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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
  • C12N5/0623—Stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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  • C12N2510/00—Genetically modified cells
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  • C12N2510/00—Genetically modified cells
  • C12N2510/04—Immortalised cells
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/22011—Polyomaviridae, e.g. polyoma, SV40, JC
  • C12N2710/22051—Methods of production or purification of viral material

Definitions

• the present invention generally relates to stem cells, including progenitor and precursor cells, which are preferably immortalized, and have been derived from the human fetal central nervous system (CNS); methods for treating a host by implanting the disclosed cells, and genetically altered forms of the disclosed cells in the host. More particularly, the present invention provides CNS derived cell lines, such as fetal CNS derived cell lines, such as human fetal CNS derived cell lines, and methods of treating a host by implantation of these immortalized CNS derived cells into the host. Also disclosed are methods of isolating immortalized CNS derived cells useful in therapeutic applications. Cell transplant therapy is particularly appealing for treatment of neurological diseases.
• Solid tissue transplantation is especially inappropriate for neurological diseases for several reasons. Open surgical exposure of the brain, as required for solid tissue transplantation, can cause irreparable damage to nervous system pathways resulting in clinical neurological deficits. Also, neurological function often depends on complex intercellular connections that can not be surgically established. Further, cells of the central nervous system are exquisitely sensitive to anoxia and nutrient deprivation. Rapid vascularization of solid tissue transplants is critical as cells in the interior of solid tissue transplants often lack sufficient perfusion to maintain viability. Stenevi et al., Brain Res., 114:1-20 (1976).
• Parkinsonism has been the object of attempts at cell transplant therapy. Bjorklund et al.,Brain Res., 177:555-560 (1979); Lindvall et al., Science, 247:574-577 (1990); Freed, Restor. Neurol. Neurosci., 3:109-134 (1991). Parkinsonism is caused by a loss of dopamine-producing neurons in the substantia nigra of the basal ganglia. Burns et al., N. Engl. J. Med., 312:1418-1421 (1985); Wolff et al., Neurobiology, 86:9011-9014 (1989).
• Parkinson's disease a disease of unknown etiology that is characterized by the clinical manifestations of Parkinsonism, is caused by idiopathic destruction of these dopamine-producing neurons.
• Parkinsonism may be caused by a variety of drugs, e.g., antipsychotic agents, or chemical agents, e.g., l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine. Burns et al., Proc. Natl. Acad. Sci. USA, 80:4546-4550 (1983) and Bankiewicz et al., Life Sci., 39:7-16 (1986).
• the fetal cells must be freshly harvested p ⁇ or to transplantation This requires coordinating the implantation procedure with elective abortions. Even then, fetal tissue has not been widely available in the United States. Also, the gestational age of the fetus from which cells are obtained influences graft survival. Gage and Fisher, supra Obtaining fetal tissue of only certain gestational ages adds additional limitations to the availability of fetal cells for transplant. Further, ethical considerations make some potential transplant recipients reluctant to undergo the procedure when fresh fetal cells are implanted.
• the SVG cells have been desc ⁇ bed as an example of a permanently established line of fetal glial cells. See, Major et al, Proc Natl Acad Sci U S A, 82: 1257-61 (1985), which was the first report of the use of an immortalizing gene to produce potentially immortal cells of the human central nervous system This population of SVG cells has also been characterized as glial or neuro -glial .
• the CNS like the hematopoietic system, contains cells that are capable of developing into more phenotypically defined cells. That is, it is now more readily appreciated that the CNS contains progenitor or stem cells which, if pu ⁇ fied, maintained and stored, may contain the potential to develop a transplantable product which could be developed, either pre- or post-transplantation, into multiple cell types, for multiple uses. These cells would be, therefore, multipotent and may reduce the requirement for production and maintenance of multiple cell types. Moreover, transplanting such a multipotent cell may allow replacement and re-population of the cell type needed by the patient, without need for external effectors as the cells may be able to develop to replace lost or damaged tissue types.
• the present invention provides methods for treating a host comp ⁇ sing implanting multipotent cells of an immortalized human neuro-de ⁇ ved cell line, preferably a fetal cell line, into the host.
• the cells for implantation and methods of their identification and purification are also provided Generally the cell line, and cell types within those cell lines, will be de ⁇ ved from CNS cells, such as human fetal CNS cells, such as the SVG cell line.
• the cells may be implanted into the central nervous system of the host without further differentiation into either specifically a neural or glial cell type.
• the multipotent cells of the present invention may be further differentiated p ⁇ or to use in the treatment methods of the present invention.
• the cells may be encapsulated by membranes which are impermeable to antibodies of the host.
• the multipotent cells may be transfected with a nucleic acid sequence encoding a peptide, ammo acid sequence or protein.
• the peptides, ammo acid sequences, or proteins will generally be enzymes, such as tyrosine hydroxylase, or growth factors, such as nerve growth factor, or portions thereof, including glycosylated and non- glycosylated peptides and proteins.
• the peptide, ammo acid sequences and proteins hereinafter generally referred to by any one of these terms, may also be a disease-associated antigen.
• the cells may be implanted for purposes of treatment or prophylaxis. In some instances, the cells may be removed following implantation.
• the present invention provides a multipotent immortalized human fetal CNS-de ⁇ ved cell line, and specific cell types de ⁇ ved from these cell lines, which contains a heterologous nucleic acid sequence, wherein the cell line is capable of expressing the heterologous nucleic acid sequence.
• Particularly preferred cell lines and cell types are capable of expressing a nucleic acid that encodes tyrosine hydroxylase, serotonin or aromatic amino acid decarboxylase.
• the present invention provides a transplantable composition that contains at least one of the multipotent cell types of the invention, or de ⁇ vatives thereof, with a pharmaceutically acceptable earner
• the present invention provides therefore an isolated fetal central nervous system cell line containing a multipotent cell that has the ability to divide, without limit, and the potential to differentiate toward a neuronal cell or a glial cell.
• the cell line of the present invention perferably contains a stem, progenitor or precursor cell.
• the fetal central nervous system de ⁇ ved cell line is de ⁇ ved from a human fetal central nervous system.
• the present invention provides a cluster of cells, preferably isolated and/or pu ⁇ fied, of the invention, preferably in the form of a neurosphere
• the present invention provides an isolated and/or pu ⁇ fied multipotent cell.
• the cells of the present invention may be characte ⁇ zed by any of the following marker combinations. TnTxJChTx-, TnTx+/ChTx+, TnTx+/ChTx-, TnTx+/ChTx+, A2B5-/TnTx-, A2B5+/TnTx-, A2B5JTnTx+, A2B5JChTx-, A2B5+/ChTx+, A2B5-/ChTx+, A2B5+/ChTx-, TnTxJChT -/nest ⁇ n+, and/or TnTxJChT Jnestin-, as further desc ⁇ bed and defined herein.
• the present invention further providfes an isolated and/or pu ⁇ fied cell and/or tissue de ⁇ ved from the cell line of the present invention.
• the cells and/or tissues of the present invention may further contain a heterologous nucleic acid sequence which encodes a biologically active peptide or protein which may be, for example, a disease associated peptide or protein, an enzyme, a trophic factor, and/or a cytokine.
• the enzymes encoded m the cells and/or tissues of the present invention may be, for example, tyrosine hydroxylase, GTPCH1, AADC or VMAT2.
• the trophic factors encoded for may be, for example, GDNF, VEGF, BDNF, NGF, bFGF, TGF5 (including the TGF3 family of peptides), CNTF, PDGF, BMP, LIF, Neurtu ⁇ n, Persephin, Neublastin, NT4/5, NT3, or Midkine.
• the cytokine may be, for example, EL- 10 or EL-6.
• the heterologous nucleic acid may be operably linked to a transc ⁇ ptional promoter, which may be, for example, a regulatable promoter.
• the prepsent invention provides populations of cells defined herein as NG1, NG2, and NG3 populations of cells.
• the present invention provides a method of identifying a multipotent cell which includes measu ⁇ ng for the presence or absence of the binding partners for TnTx and ChTx and the A2B5 antibody in a cell sample which is believed to contain a multipotent cell.
