Classifications

• • 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
  • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1808—Epidermal growth factor [EGF] urogastrone
• • 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
• • 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
  • A61P25/08—Antiepileptics; Anticonvulsants
• • 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
• • 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
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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

Definitions

• This invention relates to isolation of human central nervous system stem cells, and methods and media for proliferating, differentiating and transplanting them.
• neural stem cells During development of the central nervous system (“CNS”), multipotent precursor cells, also known as neural stem cells, proliferate, giving rise to transiently dividing progenitor cells that eventually differentiate into the cell types that compose the adult brain. Stem cells (from other tissues) have classically been defined as having the ability to self-renew (i.e., form more stem cells), to proliferate, and to differentiate into multiple different phenotypic lineages. In the case of neural stem cells this includes neurons, astrocytes and oligodendrocytes.
• Potten and Loeffier (Development, 110:1001, 1990) define stem cells as "undifferentiated cells capable of: (a) proliferation, (b) self-maintenance, (c) the production of a large number of differentiated functional progeny, (d) regenerating the tissue after injury, and (e) a flexibility in the use of these options.”
• neural stem cells have been isolated from several mammalian species, including mice, rats, pigs and humans. See, e.g., WO 93/01275, WO 94/09119, WO 94/10292, WO 94/16718 and Cattaneo et al., Mol. Brain Res., 42, pp. 161-66 (1996), all herein incorporated by reference.
• Human CNS neural stem cells like their rodent homologues, when maintained in a mitogen-containing (typically epidermal growth factor or epidermal growth factor plus basic fibroblast growth factor), serum-free culture medium, grow in suspension culture to form aggregates of cells known as "neurospheres.” Human neural stem cells have been shown to have doubling rates of about 30 days. See, e.g., Cattaneo et al., Mol. Brain Res., 42, pp.
• the stem cells Upon removal of the mitogen(s) and provision of a substrate, the stem cells differentiate into neurons, astrocytes and oligodendrocytes. In the prior art, the majority of cells in the differentiated cell population have been identified as astrocytes, with very few neurons ( ⁇ 10%) being observed. There has been recent interest in a population of cells within the adult central nervous system (CNS) which exhibit stem cell properties, in their ability to self-renew and to produce the differentiated mature cell phcnotypcs of the adult CNS.
• CNS central nervous system
• EGF epidermal growth factor
• the invention provides methods for inducing the in vivo migration and proliferation of progenitor cells transplanted to the brain.
• a method for inducing in vivo migration of progenitor cells transplanted to the brain by transplanting progenitor cells to a first locus of the brain of a subject, and inducing in vivo migration of the transplanted cells by infusing a mitogenic growth factor at a second locus of the brain.
• the first locus is in the striatum of the brain
• the second locus at which a mitogenic growth factor is infused is the lateral ventricle of the brain.
• a mitogenic growth factoi infusion induces migration towards the second locus (e g locus of infusion) but docs not induce differentiation of the progenitoi cells
• the locus of transplantation is in the st ⁇ atum of the brain, and a mitogenic growth factor is infused in the lateral ventricle of the brain
• the progenitor cells are mammalian embryonic progenitor cells, and the progenitor cells are cultured in media containing a mitogenic growth factor p ⁇ or to transplantation
• the invention further provides novel human central nervous system stem cells, and methods and media for proliferating, differentiating and transplanting them
• this invention provides novel human stem cells with a doubling rate of between 5-10 days, as well as defined growth media for prolonged proliferation of human neural stem cells
• this invention provides a defined media for differentiation of human neural stem cells so as to enrich for neurons, oligodendrocytes, astrocytes, or a combination thereof
• the invention also provides differentiated cell populations of human neural stem cells that provide previously unobtainable large numbers of neurons, as well as astrocytes and oligodendrocytes
• This invention also provides novel methods for transplanting neural stem cells that improve the viability of the graft upon implantation m a host
• Methods of the present invention can be used in preparation of a medicament for inducing in vivo proliferation and migration of transplanted progenitor cells in the bram
• FIG 1 shows a representation of spheres of proliferating 9FBr human neural stem cells (passage 6) derived from human forebram tissue
• FIG 2 Panel A, shows a growth curve for a human neural stem cell line designated 6.5Fbr cultured m (a) defined media containing EGF, FGF and leukemia inhibitory factor (“LIF”) (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds),
• Panel B shows a growth curve for a human neural stem cell line designated 9Fbr cultured in (a) defined media containing EGF, FGF and LIF (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds);
• Panel C shows a growth curve for a human neural stem cell line designated 9.5Fbr cultured in (a) defined media containing EGF, FGF and LIF (shown as closed diamonds), and (b) the same media but without LIF (shown as open diamonds);
• Panel D shows a growth curve for a human neural stem cell line designated 10.5Fbr cultured in (a) defined media containing EGF, FGF and leuk
• FIG. 3 shows a growth curve for a human neural stem cell line designated 9Fbr cultured in (a) defined media containing EGF and basic fibroblast growth factor (“bFGF”) (shown as open diamonds), and (b) defined media with EGF but without bFGF (shown as closed diamonds).
• bFGF basic fibroblast growth factor
• FIG. 4 shows a graph of cell number versus days in culture for a Mx-1 conditionally immortalized human glioblast line derived from a human neural stem cell line.
• the open squares denote growth in the presence of interferon; the closed diamonds denote growth in the absence of interferon.
• Panels H-K show double labeling with M2 (red) and vimentin (VIM; green) of H), vehicle-infused and I-K)
• EGF-infused with J high power of the region between transplant core and lateral ventricle and K) increased expression of VIM in the SVZ.
• CC corpus callosum
• Str striatum
• LV lateral ventricle
• SM stria medullaris.
• Asterisk indicates the level of cannulae placement and associated damage to the cortex.
• FIG. 8 shows images of ⁇ -galactosidase ( ⁇ gal) labeling of a typical transplant.
• This invention relates to isolation, characterization, proliferation, differentiation and transplantation of CNS neural stem cells.
• the invention further relates to inducing the in vivo migration or proliferation of progenitor cells transplanted to the brain.
• the neural stem cells described and claimed in the applications may be proliferated in suspension culture or in adherent culture.
• human nestin antibody may be used as a marker to identify undifferentiated cells.
• the proliferating cells show little GFAP staining and little ⁇ -tubulin staining (although some staining might be present due to diversity of cells within the spheres).
• most of the cells lose their nestin positive immunoreactivity.
• antibodies specific for various neuronal or glial proteins may be employed to identify the phenotypic properties of the differentiated cells.
• Neurons may be identified using antibodies to neuron specific enolase (“NSE”), neurofilament, tau, beta-tubulin, or other known neuronal markers.
• NSE neuron specific enolase
• Astrocytes may be identified using antibodies to glial fibrillary acidic protein ("GFAP"), or other known astrocytic markers.
