistry Cells were fixed with 4% paraformaldehyde for 30 minutes followed by three washes in PBS for 10 minutes each. The cells were coated with a primary anti-TH antibody (polyclonal rabbit, 1: 1,000, Engene Tech International Inc .: or 1: 100. Pel-freeze) prepared in PBS / 10% normal goat serum / 0.3 Triton X -100 for 2 hours at 37 ° C. After three rinses in PBS, rhodamine anticonejo de goat (Jackson) was applied in PBS for 30 minutes at room temperature. In some cases, MAP-2 (Boehringer-Mannhei) was used to identify the neurons. The cells were then washed three times for 10 minutes each time in PBS, rinsed with water, placed on glass slides and covered with strips using Fluorosave (Calbiochem) as a counting medium. The number of dopaminergic neurons was determined by counting all immunoreactive TH (TH +) per cm2, with magnification of 200x. Example 12: Transplantation of Dopaminergic Cells A: Transplantation of Cells Derived from Human Nervous Germ Cells Cultivated to a Mouse Model of Parkinson's Disease Fetal human pre-germinal germ cells are proliferated in culture as indicated in Example 3B and are made pass 35 times. Three days before transplantation, the floating neurospheres are removed and treated in one of two methods to improve neuronal differentiation to a phenotype.
TH. With the first TH enhancement method, the cells are treated as described in Example 10 above (adding 1 μM of BrdU to the medium). On the day of transplantation, the cells are rinsed, separated with trypsin / EDTA, and then treated with trypsin inhibitor. The cells are suspended in HBSS at a density of 20 x 106 cells / ml for transplantation. For the second method of TH improvement, the cells are placed in untreated flasks, which causes the neurospheres to remain floating. The components of the culture medium are the same as in the first method. On the day of transplantation, the cells are rinsed, lightly ground, and then suspended in HBSS at a density of 20 x 106 cells / ml for transplantation. All cells are stored at TH during the transplant period. To be the model of Parkinson's disease, the Wistar triple rats (approximately 275 gm, Charles River) receive unilateral administration of 4 μl of a solution of 2 μg / μl of 6-OHDA (6-hydroxydopamine, Sigma) to injure the neurons dopaminergic in the compact black substance on the same side. Sixteen days later they receive the transplants of the human germ cell progeny in the striated tissue on the same side. 5 animals receive the treated cells as in the first TH improvement method, and 5 animals receive the treated cells as in the second improvement method. The animals are sacrificed after 1 week, 6 weeks and 3 months. The animals infiltrate trans, cardially with aldehydes, the brain tissue is removed and cut into 10 μm thick sections and then the tissue is mounted directly on the microscope slides. They use double immunostaining techniques for light microscopy to identify transplanted cells (BrdU +) that have tyrosine hydroxylase (TH +) using antibodies against BrdU and TH. Substrates of different color allow the identification of cells labeled as double indicating a population of differentiated transplanted cells in neurons with a dopaminergic phenotype. B_ Parkinson's disease is a disease characterized by the degeneration of the dopaminergic pathway to the striated tissue and is the result of lower levels of dopamine in this region. The disease is characterized by muscle stiffness, tremor and other motor abnormalities. The precursor cells, prepared from human fetal tissue, are proliferated (Example 3B) and their differentiation is induced in dopaminergic cells.
(Example 11). Dopaminergic cells are injected stereotactically into the striated tissue of a patient Parkinsonian. Reinforcement injections may be performed if required. Alternatively, the precursor cells are prepared from nerve tissue obtained from a brain biopsy of a Parkinsonian patient and are induced to differentiate into dopaminergic cells (Example 7 or 8) and injected stereotactically into the striated tissue of a patient. Alternatively, the precursor cells can be cultured in the presence of a culture medium that induces the formation of dopaminergic cells and the striated tissue of a Parkinsonian patient is implanted wherein the precursor cells differentiate, in vivo, into dopaminergic cells. The best motor control is used to measure the success of the transplant. Example 12: Drug Testing Using Cultured Dopaminergic Cells Prozac, a drug widely used in the treatment of psychiatric diseases, is added to the cultured dopaminergic cells prepared as set forth in Examples 6, 7, 8, 9, 10 in concentrations ranging from 1 ng / ml to 1000 ng / ml. The effects of the drug at various concentrations are monitored with respect to cellular metabolism. All references, patents and patent applications cited in the present document are incorporated herein by reference.

Claims (37)