• the method of the present invention may be used to identify multipotent cells de ⁇ ved from the fetal central nervous system de ⁇ ved cells.
• the present invention provides a method wherein these cells are identified by mixing a sample containing the same with at least one factor which specifically binds to at least one cell-specific binding partner selected from an A2B5 anti body-binding cell binding partner, TnTx receptor, and ChTx receptor, or fragments thereof, under conditions where the factor binds to the cell, followed by detecting of the binding, as an indication of the presence of the multipotent cell.
• the factors used in the methods of the present invention include an A2B5 antibody or fragment, a ChTx or a fragment thereof, and a TnTx or a fragment thereof.
• binding partners and factors in this context may include an antibody, an antibody fragment, a ligand, a ligand fragment, a receptor or receptor fragment.
• the method of the present invention may also include identifying the cells by their ability to specifically bind to a human nestin antibody.
• the binding partner, factors, hgands and antibodies used for detection in the present method may include or contain a detectable label which may be, for example, fluorescent, chemiluminescent, radioactive, immunologically detectable, and/or an enzymatically active component of a detection system.
• a detectable label which may be, for example, fluorescent, chemiluminescent, radioactive, immunologically detectable, and/or an enzymatically active component of a detection system.
• the method of identifying cells according to the present invention may include analyzing the cells with a fluorescence activated cell sorter which may also be used to pu ⁇ fy, en ⁇ ch and/or separate specific cell populations.
• the present invention provides a method of enriching a population of cells the multipotent fetal nervous system de ⁇ ved cells which includes cultu ⁇ ng the population in the presence of serum, preferably through c ⁇ sis, followed by cultu ⁇ ng the population in a non-serum containing media.
• the present invention provides a method of treating a mammal having a neurological syndrome or disease which includes implanting in to the mammal a therapeutically effective amount of a composition containing at least one cell or cells population of the present invention
• FIG. 1 Immunoidentification of SVG P50 cells cultured in fetal calf serum-containing or serum-free media is shown wherein a compa ⁇ son is provided between control and forskohn- treatment
• analysis was gated on live cells.
• T ⁇ ple label characte ⁇ zation of live NGl and NG3 cells is shown.
• Results of fluorescent emission of labeled NGl cells are shown in panels A-F (panels A-C being control and panels D-F being forskohn treated) and labeled NG3 cells are shown in in panels G-L (panels G-I being controls and panels J-L being forskohn treated)
• Panels A, D, G and J show show results of A2B5-phycoeryt ⁇ n (PE) labeling versus TnTx -TC labeling
• panels B, E, H and K show results of ChTx-FTTC labeling versus TnTx- TC labeling
• panels C, F, I and L show results of ChTx
• NG3 cells had an increase in TnTx- cells (UL in A versus G) and a decrease in A2B5+ cells (LR in A versus G) compared to the NGl cells. Notably, there was no major change due to forskohn in the NGl or NG3 cells. Specifically, there was no increase in the TnTx+ cells (UR in B versus E and H versus K) indicating that there was no increase m the neuronal lineage cells due to forskohn.
• Figure 2 Immunoidentification of SVG P50 cells cultured in serum-free media with anti- ChTx-FIT C, ant ⁇ -TnTx-PE/Cy5 and anti-nestin-PE.
• T ⁇ ple label characte ⁇ zation of NG3 cells was performed with labels for ChTx, TnTx and nestin.
• Panels A-C indicate the relative intensity of the fluorescent signal for each marker (TnTx, ChTx or nestin) individually. Therefore, there are predominantly ChTx-i- cells (A), rare nest ⁇ n+ cells (B) and mostly TnTx- cells (C) observed in the general population of NG3 cells.
• Panels D-F show results of live cells labeled with the indicated markers (i.e., panel D showing TnTx-TC (fragment C) labeling versus nestin-PE labeling; panel E showing TnTx-TC and ChTx-FTTC labeling; and paenl F showing nestin-PE and ChTx-FTTC labeling) wherein the percent of cells in each quadrant, as desc ⁇ bed above in relation to Figure 1, is provided to the left of each panel.
• markers i.e., panel D showing TnTx-TC (fragment C) labeling versus nestin-PE labeling; panel E showing TnTx-TC and ChTx-FTTC labeling; and paenl F showing nestin-PE and ChTx-FTTC labeling
• the present invention generally relates to immortalized multipotent human cell-lines de ⁇ ved from cells of the central nervous system, preferably de ⁇ ved from a fetal central nervous system, such as human fetal central nervous system, and methods of using these cell lines in treatment of disorders of the central nervous system.
• the cell lines and methods of the present invention may be used in the treatment of disorders caused by neurodegeneration in the central nervous system, such as Parkinsonism.
• the present invention provides methods of making the immortalized multipotent cells of the present invention as well as methods of identifying same.
• the cells, cell lines and cell types of the present invention are preferably at least partially isolated, isolated, and/or pu ⁇ fied.
• the present invention provides methods of treating a host suffe ⁇ ng from a central nervous system disorder, or alleviating the symptoms of such a disorder, by implanting immortalized human fetal cells de ⁇ ved from cells of the central nervous system.
• the cells of the present invention will not produce graft rejection, intense intracerebral inflammation, or tumor formation following implantation of such cells into the central nervous system.
• the cells, after further differentiation, will preferably induce neuron migration and neu ⁇ te extension, which will demonstrate that the cells are functioning to produce trophic factors that stimulate neuronal responses.
• CNS such as are provided by the present invention
• Parkinson's disease will be treatable by implantation of these cells, or further differentiated cells de ⁇ ved from them, into the basal ganglia of an affected host.
• the trophic factors that should be produced by the differentiated cells de ⁇ ved from the implanted cells of the present invention, or implanted de ⁇ ved cells of the present invention may inhibit dopaminergic neuron demise and even induce dopaminergic neuron regeneration or allow increased neu ⁇ te outgrowth from existing neurons.
• the increased population of dopaminergic neurons can provide clinical improvement of persons suffe ⁇ ng from Parkinsonism
• multipotent cells of the present invention may develop into glial cells, it may be possible to provide regeneration of myeltnating o godendrocytes or multifunctional astrocytes
• the implanted cells may be transfected, in vivo or in vitro, with a nucleic acid that encodes a neurologically relevant polypeptide
• the term "neurologically relevant peptide” generally refers to a peptide or protein that catalyzes a reaction within the tissues of the central nervous system
• Such peptides may be naturally occumng neural peptides, proteins or enzymes, or may be peptide or protein fragments that have therapeutic activity within the central nervous system. Examples include neural growth factors, trophic factors, and cytokines, and enzymes used to catalyze the production of important neuro-chemicals, or their intermediates.
• the cells will be transfected with a nucleic acid that encodes, for example, TH (tyrosine hydroxylase), GTPCHl (GTP cyclohydrolase 1), AADC (aromatic amino acid decarboxylase), VMAT2 (vesicular monoamine transporter 2), GDNF (ghal-de ⁇ ved neurotrophic factor), VEGF (vascular endothe al growth factor), BDNF (bram- de ⁇ ved neurotrophic factor), NGF (nerve growth factor), bFGF (also known as FGFII or basic fibroblast growth factor), CNTF (ciliary neurotrophic factor), PDGF (platelet-de ⁇ ved growth factor), BMP (which is known as a family of bone morphogemc proteins), LIF (Leukemia inhibitory factor), Neurtu ⁇ n, Persephin, Neublastin, NT4/5 (neurotrophin 4/5), NT3 (neurotrophin 3), Midkine, EL-
• Tyrosine hydroxylase is the enzyme that converts tyrosine to L-DOPA, which is also the rate-limiting step in the production of dopamine. Therefore, expression of tyrosine hydroxylase by the implanted cells allows these cells to produce and secrete dopamine.
• the implanted cells may increase the dopamine concentration in the substantia nigra and limit or reverse the effect of dopaminergic neuron loss.
• the peptide When applied to the treatment of stroke, the peptide may aid in the revascula ⁇ zation of damaged nervous tissue or supply of neurotrophic factors that could enhance survival and regeneration of damage nervous tissue.
