WO2004011021A1 - Procede permettant d'accroitre et/ou d'induire une migration neuronale au moyen de l'erythropoietine - Google Patents

Procede permettant d'accroitre et/ou d'induire une migration neuronale au moyen de l'erythropoietine Download PDF

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WO2004011021A1
WO2004011021A1 PCT/CA2003/001181 CA0301181W WO2004011021A1 WO 2004011021 A1 WO2004011021 A1 WO 2004011021A1 CA 0301181 W CA0301181 W CA 0301181W WO 2004011021 A1 WO2004011021 A1 WO 2004011021A1
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neural stem
growth factor
multipotent neural
erythropoietin
stem cells
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Linda B. ANDERSEN
Tetsuro Shingo
Samuel Weiss
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Stem Cell Therapeutics Inc
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Stem Cell Therapeutics Inc
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Priority to CA002492434A priority Critical patent/CA2492434A1/fr
Priority to AU2003250704A priority patent/AU2003250704A1/en
Priority to US10/558,661 priority patent/US20070111932A1/en
Publication of WO2004011021A1 publication Critical patent/WO2004011021A1/fr
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    • 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
    • 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
    • A61K38/1816—Erythropoietin [EPO]

Definitions

  • This invention relates to methods of enhancing and/or inducing the migration of multipotent neural stem cells and their progeny by exposing the stem cells and their progeny to erythropoietin.
  • additional growth factors are also utilized.
  • S. Bernichtein et al. "S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist," Endocrinology 142(9) :3950-3963 (2001).
  • the role of neural stem cells in the adult is to replace cells that are lost by natural cell death, injury or disease.
  • the low turnover of cells in the mammalian CNS together with the inability of the adult mammalian CNS to generate new neuronal cells in response to the loss of cells following an injury or disease had led to the assumption that the adult mammalian CNS does not contain multipotent neural stem cells.
  • the critical identifying feature of a stem cell is its ability to exhibit self-renewal or to generate more of itself.
  • the simplest definition of a stem cell would be a cell with the capacity for self-maintenance.
  • a more stringent (but still simplistic) definition of a stem cell is provided by Potten and Loeffler (1990) who have defined 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. "
  • CNS disorders encompass numerous afflictions such as neurodegenerative diseases (e.g., Alzheimer's and Parkinson's), brain injury (e.g., stroke, head injury, cerebral palsy) and a large number of CNS dysfunctions (e.g. , depression, epilepsy, and schizophrenia).
  • neurodegenerative diseases e.g., Alzheimer's and Parkinson's
  • brain injury e.g., stroke, head injury, cerebral palsy
  • CNS dysfunctions e.g. , depression, epilepsy, and schizophrenia
  • CNS dysfunctions e.g. , depression, epilepsy, and schizophrenia.
  • the basal ganglia consists of many separate regions, including the striatum (which consists of the caudate and putamen), the globus pallidus, the substantia nigra, substantia innominate, ventral pallidum, nucleus basalis of Meynert, ventral tegmental area and the subthalamic nucleus.
  • striatum which consists of the caudate and putamen
  • Degeneration of a small region called the subthalamic nucleus is associated with violent flinging movements of the extremities in a condition called ballismus, while degeneration in the putamen and globus pallidus is associated with a condition of slow writhing movements or athetosis.
  • degeneration is seen in another area of the basal ganglia, the substantia nigra pars compacta. This area normally sends dopaminergic connections to the dorsal striatum which are important in regulating movement.
  • Alzheimer's Disease there is a profound cellular degeneration of the forebrain and cerebral cortex.
  • nucleus basalis of Meynert Upon closer inspection, a localized degeneration in an area of the basal ganglia, the nucleus basalis of Meynert, appears to be selectively degenerated. This nucleus normally sends cholinergic projections to the cerebral cortex which are thought to participate in cognitive functions including memory.
  • neurological impairment can occur as a result of neural degeneration, such as cerebral palsy, or as a result of CNS trauma, such as stroke and epilepsy.
  • CNS dysfunction In addition to neurodegenerative diseases, brain injuries often result in the loss of neurons, the inappropriate functioning of the affected brain region, and subsequent behavior abnormalities. Probably the largest area of CNS dysfunction (with respect to the number of affected people) is not characterized by a loss of neural cells but rather by an abnormal functioning of existing neural cells. This may be due to inappropriate firing of neurons, or the abnormal synthesis, release, and/or processing of neurotransmitters. These dysfunctions may be the result of well studied and characterized disorders such as depression and epilepsy, or less understood disorders such as neurosis and psychosis.
  • neurological impairment can occur as a result of neural degeneration, such as amyotrophic lateral sclerosis and cerebral palsy, or as a result of CNS trauma such as stroke and epilepsy.
  • Myelination of central and peripheral neurons occurs in a number of pathologies and leads to improper signal conduction within the nervous system.
  • Myelin is a cellular sheath, formed by glial cells, that surrounds axons and axonal processes that enhances various electrochemical properties and provides trophic support to the neuron.
  • Myelin is formed by Schwann cells in the peripheral nervous system and by oligodendrocytes in the central nervous system.
  • MS is the most notable.
  • neural grafts may avert the need not only for constant drug administration, but also for complicated drug delivery systems which arise due to the blood-brain barrier.
  • neural grafts may avert the need not only for constant drug administration, but also for complicated drug delivery systems which arise due to the blood-brain barrier.
  • cells used for transplantation which carry cell surface molecules of a differentiated cell from another host can induce an immune reaction in the host.