• Oligodendrocytes may be identified using antibodies to galactoccrebrosidc, 04, myc n basic protein (“MBP”) or other known ohgodendrocytic markers.
• Glial cells in general may be identified by staining with antibodies, such as the M2 antibody, or other known glial markers.
• the invention provides novel human CNS stem cells isolated from the forebrain.
• Four neural stem cell lines have been isolated from human forebrain, all of which exhibit neural stem cell properties; namely, the cells are self renewing, the cells proliferate for long periods in mitogen containing serum free medium, and the cells, when differentiated, comprise a cell population of neurons, astrocytes and oligodendrocytes. These cells are capable of doubling every 5-10 days, in contrast with the prior art diencephalon- derived human neural stem cells. Reported proliferation rates of diencephalon-derived human neural stem cells approximate one doubling every 30 days. See Cattaneo et al, Mol. Brain Res., 42, pp. 161-66 (1996).
• Neural stem cells can be induced to proliferate and differentiate either by culturing the cells in suspension or on an adherent substrate. See, e.g., U.S. patent 5,750,376 and U.S. patent 5,753,506 (both incorporated herein by reference in their entirety), and prior art medium described therein. Both allografts and autografts are contemplated for transplantation purposes.
• This invention also provides a novel growth media for proliferation of neural stem cells.
• a serum-free or serum-depleted culture medium for the short term and long term proliferation of neural stem cells.
• a number of serum-free or serum-depleted culture media have been developed due to the undesirable effects of serum which can lead to inconsistent culturing results. See, e.g., WO 95/00632 (incorporated herein by reference), and prior art medium described therein.
• EGF epidermal growth factor
• TGF-ot transforming growth factor alpha
• bFGF basic fibroblast growth factor
• the improved medium according to this invention which contains leukemia inhibitory factor ("LIF"), markedly and unexpectedly increases the rate of proliferation of neural stem cells, particularly human neural stem cells.
• LIF leukemia inhibitory factor
• a standard culture medium being serum-free (containing 0-0.49% serum) or serum-depleted (containing 0.5-5.0% serum), known as a "defined” culture medium, such as Iscove's modified Dulbecco's medium (“IMDM”), RPMI, DMEM, Fischer's, alpha medium, Leibovitz's, L-15, NCTC, F-10, F-12, MEM and McCoy's;
• a suitable carbohydrate source such as glucose
• a buffer such as MOPS, HEPES or Tris, preferably HEPES;
• a source of hormones including insulin, transferrin, progesterone, selenium, and putrescine
• one or more growth factors that stimulate proliferation of neural stem cells such as EGF, bFGF, PDGF, NGF, and analogs, derivatives and/or combinations thereof, preferably EGF and bFGF in combination
• LIF LIF
• Standard culture media typically contains a variety of essential components required for cell viability, including inorganic salts, carbohydrates, hormones, essential amino acids, vitamins, and the like.
• DMEM or F-12 is used as the standard culture medium, most preferably a 50/50 mixture of DMEM and F-12. Both media are commercially available (DMEM - Gibco 12100-046; F-12 - Gibco 21700-075).
• a premixed formulation is also commercially available (N2 - Gibco 17502-030). It is advantageous to provide additional glutamine, preferably at about 2 mM. It is also advantageous to provide heparin in the culture medium.
• the conditions for culturing should be as close to physiological as possible.
• the pH of the culture medium is typically between 6-8, preferably about 7, most preferably about 7.4.
• Cells are typically cultured between 30-40°C, preferably between 32-38°C, most preferably between 35-37°C.
• Cells are preferably grown in 5% CO 2 .
• Cells are preferably grown in suspension culture.
• the neural stem cell culture comprises the following components in the indicated concentrations: COMPONENT FINAL CONCENTRATION
• NaHCO 0.1-10 mM, preferably 3 mM
• Serum albumin may also be present in the instant culture medium - although the present medium is generally serum-depleted or serum-free (preferably serum-free), certain serum components which are chemically well defined and highly purified (>95%), such as serum albumin, may be included.
• the human neural stem cells described herein may be cryopreserved according to routine procedures.
• about one to ten million cells are cryopreserved in "freeze" medium that consists of proliferation medium (absent the growth factor mitogens), 10 % BSA (Sigma A3059) and 7.5% DMSO.
• Cells are centrifuged. Growth medium is aspirated and replaced with freeze medium. Cells are resuspended gently as spheres, not as dissociated cells. Cells are slowly frozen, by, e.g., placing in a container at -80°C. Cells are thawed by swirling in a 37°C bath, resuspended in fresh proliferation medium, and grown as usual.
• this invention provides a differentiated cell culture containing previously unobtainable large numbers of neurons, as well as astrocytes and oligodendrocytes.
• the differentiated human diencephalon-derived neural stem cell cultures formed very few neurons (i.e., less than 5-10%).
• neuron concentrations of between 20% and 35% (and much higher in other cases) are routinely achieved in di ilerentialcd human lorcbrain-de ⁇ vcd neural stem cell cultures. This is highlv advantageous, as it permits enrichment of the neuronal population prior to implantation in the host in disease indications where neuronal function has been impaired or lost.
• differentiated neural stem cell cultures have been achieved that are highly enriched in GABAergic neurons.
• GABAcrgic neuron enriched cell cultures are particularly advantageous in the potential therapy of excitotoxic neurodegenerative disorders, such as Huntington's disease or epilepsy.
• various cell surface or intracellular markers may be used.
• human nestin antibody can be used as a marker to identify undifferentiated cells.
• the proliferating cells should show little GFAP staining and little ⁇ -tubulin staining (although some staining might be present due to diversity of cells within the spheres).
• Neurons may be identified using antibodies to neuron specific enolase ("NSE”), neuro filament, tau, ⁇ -tubulin, or other known neuronal markers.
• NSE neuron specific enolase
• Astrocytes may be identified using antibodies to glial fibrillary acidic protein ("GFAP”), or other known astrocytic markers.
• Oligodendrocytes may be identified using antibodies to galactocerebroside, 04, myelin basic protein (“MBP”) or other known oligodendrocytic markers.
• cell phenotypes by identifying compounds characteristically produced by those phenotypes.
• neuro transmitters such as acetylcholine, dopamine, epinephrine, norepinephrine, and the like.
• GABAergic neurons may be identified by their production of glutamic acid decarboxylase ("GAD") or GAB A.
• GAD glutamic acid decarboxylase
• DDC dopa decarboxylase
• TH dopamine or tyrosine hydroxylase
• Cholinergic neurons may be identified by their production of choline acetyltransferase (“ChAT”).
• Hippocampal neurons may be identified by staining with NeuN. It will be appreciated that any suitable known marker for identifying specific neuronal phenotypes may be used.
• the human neural stem cells described herein can be genetically engineered or modified according to known methodology.