• the methods of the present invention may also be used to treat other neurological disorders such as Huntington's chorea, epilepsy, stroke, Alzheimer's disease, traumatic brain injury, spinal chord injury, epilepsy, or multiple sclerosis.
• other neurological disorders such as Huntington's chorea, epilepsy, stroke, Alzheimer's disease, traumatic brain injury, spinal chord injury, epilepsy, or multiple sclerosis.
• immortalized human fetal CNS- de ⁇ ved cells of the present invention are expected to be compatible with the CNS, it should be possible to transfect these cells with DNA sequences encoding physiologically active peptides for implantation in the CNS, to effect treatment of other disorders.
• the peptide may block excitatory neurotransmitters such as glutamate.
• the peptide When applied to the treatment of multiple sclerosis, for example, the peptide would typically be a trophic stimulator of mye nation, such as platelet de ⁇ ved growth factor or a ciliary neurotrophic factor which may block ohgodendrocyte demise.
• mye nation such as platelet de ⁇ ved growth factor or a ciliary neurotrophic factor which may block ohgodendrocyte demise.
• alternative implantation methods may be desirable.
• the cells may be implanted on a surface exposed to cerebrospinal fluid. Following expression and secretion, the peptide will be washed over the entire surface of the brain by the natural circulation of the cerebrospinal fluid. Suitable sites for implantation include the lateral vent ⁇ cles, lumbar intrathecal region, and the like.
• the cells In Alzheimer's disease, the cells may be transfected to produce nerve growth factor to support neurons of the basal forebrain as desc ⁇ bed by Rosenberg et al., Science,
• the cell lines and cell types of the present invention may therefore serve as gene vectors.
• the methods of the present invention may also be employed to treat hosts by implantation of cells in extraneural sites.
• This embodiment of the present invention is particularly useful for prophylactic treatment of a host.
• Immortalized multipotent human fetal neuro-derived cells may be transfected with DNA encoding a disease-associated antigen, e.g., TUN gpl20 polypeptides that encompass the principal neutralizing domain of HTV as described, e.g., in U.S. Pat. No. 5,166,050.
• the cells may then express and secrete the antigen encoded by the transfected DNA.
• the antigen may be continuously secreted by the implanted cells and elicit a strong immune response. Following an adequate time interval to fully immunize the host, the cells may be removed.
• treating a host includes prophylactic, palliative, and curative intervention in a disease process.
• treatment typically refers to therapeutic methods for reducing or eliminating the symptoms of the particular disorder for which treatment is sought.
• host generally refers to any warm blooded mammal, such as humans, non-human p ⁇ mates, rodents, and the like, which is to be the recipient of the particular treatment.
• host and “patient” are used interchangeably herein to refer to a human subject.
• a wide va ⁇ ety of diseases and syndromes may be treated by the methods of the present invention
• the disease will be a neurological disease including, but not limited to Parkinsonism (including Parkinson's disease), Alzheimer's disease, epilepsy, Huntington's chorea, multiple sclerosis, amyotrophic lateral sclerosis, Gaucher's disease, Tay-Sachs disease, neuropathies, brain tumors, stroke.
• the methods of the present invention may also be employed in the treatment of non-neurological diseases.
• the methods of the present invention may be used to immunize hosts against infectious diseases, such as viruses, bacte ⁇ a, protozoa, and the like as desc ⁇ bed above.
• Immortalized multipotent human fetal neuro-de ⁇ ved cells of the present invention may be transfected by DNA encoding physiologically active peptides or peptides which contain immunological epitopes.
• the methods of the present invention may be employed to implant the peptide producing cells and provide continuous in vivo delivery of other types of peptides, such as growth hormone, to the host.
• the present invention also provides cell lines suitable for transplantation into a host or patient.
• the cells implanted by the methods of the present invention are immortalized multipotent human fetal CNS-de ⁇ ved cells.
• neuro-de ⁇ ved or “CNS-de ⁇ ved” it is meant that, p ⁇ or to immortalization, the cells were harvested from the CNS and/or had a neurological cell phenotype (neuronal or glial).
• Neurological cell types include neurons, astrocytes, o godendrocytes, choroid plexus epithelial cells, and the like. Cells of the CNS have been categorized as developing from stem cells into neuronal cells and glial cells .
• a stem cell m the CNS has the ability to become a neuroblast (immature neuron) or a ghoblast (immature g a) before further differentiating into mature neuronal cells (neurons) and glial cells (astrocytes and oligodendrocytes).
• bFGF basic fibroblast growth factor
• EGF epidermal growth factor
• a CNS de ⁇ ved stem cell is defined as a cell capable of differentiating into neurons, astrocytes or oligodendrocytes and self-renewing sufficiently to populate the brain . McKay, Science, 276. 66-71 (1997).
• a progenitor cell has a more rest ⁇ cted potential cellular diversity than a stem cell, but may still form neurons or gha.
• a precursor cell has an even more rest ⁇ cted phenotype, such as a neuroblast, that can only become a neuron.
• One of ordinary skill in the art will appreciate however that the development of cells from multipotent to differentiated cell types may be defined by the appearance and disappearance of a number of histological markers, some of which are desc ⁇ bed herein.
• stem cells, progenitor cells and precursor cells are used herein, these terms should not limit the characte ⁇ zation of the multipotent immortalized cells desc ⁇ bed herein and are not meant to desc ⁇ be terminal steps along the pathway to complete differentiation. That is, for example, it is possible that progenitor cells may be capable of exhibiting characte ⁇ stics of stem cells, depending on environmental conditions, such as culture media. These are relative descriptors on a relative scale of specialization with each differentiating step leading to further restrictions of potential cellular phenotypes.
• a multipotent cell therefore, as referred to herein is a cell with the potential to express multiple phenotypes and includes stem, progenitor and precursor cells.
• Identification of stem cells in the CNS has routinely been performed using an antibody to intermediate filament protein nestin . Lendahl et al, Cell, 60: 585-95 (1990); Reynolds et al, J Neurosci, 12: 4565-74 (1992). Nestin is expressed in proliferating CNS stem/progenitor cells .
• CNS derived stem cells may be used to form differentiated hematopoietic cells .
• stromal marrow stem cells have been used to create cells of the brain, including neurons and astrocytes .
• multipotent immortalized cells it may be possible to also treat diseases associated with or requiring hematopoietic cell replacement, such as a part of chemo- or radiation therapy. It may be possible for the presently provided multipotent immortalized cells to repopulate bone marrow cells and differentiate in vivo or be differentiated in vivo or in vitro to produce hematopoietic cells.
• Preparation of the immortalized multipotent fetal cell lines may generally be earned out according to the following procedures Fetal cells may be collected following elective abortion. Women donating fetuses following abortion will typically be serologically screened for a va ⁇ ety of infectious diseases, including human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, and herpes viruses Types 1 and 2.
• the fetal brain is identified and collected.
• the cells may be prepared as follows: brain tissue is aspirated through a 19 gauge needle and washed twice in Eagle's minimum essential media (EMEM, Gibco, New York, N.Y.). Cells are plated on culture dishes treated with poly-D- lysine (0 1 mg/ml for 5 minutes). The cells are grown on EMEM supplemented with 20% fetal bovine serum, 75 g/ml streptomycin, 75 units/ml penicillin, 1% dextrose and 2 g/ml fungizone
• tissue source for obtaining these populations of cells can be nonfetal (Takahashi, J., Palmer, T. D , Gage, F. H. "Retinoic acid and neurotrophms collaborate to regulate neurogenesis in adult-de ⁇ ved neural stem cell cultures". J. Neurobiol 38:65-81, 1999).
• the cells to be implanted by the methods of the present invention can be immortalized by a variety of techniques. Typically, the cells will be immortalized by introduction of an immortalizing gene followed by passage of the cells through cnsis. This cell population is referred to herein as a NGl population of cells.
• the o ⁇ ginal cell cultures that contain an immortalizing gene, but have not passed through crisis, are expected to contain and produce multipotent, stem and or progenitor cells, neuronal and glial cells. It should be possible to purify these progenitor or multipotent cells from this original cell culture, with the methods described herein, prior to immortalization or even possibly prior to introduction of the immortalizing gene.