  1. CLAIMS 1. A method for inducing the expression of tyrosine hydroxylase in nerve cells which comprises contacting said nerve cells with a culture medium comprising at least one member of the fibroblast growth factor family and at least one additive selected from a group consisting of a conditioned medium and members of the transforming growth factor beta family.
  2. 2. The method of claim 1 wherein the culture medium is defined, the member of the fibroblast growth factor family is FGF-2 and the additive is a member of the beta family of the transformation growth factor selected from a group consisting of activin and orphagenic bone protein-2.
  3. The method of claim 1 wherein the nerve cells are cultured on a charged surface and solely selected from a group consisting of poly-D-lysine, poly-L-ornithine, Matrigel, laminin, fibroin. .
  4. The method of claim 3 wherein the charged surface is poly-L-ornithine.
  5. The method of claim 1 wherein the nerve cells are obtained from normally non-dopaminergic nerve tissue.
  6. The method of claim 5 wherein the normally non-dopaminergic nerve tissue is selected from a group consisting of striated tissue and cortex.
  7. The method of claim 1 wherein the additive is the conditioned medium selected from a group consisting of astrocyte-conditioned medium and conditioned medium per glial cell.
  8. The method of claim 7 wherein the conditioned medium is derived from the rat glial B49 cell line.
  9. The method of claim 1 wherein the culture medium further comprises serum.
  10. The method of claim 1 wherein the nerve cells are cultured in the absence of a feeder layer.
  11. The method of claim 1 wherein the nerve cells are primary cells obtained from embryonic tissue.
  12. The method of claim 1 wherein the nerve cells are the progeny of at least one multipotent germline nerve cell proliferated in the presence of a growth factor that induces proliferation.
  13. The method of claim 12 wherein the multipotent germline nerve cell is derived from adult nerve tissue.
  14. 14. A method for inducing the expression of tyrosine hydroxylase in primary nerve cells in vi tro comprising contacting the nerve cells with a charged surface and only selected from a group consisting of poly-D-lysine, poly-L- ornithine, Matrigel, laminin, fibronectin, in a defined culture medium comprising a member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family.
  15. 15. The method of claim 14 wherein the member of the fibroblast growth factor family is FGF-2 and the transforming growth factor beta family member is selected from a group consisting of activin and morphogenic protein. bone-2
  16. 16. The method of claim 14 wherein the primary nerve cells are derived from the normally non-dopaminergic nerve tissue.
  17. 17. A method for inducing the expression of tyrosine hydroxylase in nerve cells obtained from normally non-dopaminergic nerve tissue comprising contacting the nerve cells with a feeder layer and a culture medium comprising the fibroblast growth factor.
  18. 18. The method of claim 17 wherein the feeder layer is derived from normally non-dopaminergic nerve tissue.
  19. 19. The method of claim 17 wherein the culture medium further comprises serum.
  20. The method of claim 17 wherein the nerve cells are the progeny of at least one multipotent germ cell nerve will proliferate in vi tro in the presence of a growth factor.
  21. The method of claim 17 wherein the normally non-dopaminergic tissue is selected from a group that spans the striated tissue and cortex.
  22. 22. The method of claim 17 wherein the culture medium further comprises conditioned medium.
  23. 23. The method of claim 22 wherein the conditioned medium is derived from the same cells.
  24. The method of claim 22 wherein the conditioned medium is derived from astrocytes.
  25. 25. The method of claim 22 wherein the conditioned medium is derived from the same cell line B49 of rat.
  26. 26. The method of claim 18 wherein the culture medium further comprises serum.
  27. 27. A method for treating a neurological disease in a patient that requires dopaminergic cells that comprises transplanting differentiated or differentiated dopaminergic cells to the patient in a region of the patient. CNS requiring dopaminergic cells, the differentiating or differentiated dopaminergic cells being formed by contacting nerve cells with a culture medium comprising a medium of the fibroblast growth factor family and at least one member of the beta factor family of cells. transformation growth.
  28. 28. The method of claim 27 wherein the patient has Parkinson's disease and the differentiating or differentiated dopaminergic cells are administered to the patient's striated tissue.
  29. The method of claim 27 wherein the member of the fibroblast growth factor family is FGF-2 and the member of the transforming growth factor beta family is selected from a group consisting of activin and morphogenic protein that is.
  30. 30. The method of claim 27 wherein the culture medium is defined.
  31. 31. The method of claim 27 wherein the nerve cells are obtained from the patient's CNS.
  32. 32. A method for testing the effects of a drug on the dopaminergic cells comprising a) culturing nerve cells derived from nervous tissue in a culture medium comprising at least one member of the fibroblast growth factor family and therefore less an additive selected from a group consisting of conditioned medium, and members of the transforming growth factor beta family to produce dopaminergic cells, b) administering a drug to said dopaminergic cells, and c) observing the effects of the drug on the cells dopaminergic
  33. 33. The method of claim 32 wherein the effects are determined by measuring the ability of dopaminergic cells to produce or metabolize dopamine.
  34. 34. A therapeutic composition for the treatment of a neurological disease or condition, comprising the composition of differentiated or differentiated dopaminergic cells derived from nerve cells cultured in vi tro in a culture medium comprising a member of the fibroblast growth factor family and at least one member of the transforming growth factor beta family.
  35. 35. The therapeutic composition of claim 34 wherein the member of the fibroblast growth factor family is FGF-2 and the member of the beta family of the transforming growth factor is selected from a group consisting of activin and protein. morphogenic bone
  36. 36. The therapeutic composition of claim 34 and 35 wherein the culture medium is defined.
  37. 37. The therapeutic composition of any of claims 34 and 26 wherein the nerve cells are obtained from the patient's CNS.
MXPA/A/1997/003493A 1994-11-14 1997-05-13 In vitro induction of dopaminergi cells MXPA97003493A (en)

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US33909094A 1994-11-14 1994-11-14
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US08482079 1995-06-07
US08/482,079 US5981165A (en) 1991-07-08 1995-06-07 In vitro induction of dopaminergic cells
PCT/CA1995/000636 WO1996015224A1 (en) 1994-11-14 1995-11-14 In vitro induction of dopaminergic cells

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Class Patent application title: COMPOSITIONS AND METHODS FOR ISOLATION, PROPAGATION, AND DIFFERENTIATION OF HUMAN STEM CELLS AND USES THEREOF Inventors: Toomas Neuman (Santa Monica, CA, US) Michel Levesque (Beverly Hills, CA, US) Assignees: LEVESQUE BIOSCIENCES, INC.