• genetic modification refers to the stable or transient alteration of the genotype of a cell by intentional introduction of exogenous DNA.
• DNA may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful DNA sequences.
• genetic modification is not meant to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like.
• a gene of interest i.e., a gene that encodes a biologically active molecule
• a gene of interest can be inserted into a cloning site of a suitable expression vector by using standard techniques. These techniques are well known to those skilled in the art. See, e.g., WO 94/16718, incorporated herein by reference.
• the expression vector containing the gene of interest may then be used to transfect the desired cell line.
• Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection, electroporation, biolistics, or viral transfection may be utilized.
• Commercially available mammalian transfection kits may be purchased from e.g., Stratagene. Human adenoviral transfection may be accomplished as described in Berg et ⁇ l. Exp. Cell Res., 192, pp. (1991). Similarly, lipofectamine-based transfection may be accomplished as described in Cattaneo, Mol. Brain Res., 42, pp. 161-66 (1996).
• host/expression vector combinations may be used to express a gene encoding a biologically active molecule of interest. See, e.g., U.S. Patent 5,545,723, herein incorporated by reference, for suitable cell-based production expression vectors.
• Increased expression of the biologically active molecule can be achieved by increasing or amplifying the transgene copy number using amplification methods well known in the art.
• amplification methods include, e.g., DHFR amplification (see, e.g., Kaufman et al, U.S. Patent 4,470,461) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Patent 5,122,464, and European published application EP 338,841), all herein incorporated by reference.
• the genetically modified neural stem cells are derived from transgenic animals.
• the neural stem cells are genetic modified for the production of a biologically active substance, the substance will preferably be useful for the treatment of a CNS disorder.
• genetically modified neural stem cells can be produced that are capable of secreting a therapeutically effective biologically active molecule in patients. Further contemplated is the production of a biologically active molecule with growth or trophic effect on the transplanted neural stem cells. Further contemplated is inducing differentiation of the cells towards neural cell lineages.
• the genetically modified neural stem cells thus provide cell-based delivery of biological agents of therapeutic value.
• the neural stem cells described herein, and their differentiated progeny may be immortalized or conditionally immortalized using known techniques.
• Conditional immortalization of stem cells is preferred, and most preferably conditional immortalization of their differentiated progeny.
• conditional immortalization techniques contemplated are Tet-conditional immortalization (see WO 96/31242, incorporated herein by reference), and Mx-1 conditional immortalization (see WO 96/02646, incorporated herein by reference).
• This invention also provides methods for differentiating neural stem cells to yield cell cultures enriched with neurons to a degree previously unobtainable.
• the proliferating neurospheres are induced to differentiate by removal of the growth factor mitogens and LIF, and provision of 1% serum, a substrate and a source of ionic charges (e.g., glass cover slip covered with poly-ornithine or extracellular matrix components).
• the preferred base medium for this differentiation protocol excepting the growth factor mitogens and LIF, is otherwise the same as the proliferation medium.
• This differentiation protocol produces a cell culture enriched in neurons. According to this protocol, neuron concentrations of between 20% and 35% have been routinely achieved in differentiated human forebrain-derived neural stem cell cultures.
• the proliferating neurospheres are induced to differentiate by removal of the growth factor mitogens, and provision of 1% serum, a substrate and a source of ionic charges (e.g., glass cover slip covered with poly-ornithine or extracellular matrix components), as well as a mixture of growth factors including PDGF, CNTF, IGF-1, LIF, forskolin, T-3 and NT-3.
• the cocktail of growth factors may be added at the same time as the neurospheres are removed from the proliferation medium, or may be added to the proliferation medium and the cells pre-incubated with the mixture prior to removal from the mitogens.
• This protocol produces a cell culture highly enriched in neurons and enriched in oligodendrocytes. According to this protocol, neuron concentrations of higher than 35% have been routinely achieved in differentiated human forebrain-derived neural stem cell cultures.
• bFGF is trophic for the oligodendrocyte precursor cell line. Oligodendrocytes aie induced under differentiation conditions when passaged with EGF and LIF in proliferating media, without bFGF
• the human stem cells of this invention have numerous uses, including for drug screening, diagnostics, genomics and transplantation Stem cells can be induced to differentiate into the neural cell type of choice using the appropriate media desc ⁇ bed in this invention
• the drug to be tested can be added prior to differentiation to test for developmental inhibition, or added post-differentiation to monitor neural cell-type specific reactions.
• the cells of this invention may be transplanted "naked" into patients according to conventional techniques, into the CNS, as descnbed for example, in U.S. Patents 5,082,670 and 5,618,531, each incorporated herein by reference, or into any other suitable site in the body.
• the human stem cells are transplanted directly into the CNS. Parenchymal and lntrathecal sites are contemplated. It will be appreciated that the exact location in the CNS will vary according to the disease state. Implanted cells may be labeled with bromodeoxyuridine (BrdU) prior to transplantation. As observed in various experiments, cells double stained for a neural cell marker and BrdU in the various grafts indicate differentiation of BrdU stained stem cells into the appropriate differentiated neural cell type (see Example 9). Transplantation of human forebrain derived neural stem cells to the hippocampus produced neurons that were predominantly NeuN staining but GABA negative. The NeuN antibody is known to stain neurons of the hippocampus. GABAergic neurons were formed when these same cell lines were transplanted into the striatum. Thus, transplanted cells respond to environmental clues in both the adult and the neonatal brain.
• bromodeoxyuridine BrdU
• graft viability improves if the transplanted neural stem cells are allowed to aggregate, or to form neurospheres prior to implantation, as compared to transplantation of dissociated single cell suspensions.
• small sized neurospheres are transplanted, approximately 10-500 ⁇ m in diameter, preferably 40-50 ⁇ m in diameter.
• spheres containing about 5-100, preferably 5-20 cells per sphere are preferred.
• the cells may also be encapsulated and used to deliver biologically active molecules, according to known encapsulation technologies, including microencapsulation (see, e.g., U.S. Patents 4,352,883; 4,353,888; and 5,084,350, herein incorporated by reference), (b) macrocncapsulation (sec, e.g., U.S. Patents 5,284,761 , 5,158,881 , 4,976,859 and 4,968,733 and published PCT patent applications WO92/191 5, WO 95/05452, each incorporated herein by reference). If the human neural stem cells are encapsulated, macrocncapsulation as described in
• Cell number in the devices can be varied; preferably each device contains between 10 3 -10 9 cells, most preferably 10 5 to 10 7 cells.
• a large number of macroencapsulation devices may be implanted in the patient; preferably between one to 10 devices.
• the capsular device secretes a biologically active molecule that is therapeutically effective in the patient or that produces a biologically active molecule that has a growth or trophic effect on the transplanted neural stem cells, or that induces differentiation of the neural stem cells towards a particular phenotypic lineage.