• a preferred method, and the method exemplified herein however, involves introduction of an immortalizing gene, culturing the population of CNS derived fetal cells containing the immortalizing gene through crisis to thereby obtaining a more highly enriched population of multipotent, stem or progenitor immortalized CNS derived cells.
• the CNS derived fetal cells Prior to introduction of the immortalizing gene, the CNS derived fetal cells will survive for several months with regular re-feeding, but show little cell proliferation.
• the CNS derived fetal cells are preferably transfected with a replication incompetent SV40 deletion mutant, such as is described in Major (U.S. Patent No. 4,707,448).
• a truncated SV40 can be used as an immortalizing gene .
• the cells may alternatively be immortalized by other techniques that are well known in the art.
• i mortalization by Epstein-Barr virus may be employed, as described in U.S. Pat. No. 4,464,465, incorporated herein by reference.
• Epstein-Barr virus mutants which lack OriP and OriLyt origins of replication are particularly useful.
• Another useful method of immortalization is over-expression of a cellular gene for growth control such as c-myc as described by Bartlett et al., Proc. Natl. Acad. Sci. USA, 85:3255-3259 (1988), incorporated herein by reference.
• cells suitable for further immortalization procedures according to the present invention will be anchorage dependent, will not grow in soft agar, and will not exhibit focus formation.
• the cells will also have a generation time equal to normal human cells in culture and be contact inhibited for growth.
• the present invention provides a population of immortalized multipotent CNS derived fetal cells that are generally referred to herein as a NGl population.
• the SVG cell line deposited with the American Type Culture Collection, Manassas, VA (A.T.C.C. CRL 8621) which is described in U.S. Pat. No. 4,707,448, incorporated herein by reference, is particularly useful as a starting material to make the NGl cell population of the present invention.
• SVG cells or "SVG cell line”
• SVG cell line it is meant cells or a cell line derived from cell line A.T.C.C. CRL 8621.
• derivatives is meant a subclone, replication, or genetically altered mutant of cell line A.T.C.C. CRL 8621.
• the heterogeneous pre-c ⁇ sis human cell line was established by methods provided in patent 4,707,448 and Major et al, Proc Natl Acad Sci U S A, 82: 1257-61 (1985) by using a replication incompetent Gluzman, Cell, 23. 175-82 (1981), o ⁇ gin-defective-mutant (on”) of SV40 virus to express the SV40 immortalizing gene m human fetal multipotent cells.
• SVG provide rapidly growing cultures containing human astroglial cells which are capable of reproducing infectious JC virus following infection or transfection in concentrations and at the same rate as pnmary human fetal glial cells.
• SVG cells are propagated in standard media and passed through the c ⁇ sis pe ⁇ od to be immortalized. Only a small fraction of the heterogeneous parental SVG cells survived c ⁇ sis which is typical for SV40 immortalization Bryan & Reddel, C ⁇ t Rev Oncog, 5 331-57 (1994) Those SVG cells that emerged from c ⁇ sis differed from the parental SVG cells in that they were growth immortalized and thus are considered a distinct cell population termed herein as NGl
• the onset of c ⁇ sis has been found to begin at approximately 33-38 passages of the SVG cells Du ⁇ ng c ⁇ sis, there is very little cell growth and the cells look very large and granular When SVG cells were in c ⁇ sis, there was observable cell death when cells are viewed under the microscope When cells in c ⁇ sis are fed twice weekly and kept at a cell density > 1 5xl06/T75 flask, a rare colony of cells sometimes will emerge
• SVG cells were grown in "standard medium” including Eagle Minimum Essential Medium (EMEM, BioWhittaker Cat# 12-125F) + 10% FBS (BioWhi t taker Cat# 14-501F) + 2mM 1-glutamme (Quality Biological, Gaithersburg, MD). Passaging of the cells was accomplished by removal of the media and washing 2x with Hanks' balanced salt solution (HBSS. BioWhittaker, Walkersville, MD), 3 minute incubation with 0.25% trypsin-EDTA solution (BioWhittaker) to dissociate the cells. The suspended cells were added to fresh standard media and cent ⁇ fuged at lOOx g at room temperature for 5 minutes.
• EMEM Eagle Minimum Essential Medium
• FBS BioWhi t taker Cat# 14-501F
• 2mM 1-glutamme Quality Biological, Gaithersburg, MD
• Passaging of the cells was accomplished by removal of the media and washing 2x with Hanks' balanced salt solution (HBSS. BioWhittaker, Walkersville
• the cell pellet was then brought up in fresh standard media and plated in a new 75 cm2 flask.
• Pre-c ⁇ sis SVG cells were subcultured every 3-4 days when the flask was confluent, but du ⁇ ng cnsis they only needed to be subcultured every 8-10 days due to slow growth and cell death. While in c ⁇ sis, the SVG cells did not need to be subcultured as frequently, but continued to be seeded at 1.5x106 per 75 cm2 flask. The SVG cells were able to remain in c ⁇ sis for about 2 months before one or more colonies of cells emerged and began growing m the flask.
• NGl post-c ⁇ sis cell line was generated by seeding SVG cells into a designated flask and treating it as a separate cell line as it went through and emerged from c ⁇ sis The NGl cell line is not considered clonal, since it represents a pool of all the colonies that emerged from c ⁇ sis within the flask.
• the present invention provides methods of en ⁇ ching mixed populations of CNS-de ⁇ ved fetal cells for different cell types Specifically, it has been unexpectedly found that the removal of serum from the culture conditions en ⁇ ches the population of cells with a greater number of cells identified as cells which are expected to differentiate to a neuronal lineage While this effect has been demonstrated for the post-c ⁇ sis, NGl cell population, it is also expected to be equally applicable to the pre-c
• the present invention provides methods of identifying and pu ⁇ fying the multipotent fetal CNS-de ⁇ ved cells descnbed herein, as well as the specific cell types.
• a flow cytometer equipped with dual and t ⁇ ple emission filter sets to detect fluorescence is ideally suited to access this complexity and diversity of specific CNS populations in a very rapid and precise manner. While the preferred method of the present invention involves the use of a flow cytometer, other methods of identifying and sorting cells including panning and magnetic beads may be used.
• Tetanus toxin fragment C (TnTx) receptor is a marker of terminally post-mi totic developing neurons (Koulakoff et al, Dev Biol, 100: 350-7 (1983)) that express the tetanus toxin receptor.
• A2B5 antibodies to recognize a mixture of siahc acid residues on the cell surface of specific cell populations. Due to the heterogeneity of the siahc acid residues with which the A2B5 antibodies bind, the designation for A2B5 positive (A2B5+) cells indicated those cells that bind with the A2B5 antibody
• A2B5 antibodies are used as a neuronal and glial progenitor cell marker .
• Neural stem/progenitor cells were identified by the expression of the intermediate filament protein nestin Lendahl et al , Cell, 60 585-95 (1990). Identification of stem cells from the CNS has routinely been performed using an antibody to intermediate filament protein nestin. Lendahl et al , Cell, 60 585-95 (1990), Reynolds et al , J Neurosci, 12 4565-74 (1992) Nestin has been found to also be expressed in proliferating neural stem/progenitor cells . Frede ⁇ ksen & McKay. J Neurosci, 8 1144-51 (1988), Reynolds et al , J Neurosci, 12 4565-74 (1992), Stemple & Anderson. Cell, 71 973-85 (1992, Vescovi et al, Neuron, 11 951-66 (1993).
• the antibody to nestin used in the present expe ⁇ ments is a rabbit polyclonal immunoglobulin designated as nest ⁇ n-331B identifies human nestin (Messam et al, Exp Neurol., 161-585-596 (2000))
• the antibody to human nestin has been characte ⁇ zed to be a marker for CNS stem/progenitor cells that have the potential to become neurons or gha (Messam, et al, Exp Neurol 161 585-596, 2000)
• Antibodies to nestin are available from Boeh ⁇ nger Mannheim Biochemicals, Indianapolis, IN (antibody to rat nestin) or an antibody that recognizes human nestin may be requested from R McKay (NEH, Bethesda, MD)
• the human nestin gene sequence has been cloned Dahlstrand et al , J Cell Sci, 103 589-97 (1992) and is available through publicly available databases such that antibodies to human nestin may be made by means known in the art For example, with a known nestin sequence, it is straightforward to use an epitope mapping program algo ⁇ thm to obtain a highly antigenic peptide fragment.
• 6 up to >50 amino acid length peptide can be synthesized by commercial vendors (Peptides International, Louisville, KY) and conjugated to KLH or another earner molecule to enhance lmmunogenicity of the peptide.
• Adequate amounts > 1 mg / inoculation can be given to rabbits subcutaneously and 3 booster inoculations may be provided typically at 4, 8, 12-16 weeks.
• Jennes & Stumpf Neuroendoc ⁇ ne Peptide Methodology, 42: 665 (1989; Youngblood & Kizer, Neuroendocrine Peptide Methodology, 38: 605 (1989).
• JC virus preferentially infects glial cells, as opposed to neuronal cells. JC virus more preferentially infects oligodendrocytes, as opposed to astrocytes .
• Table 1 NGl and NG3 cell populations, as de ⁇ ved from SVG population
• ChTx+ Cholera toxin
• TnTx+ Tetanus toxin
• A2B5+ glial or neuronal progenitor
• JC virus infects cells of glial lineage
• the 14 specific cell types (type number indicated in parentheses) of the present invention are defined as follows with regard to their expression of the markers described above that could arise from NGl or NG3 cell populations.
• Type 1 cells above can be further divided into nestin+ (Type 13) and nestin- (Type 14) cells that are also TnTxJChTx-.
• TnTxJChTx- type 1 cells in Neurobasal medium + N2 supplements have about 1% A2B5+ cells in the total population ( Figure 3).
• Figure 3 When these type 1 cells are gated out, 95% of the remaining cells are nest ⁇ n+.
• This nest ⁇ n+/TnTx-/ChTx- cell phenotype likely represents stem/progenitor cells that have not entered into either a glial or neuronal pathway. These cells have characteristics consistent with stem/progenitor cells that may have the potential to differentiate into types of neurons or different types of glial cells i.e. astrocytes or oligodendrocytes.
• ChTx+/TnTx- (cell-type 2 or possibly 11) cell phenotype is generally considered a pre- neuronal marker.
• ChTx+/TnTx+ type 4 cells identifies cells as post mitotic neurons. These type 4 cells predominantly go along a neuronal pathway, particularly if instructed in the presence of factors which potentiates neuronal growth.
• A2B5 positive cells can be either neurons or glial cells.
• ChTx+ and/or TnTx+ (cell types
• A2B5+/ChTx- or A2B5+/TnTx- cells are likely in a glial lineage unless instructed to become neuronal.
• A2B5+/ChTx+ cells (cell type 10) can differentiate into either glial or neuronal pathways.
• A2B5JTnTx+ (cell type 8) identifies cells as likely to be neurons.
• the NGl cells maintained on EMEM 10% sustains cells that are phenotypically neuronal or glial, but when placed into a neuron environment i.e. Neurobasal + N2 supplements, will preferentially develop into neurons.
• Neurobasal + N2 supplements There were also the TnTxJChTx- type 1 cells that were nest ⁇ n+. This population is enhanced in the Neurobasal + N2 supplements condition.
• the shift in phenotypes in these cultures, as instructed by metabolic conditions or factors in the medium, indicates that the NGl cells in culture are maintained as a heterogeneous population of cells with precursor properties able to differentiate by specific cues into either more mature glial or neuronal pathways.
• the present invention is based, in part, on the discovery that the cells of the populations desc ⁇ bed herein are not a single phenotype and that multiple phenotypes can anse by intentional alteration ('instruction') of the environment or culture of the populations.
• a method of the present invention includes analysis of the phenotypes of the cells of these populations with at least 3 markers, preferably in combination, to define preferred phenotypes of cells of the invention (Figure 2)
• the present invention provides a composition containing one or more of these cell types, preferably in a pharmaceutically acceptable excipient. These markers have also been useful in demonstrating the method of ennchment of cell populations, which is a further embodiment of the present invention. Specifically, as further detailed in the examples, a NGl population of cells was charactenzed as shown in Table 2 as a function of the culture conditions.
• the cells of the present invention may be prepared for implantation by suspending the cells in a physiologically compatible carrier, such as cell culture medium (e.g., Eagle's minimal essential media) or phosphate buffered saline.
• a physiologically compatible carrier such as cell culture medium (e.g., Eagle's minimal essential media) or phosphate buffered saline.
• Cell density may generally be about 10 ⁇ to 10 ⁇ cells/ml.
• the cell suspension is gently rocked prior to implantation.
• the volume of cell suspension to be implanted will vary depending on the site of implantation, treatment goal, and cell density in the solution.
• the amount of cells transplanted into the patient or host will be a "therapeutically effective amount.”
• a therapeutically effective amount refers to the number of transplanted cells which are required to effect treatment of the particular disorder for which treatment is sought.
• transplantation of a therapeutically effective amount of cells will typically produce a reduction in the amount and/or severity of the symptoms associated with that disorder, e.g., rigidity, akinesia and gait disorder.
• the amount of cells to be administered in each injection will be sufficient to achieve this effective amount.
• Several injections may be used in each host. Persons of skill will understand how to determine proper cell dosages.
• the cells that are useful for transplantation may be transfected with, and capable of expressing, a heterologous nucleic acid sequence which encodes a neurologically relevant peptide.
• heterologous as used to describe the nucleic acids herein, generally refers to a sequence which, as a whole, is not naturally occurring within the cell line transfected with that sequence.
• the heterologous sequence may comprise a segment which is entirely foreign to the cell line, or alternatively, may comprise a native segment which is incorporated within the cell line in a non-native fashion, e.g., linked to a non-native promoter/enhancer sequence, linked to a native promoter which is not typically associated with the segment, or provided in multiple copies where the cell line normally provides one or no copies.
• the nucleic acid sequence will be operably linked to a transcriptional promoter and a transcriptional terminator.
• a DNA segment is operably linked when it is placed into a functional relationship with another DNA segment.
• a promoter or enhancer is operably linked to a coding sequence if it stimulates the transcription of the sequence;
• DNA for a signal sequence is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates m the secretion of the polypeptide.
• DNA sequences that are operably linked are contiguous, and in the case of a signal sequence both contiguous and in reading phase
• enhancers need not be contiguous with the coding sequences whose transcnption they control.
• Linking is accomplished by hgation at convenient rest ⁇ ction sites or at adapters or linkers inserted in lieu thereof
• the DNA sequence may also be linked to a transc ⁇ ptional enhancer.
• Expression of the DNA in the implanted cells may be constitutive or inducible
• a vanety of expression vectors having these characte ⁇ stics may cany the DNA for transfection of the cells, such as plasmid vectors pTK2, pHyg, and pRSVneo, simian virus 40 vectors, bovine papillomavirus vectors or Epstein-Barr virus vectors, as desc ⁇ bed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Sp ⁇ ng Harbor Press, 1988.
• vectors are may also include the vectors pcDNA3 1 and EF-1 that are commercially available (Invitrogen, Carlsbad, CA) or previously incorporated herein by reference.
• the vectors may be introduced into the cells by standard methods, such as electroporation, calcium phosphate - mediated transfection, polybrene transfection, cationic hpids Feigner & Gadek, Proc Natl Acad Sci USA, 84 7413 (1987) and the like, as desc ⁇ bed in Sambrook et al , Molecular Cloning, A Laboratory Manual, Cold Sp ⁇ ng Harbor Press, 1988
• the present invention contemplates the use of promoter systems that can be regulated by the exogenous addition of compounds
• regulatable promoter systems allow some control of the dose of the encoded peptide/protein
• Four separate leading technologies currently exist and have been developed for regulating the expression of target genes in vitro and in vivo include (1) the tetracyc ne based gene switch, (2) the RU486 based gene switch, (3) the ecdysone based gene switch and (4) the FK1012 (rapamycin) based gene switch.