• the invention further provides methods of inducing the in vivo migration and proliferation of progenitor cells transplanted to the brain. In one embodiment, in vivo migration of progenitor cells transplanted to a first locus of the brain of a subject is induced by infusing EGF at a second locus of the brain.
• the first locus is in the striatum of the brain
• the second locus at which EGF is infused is the lateral ventricle of the brain.
• EGF infusion induces migration towards the second locus (e.g. locus of infusion) but does not induce differentiation of the progenitor cells.
• in vivo proliferation of progenitor cells transplanted to a locus of the brain of a subject is induced by infusing EGF at or near the locus of transplantation.
• the locus of transplantation is in the striatum of the brain, and EGF is infused in the lateral ventricle of the brain.
• the progenitor cells are mammalian embryonic progenitor cells, and the progenitor cells are cultured in media containing EGF prior to transplantation.
• Any EGF -responsive neural stem cell suitable for treatment of a given neural disease state may be utilized.
• EGF -responsive stem cells may be dissected from the striatal strom, e.g.
• Progenitor cells may be cultured and propagated as described above.
• the cells may be cultured in growth medium containing EGF, and arc prepared for transplantation by collecting small "spheres" of cells, typically of about 15-30 cells, as described above, by ccntrifugation and rcsuspcnding to a desired final concentration, typically 250,000 cclls/ ⁇ L.
• Progenitor cells may also be encapsulated for transplant, as described above. Transplantation of cells to the brain of a subject is performed by stereotaxic surgery under anesthesia. Multiple deposits of cell sphere suspension may be made, for example 500,000 cells per deposit, in the striatum of the brain.
• an infusion cannulae is placed in the ventricle, e.g. lateral ventricle, for EGF infusion, and may be secured using dental cement.
• a minipump may be used to infuse EGF (e.g. dissolved in serum/gentamycin/saline solution) over a period of days.
• EGF e.g. dissolved in serum/gentamycin/saline solution
• the total dose of EGF required to induce migration and proliferation of transplanted cells will vary somewhat from subject to subject, but may be, for example, around about 400 ng/day of EGF infused.
• Diving cells may be labeled for study by BrdU, for example by intraperitoneal injection of BrdU subsequent to cell transplantation.
• encapsulated EGF-producing cells may be implanted in the ventricle adjacent to the progenitor cell transplant.
• EGF-responsive neural progenitor cells are able to respond to EGF after transplantation in vivo.
• Cells transplanted to the adult rat striatum are able to proliferate and migrate toward the source of intraventricular EGF and this response is maintained over the multiple days of EGF infusion.
• Some of these newly generated cells subsequently differentiate into glia, expressing the astrocytic marker GFAP.
• Newly generated BrdU-positive cells within the sub-ventricular zone (SVZ) may be found at a maximal distance of 1 mm rostral to the infusion cannulae, and not further away in the rostral migratory stream on route to the olfactory bulb.
• Transplanted progenitor cells show an active response to EGF in vivo, with proliferation and directed migration of cells away from the graft core toward the EGF source.
• EGF protein is able to penetrate and diffuse through the striatal parenchyma in order to exert an effect on the transplanted cells, which retain their responsiveness to EGF after transplantation in vivo.
• the present invention therefore, provides for the intraventricular delivery of neural growth factors, e.g. EGF, as a promising system by which to manipulate cells after transplantation.
• EGF EGF-induced fibroblast growth factor
• the infusion of EGF in vivo provides a means to manipulate progenitor cells after transplantation, at least in the short term, to direct the cells towards specific differentiation, or directed migration, or to increase their survival.
• This technique will play an important role in overcoming problems associated with the limited migration and differentiation of transplanted cells, and therefore could increase the ability of transplanted neurons to reinnervate host tissue in neural transplantation paradigms.
• the cells and methods of this invention may be useful in the treatment of various neurodegenerative diseases and other disorders. It is contemplated that the cells will replace diseased, damaged or lost tissue in the host. Alternatively, the transplanted tissue may augment the function of the endogenous affected host tissue.
• the transplanted neural stem cells may also be genetically modified to provide a therapeutically effective biologically active molecule.
• Neural stem cells may provide one means of preventing or replacing the cell loss and associated behavioral abnormalities of these disorders.
• Neural stem cells may replace cerebellar neurons lost in cerebellar ataxia, with clinical outcomes readily measurable by methods known in the medical arts.
• Huntington's disease is an autosomal dominant neurodegenerative disease characterized by a relentlessly progressive movement disorder with devastating psychiatric and cognitive deterioration. HD is associated with a consistent and severe atrophy of the neostriatum, which is related to a marked loss of the GABAergic medium-sized spiny projection neurons, the major output neurons of the striatum.
• Intrastriatal injections of excitotoxins such as quinolinic acid (QA) mimic the pattern of selective neuronal vulnerability seen in HD. QA lesions result in motor and cognitive deficits, which are among the major symptoms seen in HD.
• intrastriatal injections of QA have become a useful model of HD and can serve to evaluate novel therapeutic strategies aimed at preventing, attenuating, or reversing neuroanatomical and behavioral changes associated with HD.
• GABAergic neurons arc characteristically lost in Huntington's disease
• treatment of Huntington's patients can be achieved by transplantation of cell cultures enriched in GABAergic neurons derived according to the methods of this invention.
• Epilepsy is also associated with excitotoxicity. Accordingly, GABAergic neurons derived according to this invention are contemplated for transplantation into patients suffering from epilepsy.
• the cells of the present invention can be used in the treatment of various demyelinating and dysmyelinating disorders, such as Pelizaeus-Merzbacher disease, multiple sclerosis, various leukodystrophies, post-traumatic demyelination, and cerebrovascular (CVS) accidents, as well as various neuritis and neuropathies, particularly of the eye.
• the present invention contemplates the use of cell cultures enriched in oligodendrocytes or oligodendrocyte precursor or progenitors, such cultures prepared and transplanted according to this invention to promote remyelination of demyelinated areas in the host.
• the cells of the present invention can also be used in the treatment of various acute and chronic pains, as well as for certain nerve regeneration applications (such as spinal cord injury).
• the present invention also contemplates the use of human stem cells for use in sparing or sprouting of photoreceptors in the eye.
• the local delivery of a neurotrophic factor, such as EFG, to newly transplanted cells in accordance with the invention, to provide a means of regulation in vivo, to guide undifferentiated progenitor cells to divide, migrate or differentiate into specific phenotypes, and may provide a controlled means to increase graft survival, reinnervation of host tissue and associated behavioral recovery, to enhance the effectiveness of transplantation as a potential restorative therapy for neurodegenerative diseases.
• the cells and methods of this invention are intended for use in a mammalian host, recipient, patient, subject or individual, preferably a primate, most preferably a human.