• the peptide encoded by the nucleic acid may generally be a directly therapeutic compound, such as a movement inhibitor in the treatment of Huntington's chorea.
• the peptide encoded by the nucleic acid may be selected to supplement or replace deficient production of the peptide by the endogenous tissues of the host, which deficiency is a cause of the symptoms of a particular disorder.
• the cells or cell populations act as an artificial source of the peptide.
• the peptide may be an enzyme that catalyzes the production of a therapeutic, or neurologically relevant compounds. Again, such compounds may be exogenous to the host's system, or may be an endogenous compound whose synthesis pathway is otherwise impaired. In this latter case, production of the peptide within the CNS of the host provides supplemental pathways for the production of the compound.
• the immortalized human fetal neuro-derived cell lines are transfected with a nucleic acid that encodes a tyrosine hydroxylase enzyme.
• Tyrosine hydroxylase catalyzes the synthesis of L-dopa from tyrosine.
• Dopamine has been demonstrated to be effective in the treatment of Parkinsonism.
• the nucleic acid may also encode a trophic factor such as a nerve growth factor, an inhibitory growth factor, or a cytokine useful in the treatment of brain tumors. Due to their ability to enhance neural regeneration and produce and secrete L-dopa, the cells and cell populations of the present invention are particularly useful in the treatment of central nervous system disorders which are associated with the loss of dopaminergic cells in the CNS of the host, such as Parkinsonism.
• a trophic factor such as a nerve growth factor, an inhibitory growth factor, or a cytokine useful in the treatment of brain tumors. Due to their ability to enhance neural regeneration and produce and secrete L-dopa, the cells and cell populations of the present invention are particularly useful in the treatment of central nervous system disorders which are associated with the loss of dopaminergic cells in the CNS of the host, such as Parkinsonism.
• the cells or cell populations of the present invention may be implanted within the parenchyma of the brain, in a space containing cerebrospinal fluid, such as the sub-arachnoid space or ventricles, or e traneurally.
• cerebrospinal fluid such as the sub-arachnoid space or ventricles
• e traneurally is intended to indicate regions of the host which are not within the central nervous system or peripheral nervous tissue, such as the celiac ganglion or sciatic nerve. "Extraneural" regions may contain peripheral nerves.
• Central nervous system is meant to include all structures within the dura mater.
• stereotaxic methods will generally be used as described in Leksell and Jernberg, Acta Neurochir., 52: 1-7 (1980) and Leksell et al., J. Neurosurg., 66:626-629 (1987), both of which are incorporated herein by reference.
• Localization of target regions will generally include pre- implantation MRI as described in Leksell et al., J. Neurol. Neurosurg. Psychiatry, 48: 14-18 (1985), incorporated herein by reference.
• Target coordinates will be determined from the pre- implantation MRI.
• the viability of the cells may be assessed as described by Brundin et al., Brain Res., 331:251-259 (1985), incorporated herein by reference. Briefly, sample aliquots of the cell suspension are mixed on a glass slide with of a mixture of acridine orange and ethidium bromide at the prescribed ratio of each component in 0.9% saline; Sigma). The suspension is transferred to a hemocytometer, and viable and non-viable cells were visually counted using a fluorescence microscope under epi-illumination at 390 nm. combined with white light trans-illumination to visualize the counting chamber grid.
• Cell suspensions should generally contain more than about 50%, preferably 75%, more preferably 85%, alternatively 95%, viable cells.
• Injections will generally be made with ste ⁇ hzed 10 :1 Hamilton synnges having 23-27 gauge needles.
• the sy ⁇ nge, loaded with cells, are mounted directly into the head of a stereotaxic frame.
• the injection needle is lowered to predetermined coordinates through small burr holes in the cranium, 40-50 :1 of suspension are deposited at the rate of about 1-2 :l/rmn. and a further 2-5 mm. are allowed for diffusion pnor to slow retraction of the needle.
• at least two separate deposits will be made, separated by 1-3 mm, along the same needle penetration, and up to 5 deposits scattered over the target area can readily be made in the same operation.
• the injection may be performed manually or by an infusion pump.
• the host is removed from the frame and the wound is sutured.
• Prophylactic antibiotics or immunosuppressive therapy may be administered as needed.
• cells may be transfected to express a therapeutic compound and implanted in the vent ⁇ cles or lumbar theca.
• the therapeutic compound is secreted by the cells, natural circulation of the cerebrospinal fluid washes the therapeutic compound throughout the central nervous system providing a means of generalized treatment.
• Implantation into the ventncles may be accomplished by an open procedure, such as desc ⁇ bed in Madrazo et al , New Engl. J. Med., 316.831-834 (1987) or Penn et al , Neurosurgery, 22.999-1004 (1988), both of which are incorporated herein by reference.
• Implantation of cells into the lumbar theca is most conveniently accomplished by standard procedures similar to instillation of radiographic contrast media or anti-tumor medication via a lumbar puncture.
• cells may be implanted ex traneurally according to the present invention.
• the cells may be implanted percutaneous ly through a needle or endoscope or by an open procedure. Persons of skill will readily appreciate the most appropnate method of implanting cells for particular applications.
• the cells or cell populations may be encapsulated by membranes pnor to implantation.
• the encapsulation provides a barner to the host's immune system and inhibits graft rejection and inflammation.
• Several methods of cell encapsulation may be employed. In some instances, cells will be individually encapsulated. In other instances, many cells will be encapsulated within the same membrane. When the cells will be removed following implantation, the relatively large size of a structure encapsulating many cells within a single membrane provides a convenient means for retneval of the implanted cells Several methods of cell encapsulation are well known in the art, such as desc ⁇ bed in European Patent Publication No. 301,777, or U.S. Pat. Nos.
• a polyamonic seaweed extract a polyamonic seaweed extract
• a solution of divalent cations e.g., calcium chlo ⁇ de
• the gel beads are incubated with a high molecular weight (MW 60-500x 10 ⁇ ) concentration (0 03-0 1% w/v) polyamino acid, such as poly-L-lysine, for a bnef penod of time (3-20 minutes) to form a membrane.
• the intenor of the formed capsule is rehquified by treating with sodium citrate
• the single membrane around the cells is highly permeable (MW cut-off 200-400x 10 ⁇ )
• the single membrane capsule containing the cell is incubated in a saline solution for 1-3 hours to allow entrapped sodium alginate to diffuse out of the capsule and expand the capsule to an equi b ⁇ um state
• the resulting alginate-poor capsule is reacted with a low molecular weight polyamino acid (MW 10-30x 10- ⁇ ) such a poly-L-lysine
• any non-toxic water soluble substance that can be gelled to form a shape-retaining mass by a change m conditions in the medium in which it is placed may be employed.
• Such gelling mate ⁇ al generally compnses several chemical moieties which are readily ionized to form anionic or cationic groups so that the surface layers can cross link to form a permanent membrane when exposed to oppositely charged polymers.
• Most polysacchande gums, both natural and synthetic, can be cross-linked by polymers containing positively charged reactive groups such as amino groups.
• the cross-linking biocompatible polymers which may be reacted with the sodium alginate gum include polylysine and other polyamino acids.
• the degree of permeability of the membrane formed may be controlled by careful selection of a polyamino acid having the desired molecular weight.
• Poly-L-lysme (PLL) is the preferred polyme ⁇ c mate ⁇ al but others include chitosan and polyacrylate.
• Molecular weights typically vary from about 10 ⁇ to about 10 ⁇ .
• SVG cells may be obtained from the Ame ⁇ can Type Culture Collection, Manassas, VA. (Accession number ATCC CRL 8621) SVG cells were grown by means known in the art. See, U.S. Patent No. 4,707,448 and . Major et al, Proc Natl Acad Sci U S A, 82: 1257-61 (1985).
• immortalized SVG cells While immortalized SVG cells were used as a starting matenal to exemplify the present invention, it is appreciated that immortalized cells, which are cells that have an ability to divide without limit, may be made by a number of means, including immortalization by genetic modification of the cells to express oncogenes such as ras, SV40T-ant ⁇ gen, v-myc, c-myc and cells that have spontaneously immortalized.
• cells can be immortalized by genetic modification of the cells to express telomerase (Zhu, Wang et al (1999) Proc Natl Acad Sci U S A 96(7). 3723-88)
• Immortalized cells include stem cells that have the ability to divide without limit due, at least part, to their high expression of telomerase. Imortalized cells can be de ⁇ ved from a spontaneous process that is thought to be due to mutat ⁇ on(s).
• the starting mate ⁇ als of the present invention are not limited therefore to the SVG cells desc ⁇ bed here
• SV40 induced-immortalization of human cells was a two step process.
• the first step was an extension of lifespan in culture, where cells underwent a limited number of doublings beyond the point normal cells undergo senescence. After the lifespan extension, the cells entered "c ⁇ sis", a state in which cell division was balanced by cell death.
• the second step involved the rare appearance of a colony of immortalized cells, which were subcultured indefinitely .
• SVG cells were cultured to approximately passage 33-38, they entered "crisis" typical of cells expressing SV40, where there was very little cell growth and the cells looked very large and granular.
• SVG cells were grown in standard medium consisting of Eagle Minimum Essential Medium (EMEM, BioWhittaker Cat# 12-125F) + 10% FBS (BioWhittaker Cat# 14-501F) + 2mM l-glutamine (Quality Biological Cat# 118-084-060) Passaging of the cells was done by removal of the media and washing 2x with Hanks' balanced salt solution (HBSS BioWhittaker Cat#10-543F), 3 minute incubation with 0 25% trypsin (BioWhittaker Cat#17-161E) to dissociate the cells The suspended cells were added to fresh standard media and cent ⁇ fuged at lOOx g at room temperature for 5 minutes The cell pellet was then brought up in fresh standard medium and plated in a new 75 cm2 tissue culture flask
• EMEM Eagle Minimum Essential Medium
• FBS BioWhittaker Cat# 14-501F
• 2mM l-glutamine Quality Biological Cat# 118-084-060
• Pre-cnsis SVG cells were subcultured every 3-4 days when the flask was confluent, but du ⁇ ng c ⁇ sis they only needed to be subcultured every 8-10 days due to slow growth and cell death While in c ⁇ sis, the SVG cells did not need to be subcultured as frequently, but continue to be seeded at 1 5x106 per 75 cm2 flask The SVG cells remained in c ⁇ sis for about 2 months before one or more colonies of cells emerged and began growing in the flask These colonies, which were mixed together with each passage, outgrew the other cells in the flask and constitute the post-c ⁇ sis cell line
• the NGl-type post-c ⁇ sis cell line was generated by seeding SVG cells into a designated flask and treating it as a separate cell line or population as it went through and emerged from cnsis The NGl-type cell line is not considered clonal, since it represents a pool of all the colonies that emerged from c ⁇ sis within the flask
• SVG cells for flow cytomet ⁇ c analysis were propagated in standard medium through passage 50 and then trypsimzed using 0 25% trypsin in EDTA buffer These NGl-type cells were plated into tissue culture grade plastic flasks at approximately 1X104 cells/cm 2 The cells were allowed incubate for 16 hrs or overnight and then refed with either standard medium or in Neurobasal (NB, Gibco, Gaithersburg, MD) + N2 supplements (Gibco) without serum The formulation for Neurobasal medium (NB) is available from Life Technologies (1-800-847-4226; Cat. No. 21103) and has been previously desc ⁇ bed by Brewer et al. (J Neurosci Res, 35.
• inorganic salts CaCl 2 (anhydrous, 1800), Fe(NO 2 ) 3 .9H 2 O (0.2), KC1 (5360), MgCl 2 (anhydrous, 812), NaCl (51300), NaHCO 3 (26000) and NaH 2 PO 4 .H 2 O (900)); other components (D-glucose (25000), Phenol Red (23), HEPES (10000) and sodium pyruvate (230)), ammo acids (L-alamne (20), L-arganine.HCl (400), L-asparagme.H2) (5), L-cysteine (10), L-glutamine (500), glycine (400), L-h ⁇ st ⁇ d ⁇ ne.HCl.H 2 O (200), L-isoleucine (800), L-leucine (800), L-lycine.HCl (5), L
• a lOOx N2 supplement contains: insulin 500 ⁇ g/ml, human transfernn 10,000 ⁇ g/ml, progesterone 0 63 ⁇ g/ml, putrescine 0.16 ⁇ g ml, and selemte 0.52 ⁇ g/ml. Forty eight-seventy two hours later the cells were harvested by trypsmization, resuspended in PBS with 0.1% BSA (bovine serum albumin) at 106 cells/ml and processed for FACS analysis according by the protocol previously desc ⁇ bed Ma ⁇ c et al, Neuromethods, 33. 287-318 (1999) In order to achieve single cell suspensions for application for flow cytometry, trypsin was used instead of papain to obtain better results.
• BSA bovine serum albumin
• FACS fluorescence activated cell sorter
• the p ⁇ mary labels used for FACS analysis included a mixture of 1) TnTx fragment C (Sigma, St Louis, MO) combined with a mouse monoclonal class IgG anti- TnTx fragment C antibody (Boeh ⁇ nger Mannheim, Indianapolis, IN), 2) a mouse monoclonal class IgM antibody A2B5 (Boeh ⁇ nger Mannheim, Indianapolis, IN), 3) cholera toxin-conjugated with FTTC (Sigma, St Louis, MO) and/or 4) a rabbit polyclonal anti-nestin rabbit antibody (Messam CA, Hou J, Major EO Exp Neurol 161 585-596, 2000 "Coexpression of nestin in neural and glial cells in the developing human CNS defined by a human-specific anti-nestin antibody”)
• These p ⁇ mary labels were used to charactenze the cell phenotypes of the present invention
• the secondary labels used for flow cytometry included 1) goat anti-mouse
• FALS Forward angle light scatter
• fluorescence emissions were corrected for color crossover by using electronic compensation FALS properties and fluorescence intensities were each resolved into 1024 channels
• the data were analyzed using Cell Quest Analysis software operating on a FACStation Macintosh-based computer platform (Becton Dickinson, Mountain View, CA).
• NPM Normal Physiological Medium
• a solution of tetanus toxin fragment C (Sigma) and anti-TnTx (Boeh ⁇ nger Mannheim) was prepared by mixing them together at 1:1 ratio (e g., add 1 mg TnTx for every 1 mg of anti-TnTx) and allowing the reaction to occur at 4°C for 30 minutes.
• Cells were resuspended at a final density of 10 million cells/ml in a NPM solution with BSA (NPM/BSA).
• NPM/BSA NPM solution with BSA
• One :g TnTx/anti-TnTx mixture was used for every one million cells and incubated at 4°C for 1 hour.
• the cells were cet ⁇ fuged at 200 x g at 4°C for 10 minutes after which supernatant medium was decanted off and the cells resuspended in 10 ml of NPM/BSA. This wash step was repeated twice more after which the cells were resuspended at a final density of 10 million cells/ml in a NPM/BSA.
• One -g of PE/CY5 -conjugated goat anti-mouse IgGl(Caltag Laborato ⁇ es) and 1 :g FTTC -conjugated ChTx were added for every one million cells and incubated at 4°C for 1 hour.
• the cent ⁇ fuge and wash steps in NPM/BSA were then repeated three times followed by resuspension of the cells at a final density of 10 million cells/ml in a NPM/BSA
• the ChTx +, TnTx + cells went from 2% to 18% of the NGl cells as the medium changed from standard medium to Neurobasal + N2 supplements (Table 3). Consistent with a medium-induced metabolic change from a glial to a neuronal phenotype, there was a complete loss of JC virus sensitivity when the medium was changed to Neurobasal + N2 supplements.
• ChTx Cholera toxin
• TnTx Tetanus toxin
• A2B5 glial or neuronal progenitor
• JC virus infects cells of glial lineage
• the most undifferentiated multipotent cells would be expected to be the cells that do not express any of the markers specific for glial or neuronal lineage. These negative cells (ChTx -, TnTx -, A2B5-) cells were tested for their possible "stem/progenitor" status. Multi-potential stem/progenitor cells can be identified by expression of the intermediate filament protein nestin . Lendahl et al, Cell, 60: 585-95 (1990). About 95% of the TnTxJChTx- cells labeled positive for nestin (Table 2 and Figure 3).