• EXAMPLE 1 MEDIA FOR PROLIFERATING NEURAL STEM CELLS
• Proliferation medium was prepared with the following components in the indicated concentrations: COMPONENT FINAL CONCENTRATION
• EXAMPLE 2 ISOLATION OF HUMAN CNS NEURAL STEM CELLS
• Sample tissue from human embryonic forebrain was collected and dissected in Sweden and kindly provided by Huddinje Sjukhus. Blood samples from the donors were sent for viral testing. Dissections were performed in saline and the selected tissue was placed directly into proliferation medium (as described in Example 1). Tissue was stored at 4°C until dissociated. The tissue was dissociated using a standard glass homogenizer, without the presence of any digesting enzymes. The dissociated cells were counted and seeded into flasks containing proliferation medium. After 5-7 days, the contents of the flasks are centrifuged at 1000 rpm for 2 min. The supernatant was aspirated and the pellet resuspended in 200 ⁇ l of proliferation medium.
• the cell clusters were triturated using a P200 pipetman about 100 times to break up the clusters.
• Cells were reseeded at 75,000-100,000 cells/ml into proliferation medium. Cells were passaged every 6-21 days depending upon the mitogens used and the seeding density. Typically these cells incorporate BrdU, indicative of cell proliferation.
• T75 flask cultures initial volume 20 ml
• cells are "fed” 3 times weekly by addition of 5 ml of proliferation medium.
• Nunc flasks are used for culturing.
• Cells were stained for nestin ( a measure of proliferating neurospheres) as follows. Cells were fixed for 20 min at room temperature with 4% paraformaldehyde. Cells were washed twice for 5 min with 0.1 M PBS, pH 7.4. Cells were pcnneabilized for 2 min with 100% EtOH. The cells were then washed twice for 5 min with 0.1 M PBS. Cell preparations were blocked for 1 hr at room temperature in 5% normal goat serum ("NGS") diluted in 0.1 M PBS, pH 7.4 and 1% Triton X-100 (Sigma X-100) for 1 hr at room temperature with gentle shaking. Cells were incubated with primary antibodies to human nestin (from Dr.
• NGS normal goat serum
• FIG. 1 shows a picture of proliferating spheres (here called "neurospheres") of human forebrain derived neural stem cells.
• the proliferation of four lines of human forebrain derived neural stem cells were evaluated in proliferation medium as described above with LIF present of absent.
• LIF significantly increased the rate of cell proliferation in three of the four lines (6.5 Fbr, 9Fbr, and 10.5FBr). The effect of LIF was most pronounced after about 60 days in vitro.
• the proliferating neurospheres were induced to differentiate by removal of the growth factor mitogens and LIF, and provision of 1% serum, a substrate and a source of ionic glass cover slip covered with poly-ornithine).
• the staining protocol for neurons, astrocytes and oligodendrocytes was as follows: ⁇ -tubulin Staining for Neurons
• cells were also stained with DAPI (a nuclear stain) as follows. Coverslips prepared as above are washed with DAPI solution (diluted 1 : 1000 in 100%> MeOH, Boehringer Mannheim, # 236 276). Coverslips are incubated in DAPI solution for 15 min at 37°C.
• DAPI nuclear stain
• Preparations were then washed twice for 5 min with 0.1 M PBS. Cells were incubated with secondary antibodies, and further processed as described above for ⁇ -tubulin.
• This differentiation protocol produced cell cultures enriched in neurons as follows:
• the proliferating neurospheres were induced to differentiate by removal of the growth factor mitogens and LIF, and provision of 1% serum, a substrate (e.g., glass cover slip or extracellular matrix components), a source of ionic charges (e.g., poly-ornithine) as well as a mixture of growth factors including 10 ng/ml PDGF A/B, 10 ng/ml CNTF, 10 ng/ml IGF-1, 10 ⁇ M forskolin, 30 ng/ml T3, 10 ng/ml LIF and 1 ng/ml NT-3.
• This differentiation protocol produced cell cultures highly enriched in neurons (i.e., greater than 35% of the differentiated cell culture) and enriched in oligodendrocytes.
• cell suspensions were initially cultured in a cocktail of hbFGF, EGF, and LIF, were then placed into altered growth media containing 20 ng/mL hEGF (GIBCO) and 10 ng/mL human leukemia inhibitory factor (hLIF) (R&D Systems), but without hbFGF.
• hbFGF human leukemia inhibitory factor
• the cells initially grew significantly more slowly than the cultures that also contained hbFGF (see FIG. 3). Nonetheless, the cells continued to grow and were passaged as many as 22 times.
• Stem cells were removed from growth medium and induced to differentiate by plating on poly-ornithine coated glass coverslips in differentiation medium supplemented with a growth factor cocktail (hPDGF A B, hCNTF, hGF-1 , forskolin, T3 and hNT-3).
• a growth factor cocktail hPDGF A B, hCNTF, hGF-1 , forskolin, T3 and hNT-3.
• GalC immunoreactivity was seen in these differentiated cultures at levels that far exceeded the number of 04 positive cells seen in the growth factor induction protocol described in Example 4.
• this protocol produced differentiated cell cultures enrichment in oligodendrocytes. Neurons were only occasionally seen, had small processes, and appeared quite immature.
• a glioblast cell line derived from the human neural stem cells described herein was conditionally immortalized using the Mx-1 system described in WO 96/02646.
• the Mx-1 promoter drives expression of the SV40 large T antigen.
• the Mx-1 promoter is induced by interferon. When induced, large T is expressed, and quiescent cells proliferate.
• Human glioblasts were derived from human forebrain neural stem cells as follows. Proliferating human neurospheres were removed from proliferation medium and plated onto poly-omithine plastic (24 well plate) in a mixture of N2 with the mitogens EGF, bFGF and LIF, as well as 0.5% FBS. 0.5 ml of N2 medium and 1%, FBS was added. The cells were incubated overnight. The cells were then transfected with p318 (a plasmid containing the Mx-1 promoter operably linked to the SV 40 large T antigen) using Invitrogen lipid kit (lipids
• the transfection solution contained 6 ⁇ l/ml of lipid and 4 ⁇ l/ml DNA in optiMEM medium.
• the cells were incubated in transfection solution for 5 hours.
• the transfection solution was removed and cells placed into N2 and 1%> FBS and 500 U/ml A/D interferon.
• the cells were fed twice a week. After ten weeks cells were assayed for large T antigen expression. The cells showed robust T antigen staining at this time. As FIG. 4 shows, cell number was higher in the presence of interferon than in the absence of interferon.
• T expression was monitored using immunocytochemistry as follows. Cells were fixed for 20 min at room temperature with 4% paraformaldehyde. Cells were washed twice for
• Cells were incubated with secondary antibodies (goat-anti-mouse biotinylated at 1 :500 from Vector Laboratories, Vectastain Elite ABC mouse IgG kit, PK-6102) diluted in 1%> NGS for 30 min at room temperature. Preparations are washed twice for 5 min with 0.1 M PBS. Preparations are incubated in ABC reagent diluted 1 :500 in 0.1 M PBS, pH 7.4 for 30 min at room temperature. Cells are washed twice for 5 min in 0.1 M PBS, pH 7.4, then washed twice for 5 min in 0.1 M Tris, pH 7.6. Cells are incubated in DAB
• EXAMPLE 7 ENCAPSULATION If the human neural stem cells are encapsulated, then the following procedure may be used:
• the hollow fibers are fabricated from a polyether sulfone (PES) with an outside diameter of 720 ⁇ m and a wall thickness of a 100 ⁇ m (AKZO-Nobel Wuppertal, Germany). These fibers are described in U.S. Patents 4,976,859 and 4,968,733, herein incorporated by reference.
• the fiber may be chosen for its molecular weight cutoff.
• a PES#5 membrane with a MWCO of about 280 kd is used.
• a PES#8 membrane with a MWCO of about 90 kd is used.
• the devices typically comprise:
• the scmipcrmeable membrane used typically has the following characteristics.
• the components of the device are commercially available.
• the LCM glue is available from Ablestik Laboratories (Newark, DE); Luxtrak Adhesives LCM23 and LCM24).
• the tether material is available from Specialty Silicone Fabricators (Robles, CA).
• the tether dimensions are 0.79 mm OD x 0.43 mm ID x length 202 mm.
• the mo ⁇ hology of the device is as follows:
• the inner surface has a permselective skin.
• the wall has an open cell foam structure.
• the outer surface has an open structure, with pores up to 1.5 ⁇ m occupying 30 ⁇ 5% of the outer surface.
• Fiber material is first cut into 5 cm long segments and the distal extremity of each segment sealed with a photopolymerized acrylic glue (LCM-25, ICI). Following sterilization with ethylene oxide and outgassing, the fiber segments are loaded with a suspension of between 10 T0 7 cells, either in a liquid medium, or a hydrogel matrix (e.g., a collagen solution (Zyderm®), alginate, agarose or chitosan) via a Hamilton syringe and a 25 gauge needle through an attached injection port. The proximal end of the capsule is sealed with the same acrylic glue.
• the volume of the device contemplated in the human studies is approximately 15-18 ⁇ l.
• a silicone tether (Specialty Silicone Fabrication, Taunton, MA) (ID: 690 ⁇ m; OD: 1.25 mm) is placed over the proximal end of the fiber allowing easy manipulation and retrieval of the device.
• Human neural stem cells were transplanted into rat brain and assessed graft viability, integration, phenotypic fate of the grafted cells, as well as behavioral changes associated with the grafted cells in lesioned animals. Transplantation was performed according to standard techniques.
• Adult rats were anesthetized with sodium pentobarbitol (45 mg/kg, i.p.) And positioned in a Kopf stereotaxic instrument. ⁇ crline incision was made in the scalp and a hole drilled lor the injection ot cells.
• Rats received implants of unmodified, undifferentiated human neural stem cells into the left striatum using a glass capillary attached to a 10 ⁇ l Hamilton syringe.
• neural stem cells described in this invention are suitable for replacement, because only a structural function would be required by the cells.
• Neural stem cells are implanted in the spinal cord of injured patients to prevent syrinx formation. Outcomes arc measured preferably by MRI imaging. Clinical trial protocols have been written and could easily be modified to include the described neural stem cells.
• Neural stem cells are obtained from ventral mesencephalic tissue from a human fetus aged 8 weeks following routine suction abortion, which is collected into a sterile collection apparatus. A 2x4x1 mm piece of tissue is dissected and dissociated as in Example 2. Neural stem cells are then proliferated. Neural stem cell progeny are used for neurotransplantation into a blood- group matched host with a neurodegenerative disease. Surgery is performed using a BRW computed tomographic (CT) stereotaxic guide. The patient is given local anesthesia suppiemencea with intravenously administered midazolam. The patient undergoes CT scanning to establish the coordinates of the region to receive the transplant.
• CT computed tomographic
• the injection cannula consists of a 17-gauge stainless steel outer cannula with a 19-gauge inner stylet. This is inserted into the brain to the correct coordinates, then removed and replaced with a 19-gauge infusion cannula that has been preloaded with 30 ⁇ l of tissue suspension. The cells are slowly infused at a rate of 3 ⁇ l/min as the cannula is withdrawn. Multiple stereotactic needle passes are made throughout the area of interest, approximately 4 mm apart. The patient is examined by CT scan postoperatively for hemorrhage or edema. Neurological evaluations are performed at various post-operative intervals, as well as PET scans to determine metabolic activity of the implanted cells.
• EXAMPLE 12 GENETIC MODIFICATION OF NEURAL STEM CELL
• Neural stem cell progeny are propagated as described in Example 2.
• the cells are then transfected using a calcium phosphate transfection technique.
• the cells are mechanically dissociated into a single cell suspension and plated on tissue culture-treated dishes at 50% confluence (50,000-75,000 cells/cm 2 ) and allowed to attach overnight.
• the modified calcium phosphate transfection procedure is performed as follows: DNA (15-25 ⁇ g) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA, pH 7.5) diluted to 440 ⁇ l with TE, and 60 ⁇ l of 2M CaCl 2 (pH to 5.8 with 1M HEPES buffer) is added to the DNA/TE buffer. A total of 500 ⁇ l of 2 x HeBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KC1, 1.4 mM Na ll'U.,, 12 ni dextrose, 40 mM HEPES buffer powder, pll 6.92) is added dropwisc to this mix.
• 2 x HeBS HPES-Buffered saline
• 275 mM NaCl 10 mM KC1, 1.4 mM Na ll'U.
• 12 ni dextrose 40 mM HEPES buffer powder, pll 6.92
• the mixture is allowed to stand at room temperature for 20 minutes.
• the cells are washed briefly with 1 x HeBS and 1 ml of the calcium phosphate precipitated DNA solution is added to each plate, and the cells are incubated at 37°C for 20 minutes. Following this incubation, 10 ml of complete medium is added to the cells, and the plates are placed in an incubator (37°C, 9.5%) CO 2 ) for an additional 3-6 hours.
• the DNA and the medium are removed by aspiration at the end of the incubation period, and the cells are washed 3 times with complete growth medium and then returned to the incubator.
• Cells proliferated as in Examples 2 are transfected with expression vectors containing the genes for the FGF-2 receptor or the NGF receptor.
• Vector DNA containing the genes are diluted in 0.1X TE (1 mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of 40 ⁇ g/ml. 22 ⁇ l of the DNA is added to 250 ⁇ l of 2X HBS (280 mM NaCl, 10 mM KC1, 1.5 mM Na 2 HPO 4 2H 2 O, 12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5 ml plastic tube. 31 ⁇ l of 2M CaCl 2 is added slowly and the mixture is incubated for 30 minutes at room temperature.