• the populations of cells according to the invention are not limited to being characterized by only these markers.
• other cellular phenotypic markers or binding partners such as the presence or absence of cell surface receptors, neurotransmitter transporters, ion channels, and or ion exchange pumps, for example, may further charactenze the developmental stage of the cells and/or cell populations of the present invention.
• Such further markers are known to those of ordinary skill and have been desc ⁇ bed in, for example, Siegel 1994; Palmer, Takahashi et al. 1997; Sah, Ray et al. 1997; Snyder, Yoon et al.
• neurons can be identified by the expression of N-type calcium channels.
• Oligodendrocytes can be identified by the presence of Galactocerebroside C (GalC) on the cell surface.
• GalC Galactocerebroside C
• Even more specific markers for subpopulations of neurons may include the use of antibodies to receptors such as the ⁇ opioid receptor to obtain cells involved in the sensory/pain pathway.
• FACS can be performed on greater than three labels simultaneously, which allow sorting of cells based on the number of labels that can be resolved in the emission spectrum.
• A2B5JTnTx- (5) A2B5+/TnTx+ (6)
• A2B5-/ChTx- (9) A2B5+/ChTx+ (10)
• Type 1 cells above can be further divided into nest ⁇ n+ (Type 13) and nestin- (Type 14) cells that are also TnTxJChTx-.
• the cells identified above represent distinct cell populations that can be sorted and possibly cultured for further use. There may be uses for an optimal mixture of 2 or more populations of these cells. Standard methods of cultu ⁇ ng cells of the CNS are established. Banker & Goshn, Cellular and Molecular Neuroscience, (1991); Brewer et al, J Neurosci Res, 35: 567-576 (1993); Freshney Culture of Anima Cells, Wiley Liss, NY, NY 3rd Ed. 1994, and are also indicated in other references cited herein.
• medium formulations that can be obtained commercially (Gibco, Gaithersburg, MD) and used for cultunng cells of the CNS.
• Medium formulations could also be supplemented to include growth factors and cytokines including basic or acidic fibroblast growth factor, glial cell line de ⁇ ved neurotrophic factor, leukemia inhibitory factor, interleukins that are commercially available (Gibco, Gaithersburg, MD; R&D Systems, Minneapolis, MN; Sigma, St. Louis, MO) preferably including ILl , EL1V , IL13, insulin, insulin-like growth factors, ciliary neurotrophic factor, brain-denved neurotrophic factor, neurotrophin 4, neurotrophin 3. bone morphogenic proteins, nerve growth factor, epidermal growth factor, platelet de ⁇ ved growth factor, sonic hedgehog proteins and any other commercially available proteins that are known to support survival, proliferation of cells in the CNS.
• Sorting of cells can be based on the expression or lack of surface epitopes.
• Cells were double or triple immunolabeled with anti-A2B5 and/or cholera toxin and/or TnTx and/or nestin antibodies, as described previously and categorized into 14 populations based on their fluorescence signatures determined by FACS electronic gates. Based on the information cited, the 14 cell types indicated above, and mixtures thereof, could be sorted by means of electrically charged saline droplets, which could be deflected by charged plates directly into appropriate test tubes . Marie et al, Neuromethods, 33: 287-318 (1999).
• JC Virus infection of SVG cells cultured in standard medium or in Neurobasal medium + N2 supplements JC Virus has a tropism for cells of a glial phenotype in the human central nervous system.
• JCV does not multiply in cells of a neuronal phenotype either in culture or in situ in the human brain .
• NGl cells at passage 50 were trypsinized from semiconfluent monlayers and plated into multiple 75cm flasks at 1X106 cells/flasks or on glass coverslips in 35mm wells. The cells were allowed to attach overnight in E-MEM medium with 10% FBS. Half of the flasks or coverslips were then refed with Neurobasal medium with N2 supplements and the other half were refed with fresh standard medium. All cultures received 25ug/ml gentamicin as an antibiotic.
• HA assays were conducted using human type O erthryocytes in 2 fold serial dilutions according to the previously published protocol. [Neel et al., Proc Natl Acad Sci 93:2690-2695 (1996).
• the coverslips were washed in PBS, fixed with acetone/methanol and used for immunocytochemistry for JC viral antigens.
• Fixed cells were treated with rabbit polyclonal antiserum to the JCV capsid antigen for 2 hrs at 37°C, washed and treated with mouse fluorescein conjugatged anti-rabbit IgG.
• the cells were examined using a Zeiss inverted ICM 405 microscope with Xenon arc lamp and appropnate filters for UV light examination.
• NGl and NG3 cells secrete different levels of trophic factors
• SVG-denved cells are able to secrete numerous trophic factors including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), transforming growth factor ⁇ l (TGF ⁇ l), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF-2; also known aa basic FGF).
• NGF nerve growth factor
• BDNF brain-derived neurotrophic factor
• NT-3 neurotrophin-3
• TGF ⁇ l transforming growth factor ⁇ l
• VEGF vascular endothelial growth factor
• PDGF platelet-derived growth factor
• FGF-2 fibroblast growth factor 2
• Table 4 indicates that the SVG cell secretion of trophic factors changes when the medium is Neurobasal + N2 compared to EMEM + 10% FBS. Specifically, there is a decreased secretion of BDNF in Neurobasal + N2 medium compared to EMEM + 10% FBS.
• ELISA results from media conditioned with NGl cells (EMEM + 10% FBS) or NG3 cells (Neurobasal + N2). Cells were grown in 6-well plates (35-mm well diameter) in 0.9 ml of media per well. Cytokine concentrations are pg/10 5 cells x 48 hrs. "0" represents optical densities below the detectable threshold of the assay.
• the assays were performed according to the instructions of the supplier using VEGF and PDGF ELISA kits from R&D Systems (Minneapolis, MN) and BDNF and NGF ELISA kits from Promega (Madison, WI). These suppliers also have ELISA kits for NT-3, TGF ⁇ l and FGF-2.
• NGl and NG3 cells can be distinguished morphologically
• NG3 cells Neurosphere-hke clusters
• Figure 3B Normal human neural progenitor (NHNP) cells (Clonetics, Fredenck, MD) are multipotent cells that can differentiate into neuronal or glial cells. These NHNP cells ( Figure 3C) appear morphologically identical to the NG3 cells as clusters of cells.
• the morphological characte ⁇ stics of the NG3 cells are thus consistent with human multipotent stem/progenitor cells as observed previous and termed neurospheres (Reynolds and Weiss, Science, 255:1707-1710 (1992)) It was surp ⁇ sing that the NG3 cells do not require mitotic factors EGF or FGF-2 to form these neurosphere- ke clusters and that the NG3 cells maintain a proliferauve state These neurosphere- ke clusters were formed after culture in NB + N2 for a penod greater than 30 days
• FGF and EGF are mitogens for immortalized neural progenitors. J Neurobiol, 25, 797-807.
• CNS stem cells express a new class of intermediate filament protein. Cell, 60, 585-95.
• Neural stem cells in the adult mammalian forebrain a relatively quiescent subpopulation of subependymal cells. Neuron, 13, 1071-82. NATIONAL INSTITUTES OF HEALTH, Office of the Director, December 1999, Stem
• Marrow stromal cells as a source of progenitor cells for nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis lmperfecta. Proc Natl Acad Sci USA, 95, 1142-7.
• FGF-2 is sufficient to isolate progenitors found in the adult mammalian spinal cord. Exp Neurol, 148, 577-86.
• Tornatore C, Baker-Cairns, B., Yadid, G., Hamilton, R., Meyers, K, Atwood, W., Cummins, A., Tanner, V. & Major, E. (1996). Expression of tyrosine hydroxylase in an immortalized human fetal astrocyte cell line; in vitro characterization and engraftment into the rodent stnatum. Cell Transplant, 5, 145-63.

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