• 2X HBS 280 mM NaCl, 10 mM KC1, 1.5 mM Na 2 HPO 4 2H 2 O, 12 mM dextrose, 50 mM HEPES
• the cells are centrifuged at 800 g for 5 minutes at 4°C.
• the cells are resuspended in 20 volumes of ice-cold PBS and divided into aliquots of 1 x 10 7 cells, which are again centrifuged.
• Each aliquot of cells is resuspended in 1 ml of the DNA-CaCl 2 suspension, and incubated for 20 minutes at room temperature.
• the cells are then diluted in growth medium and incubated for 6-24 hours at 37°C. in 5%-7%> CO 2 .
• the cells are again centrifuged, washed in PBS and returned to 10 ml of growth medium for 48 hours.
• transfected neural stem cell progeny are transplanted into a human patient using the procedure described in Example 8 or Example 11 , or are used for drug screening procedures as described in the example below.
• BDNF Effects of BDNF on Neuronal and Glial Cell Differentiation and Survival
• Precursor cells were propagated as described in Example 2 and differentiated as described in Example 4.
• BDNF was added at a concentration of 1 ng/ml.
• DIV vitro
• cells were processed for indirect immunocytochcmistry. BrdU labeling was used to monitor proliferation of the neural stem cells.
• the effects of BDNF on neurons, oligodendrocytes and astrocytes were assayed by probing the cultures with antibodies that recognize antigens found on neurons (MAP-2, NSE, NF), oligodendrocytes (04, GalC, MBP) or astrocytes (GFAP).
• BDNF significantly increased the differentiation and survival of neurons over the number observed under control conditions. Astrocyte and oligodendrocyte numbers were not significantly altered from control values.
• BDNF BDNF-treated culture conditions
• neurons tested positive for the presence of substance P and GABA As well as an increase in numbers, neurons grown in BDNF showed a dramatic increase in neurite extension and branching when compared with control examples.
• BDNF BDNF-derived neurotrophic factor
• NSE neurotrophic factor
• NF oligodendrocytes
• GFAP astrocytes
• Exposure to BDNF resulted in a selective increase in the expression of c-fos in neuronal cells.
• Chlo ⁇ romazinc a drug widely used in the treatment of psychiatric illness, is used in concentrations ranging from 10 ng/ml to 1000 ng/ml in place of BDNF in Examples 14A to 14D above.
• the effects of the drug at various concentrations on stem cell proliferation and on stem cell progeny differentiation and survival is monitored. Alterations in gene expression and electrophysiological properties of differentiated neurons are determined.
• EGF-responsive murine progenitor cells would remain responsive to intraventricularly administered EGF after their transplantation in vivo
• embryonic cells generated from transgenic mice carrying the beta-galactosidase enzyme (lacZ) gene under the control of the promoter for myelin basic protein (MBP), and grown in medium containing EGF were transplanted in the medial striatum of the adult rat.
• EGF was administered over seven days after transplantation to assess its affects on the proliferation migration and differentiation of the transplanted cells.
• EGF-responsive stem cells were generated from transgenic mice containing the insertion of the ⁇ -galactosidase enzyme under the control of the MPB promoter (MPB-lacZ).
• the striatal strom was dissected from el4.5-e 15.5 mouse embryos as described previously. See Reynolds et al, Journal ofNeuroscience 12, pp. 4565-4574 (1992).
• the pieces of tissue were broken up into a single cell suspension by mechanical trituration using a flame-polished pasteur pipette, and the cells resuspended growth medium: N2, a defined DMEM:F12-based GIBCO medium containing 0.6%> glucose, 25 ⁇ g/ml insulin, 100 ⁇ g/ml transferrin, 20 nM progesterone, 60 pM putrescine, 30 nM selenium chloride, 2 nM glutamine, 3 mM sodium bicarbonate, 5 mM HEPES and 20 ng/ml human recombinant epidermal growth factor (EGF, R & D Systems).
• the cells grew as free-floating clusters or "spheres", and were passaged by trituration to a single cell suspension every seven days.
• the extracranial end of the cannula was attached to a minipump device (Alzet, 1007D, infusion rate 0.5:l/hour), placed dorsally under the skin of the neck.
• Infusion was over 7 days with either 400ng/day EGF dissolved in a solution of 0.1 %> rat serum and 0.01 % gentamycine in 0.9% saline, or control vehicle without EGF. This gave a total delivery of 3.2 ⁇ g EGF during the study.
• the rats were terminally anaesthetized with an overdose of chloral hydrate, and transcardially perfused with 0.1M phosphate buffered saline (PBS) followed by 250ml 4%> paraformaldehyde in PBS, over 5 minutes.
• PBS phosphate buffered saline
• the brains were removed and immersed in 4% paraformaldehyde overnight before being rinsed and transferred to a 25% sucrose solution in PBS.
• the brains were cut on a freezing microtome at a thickness of 3 ⁇ m. Fluorescence immunohistochemistry was performed on wells of sections, for different combinations of markers. Free floating sections were preincubated in blocking solution of potassium phosphate buffered saline (KPBS) containing 5% normal donkey serum (NDS) and 5% normal rabbit serum (NRS) for one hour. This solution was then replaced with the primary antibodies, made up in blocking solution, for 36 hours at 4°C. For M2, no triton was included in the procedure, antibodies used in this study were: M2 (a mouse-specific glial marker a gift from Dr.
• KPBS potassium phosphate buffered saline
• NDS normal donkey serum
• NRS normal rabbit serum
• a further series of sections were stained for BrdU, but with diamino-benzidine (DAB) as the chromogen. These were mounted, delipidized and dipped in Kodak50 emulsion for 6 weeks to assess thymidine labeling. The sections were then counterstained with cresyl violet before dehydrating and coverslipping with DPX. Using a similar immunohistochemistry protocol, expression of the lacZ transgene was investigated, using an antibody to ⁇ -galactosidase ( ⁇ gal, 1 :500, 5'3'Inc).
• Fluorescent sections were viewed in a Bio-Rad MRC1024UV confocal scanning microscope to enable exact definition of each of the antibodies. Double-labeled cells were verified by collecting serial sections of 1 -2 ⁇ m throughout the specimen.
• volumes of the graft cores were measured using M2 positivity. A full substitutes of 1 :8 sections was taken through each graft, and the area of the densely stained graft core was outlined in each section, and the area calculated using an image analysis system. The areas were then converted to volumes for comparison, using a standard ANOVA test (Statview software).
• transplanted murine progenitors were able to respond to EGF in vivo, in the same manner that they respond under culture conditions.
• the astrocyte marker, 3FAP was used to identify the host glial reaction to the EGF infusion.
• GFAP reactive astrocytes were observed in the periphery of the transplant core, intermingled with M2-positive profiles ( ⁇ I J. 3U
• High power microscopy revealed that a number of the GFAP-positivc cells were also labeled with BrdU, both within the graft core (FIG. 5F) and in the area of striatum between the transplant and lateral ventricle (FIG. 5G), indicating that these cells had divided in the last 16 hours prior to perfusion.
• Vimentin (VIM)-positivity was used to delineate both the immature cells of the SVZ and reactive immature astrocytes present in the host striatum.
• VIM staining of immature cells was restricted to the SVZ and scattered immature astrocytes surrounding the graft core (FIG. 5H).
• EGF-infused animals the VIM-positive SVZ appeared thickened (FIG. 51), indicative of an increase in cell number, with extension of radial-like VIM-positive processes emanating from the SVI into the adjacent striatum (FIG. 5K). Slightly further away from the SVZ (200-400 ⁇ m), individual immature VIM-positive glia were observed (FIG. 5J).
• the antibody nestin was used as a marker of immature progenitor cells. See Lendahl et al, Cell 60, pp. 585-595 (1990). Nestin immunoreactivity showed a similar distribution to vimentin. In vehicle-infused animals nestin-positive cells were restricted to the SVZ, while in EGF-infused this region was thickened indicative of cell division (FIG. 5L). In addition, in animals receiving EGF, numerous nestin-positive profiles were observed in the region between the lateral ventricle and the transplants. High power microscopy revealed double-labeled. BrdU/nestin-positive cells within this area (FIG. 5M).
• the grafts were characteristically dense with very little M2-posltivc staining outside the graft core. M2-positivc profiles were observed emanating from the graft in all directions to a limited extent into the surrounding parenchyma (FIG. 5 B,D,H).
• BrdU/ 3 H-Thymidine double-labeled sections were assessed for colocalization of these two markers.
• the majority of BrdU-labeled cells in the zone between the graft deposit and lateral ventricle did not contain a significant number of silver grains, above background levels.
• scattered BrdU/ 3 H-Thymidine double-labeled cells were occasionally observed (arrowhead in FIG. 7A).
• fluorescence immunohistochemistry showed there was an increase in the number of BrdU-positive cells found within the M2-positivc area in the EGF-infused animals.
• BrdU/M2 double-labeled cells could be found in the graft core (FIG.
• BrdU-labeled cells were double-labeled with M2, however a proportion of these were positive for GFAP as described above, and the remainder did not label with either M2 or GFAP.
• BrdU-positive cells were often found interspersed with GFAP or M2-positive profiles, with only a few occasional cells double-labeled for either marker.
• the cells used in this study were derived from transgenic mice carrying the ⁇ -galactosidase enzyme (lacZ) under the myclin basic protein (MBP) promoter. Sections were stained for ⁇ -galactosidase ( ⁇ gal) to look for expression of the gene. In all animals, regardless of infusion media, there was very low ⁇ gal expression within the graft core. Where positive staining was seen at this site, the expression was punctuate, giving the cells a spherical immature appearance (FIG. 8K). In cases where positive ⁇ gal staining was observed away from the graft core, good expression was seen throughout the cells and primary processes.
• lacZ ⁇ -galactosidase enzyme
• MBP myclin basic protein
• EGF-responsive murine neural progenitor cells are able to respond to EGF after transplantation in vivo.
• Cells transplanted to the adult rat striatum are able to proliferate and migrate toward the source of intraventricular EGF and this response is maintained over the 7 days of EGF infusion.
• Transplanted murine progenitor cells showed an active response to EGF in vivo, with proliferation and directed migration of cells away from the graft core toward the EGF source.
• Two conclusions that can be drawn from these results are that the EGF protein is able to penetrate and diffuse through the striatal parenchyma in order to exert an effect on the transplanted cells, and that the murine cells retained their responsiveness to EGF even after transplantation in vivo. It is possible that addition of neurotrophic factors (see e.g. Ahmed et al, supra; Kirschenbaum et al, Cerebral Cortex 6, pp.
• EGF-responsive progenitor cells are multipotent in vitro; they differentiate preferentially into a glial phenotype after transplantation in either the developing or adult rat brain. See e.g., Hammang et al, Experimental Neurology 147, pp. 84-95 (1997); Winkler et al, Molecular and Cellular Neuroscience 1 1 , pp. 99-116 (1998).
• double-labeling with BrdU and M2 revealed newly generated murine glial cells in the animals which received EGF infusions when compared to the vehicle-infused group, suggesting that EGF stimulated the division of those transplanted progenitors which were committed to a glial phenotype. It is likely, therefore, that the EGF infusion stimulated cell division and migration, but not differentiation of the grafted cells, in vivo in a similar manner to its actions in vitro.
• a number of BrdU-positive cells within the graft area did not express either M2 or GFAP. These cells may belong to one of two populations, either host progenitor cells, or transplanted progenitors, both of which have a more undifferentiated, immature phenotype. It is possible that EGF may also play a role in maintaining the transplanted donor cells in progenitor-like state, similar to its role in culture. See, e.g. Reynolds et al. , Developmental Biology 175, pp. 1-13 (1996).
• a third population of cells found within the graft and region adjacent to the lateral ventricle could be double-labeled with BrdU and GFAP, but not with M2.
• this population represents newly divided glial cells which originate from the host SVZ, as we have previously observed that all GFAP -positive murine progenitors simultaneously express M2 after their differentiation in vitro. See e.g., Winkler et al (1998), supra. Further evidence for this may come from BrdU/nestin double-labeled cells found within the region between the transplant and the lateral ventricle. These cells may have been derived from either the murine or host progenitor cells, which have divided in response to the EGF infusion. The cells used in this transplantation study were obtained from transgenic mice, therefore carried the transgene lacZ under the control of the MBP promoter.
• ⁇ -galactosidase as a sign of oligodendrocyte formation in vivo, revealed a small number of transplanted cells with an immature oligodendrocyte mo ⁇ hology, mainly within the white matter tracts, e.g. the co ⁇ us callosum.
• the small number of lacZ-positivc cells found within the transplants suggests that the majority of the cells had differentiated into astrocytes rather than oligodendrocytes as has been seen previously. See e.g., Winkler et al. (1998), supra.; Winkler et al, Society for Neuroscience Abstracts (1995).
• lacZ-positive cells within the rat brain indicates that the transgene can still be expressed under appropriate conditions after xenotransplantation, and supports the efficacy of using these cells as a tool to enable the introduction of relevant genes to the brain. It remains to be seen whether more differentiated oligodendrocytes are observed after longer survival times.
• neural growth factor infusion can stimulate murine progenitor cells in vivo, after transplantation to the adult rat brain.
• This technique of local delivery of a neurotrophic factor to newly transplanted cells provides a novel means of regulation in vivo, to guide undifferentiated progenitor cells to proliferate, migrate or differentiate into specific phenotypes, and further provides a controlled means to increase graft survival, reinnervation of host tissue and associated behavioral recovery, to enhance the effectiveness of transplantation as a potential restorative therapy for neurodegenerative diseases.

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