  • the cells must be at a stage of development where they are able to form normal neural connections with neighboring cells. For these reasons, initial studies on neurotransplantation centered on the use of fetal cells. Several studies have shown improvements in patients with Parkinson's Disease after receiving implants of fetal CNS tissue.
  • the tissue may already be infected with a bacteria or virus, thus requiring expensive diagnostic testing for each fetus used.
  • diagnostic testing might not uncover all infected tissue.
  • the successful diagnosis of HlV-free tissue is not guaranteed because antibodies to the virus are generally not present until several weeks after infection.
  • the repair of damaged neural tissue may potentially be replaced in a relatively non-invasive fashion, by inducing neural cells to proliferate and differentiate into neurons, astrocytes, and oligodendrocytes in vivo, averting the need for transplantation.
  • simply inducing neural cells to proliferate and differentiate is not always sufficient to treat a neurodegenerative disease or brain injury if the new neurons are not able to reach the lesioned or damaged area.
  • neurons in many regions of the brain are directed to their appropriate destinations by migrating along radial glia. For example, developing neurons migrate outward from the ventricular zone to the cortical plate.
  • neural stem cells in the adult nervous system are in the localized areas, which may be remote from the affected areas, it is particularly desirable to be able to elicit migration of these cells to other affected areas of the brain to replace lost neurons, e.g. , the basal ganglia in Parkinson's Disease.
  • a major object of the present invention is to provide both in vivo and in vitro techniques of enhancing or inducing migration of multipotent neural stem cells or multipotent neural stem cell progeny.
  • the current invention provides a method of enhancing or inducing the migration of multipotent neural stem cell and/or multipotent neural stem cell progeny in a subject comprising administering erythropoietin to a subject in an amount effective to enhance neural stem cell migration.
  • at least one other growth factor besides erythropoietin is administered.
  • the other growth factor is epidermal growth factor.
  • the other growth factor is prolactin.
  • the erythropoietin and growth factors can be ad ⁇ iinistered in a different order.
  • the erythropoietin is administered concurrently with at least one other growth factor.
  • the erythropoietin is administered sequentially with at least one other growth factor.
  • at least one other growth factor is administered prior to the administration of erythropoietin.
  • the at least one other growth factor is administered after the erythropoietin.
  • the subject is suffering from a neurodegenerative disease or brain injury.
  • the subject is suffering from Alzheimer's Disease, Multiple Sclerosis, Huntington's Disease, Amyotrophic Lateral Sclerosis, Parkinson's Disease, surgery, stroke, a physical accident, depression, epilepsy, neurosis, or psychosis.
  • the subject is suffering from a stroke.
  • the multipotent neural stem cells and/or progeny migrate towards a lesioned or damaged area of the brain of the subject. In a particularly preferred embodiment, the multipotent neural stem cells and/or progeny migrate to the basal ganglia.
  • the subject is a mammal. In a preferred embodiment, the subject is a human. In a particularly preferred embodiment, the mammal is an adult.
  • the multipotent neural stem cells and/or progenitor cells which are derived from the multipotent neural stem cells are transplanted into the subject. In a preferred embodiment, the multipotent neural stem cells and/or progenitor cells are incubated with erythropoietin and at least one growth factor before being transplanted into the subject.
  • Another aspect of the invention provides a method of enhancing or inducing the migration of multipotent neural stem cells and/or multipotent neural stem cell progeny comprising exogenously adding to the multipotent neural stem cells and/or multipotent neural stem cell progeny an amount of erythropoietin effective to cause the multipotent neural stem cells and/or multipotent neural stem cell progeny to migrate.
  • at least one other growth factor is added.
  • the other growth factor is epidermal growth factor.
  • the at least one other growth factor is prolactin.
  • the erythropoietin is added concurrently with the at least one other growth factor. In an alternative embodiment, the erythropoietin is added sequentially with the at least one other growth factor. In a particularly preferred embodiment, the other growth factor is added prior to the addition of erythropoietin. In another embodiment, the other growth factor is added after the addition of erythropoietin.
  • Another aspect of the invention provides a method for enhancing or inducing migration of multipotent neural stem cells and/or multipotent neural stem cell progeny, comprising exposing said multipotent neural stem cells and/or multipotent stem cell progeny to hypoxic conditions to induce expression of erythropoietin in order to enhance or induce migration.
  • at least one other growth factor is exogenously added.
  • the other growth factor is epidermal growth factor.
  • the other growth factor is prolactin.
  • the other growth factor is added to said multipotent neural stem cells and/or multipotent neural stem cell progeny concurrently with hypoxic conditions.
  • the other growth factor is added to the multipotent neural stem cells and/or multipotent neural stem cell progeny sequentially with hypoxic conditions.
  • the other growth factor is added to the multipotent neural stem cells and/or multipotent neural stem cell progeny prior to exposure to hypoxic conditions.
  • the other growth factor is added after exposure to hypoxic conditions.
  • Figure 1 Distribution of total BrdU+ cells between the subventricular zone (SVZ) and the striatum (Str) in mice lesioned with ibotenic acid and treated with epidermal growth factor (EGF) and Erythropoietin (Epo). When administered to animals treated with EGF, Epo enhanced the number of neural progenitors in the stratium. (* p ⁇ 0.05).
  • Figure 2 Number of NeuN+/BrdU+ cells (mature neurons) in the striatum of mice lesioned with ibotenic acid and treated with EGF and Epo.
  • EGF enhanced the number of NeuN- /BrdU-h cells in the striatum.
  • Epo further enhanced this effect.
  • Figure 3 A Number of Dcx+/BrdU+ cells (immature neurons or neuronal precursors) in the subventricular zone (SVZ) in mice lesioned with ibotenic acid and treated with EGF and Epo.
  • 3B Number of Dcx+/BrdU+ cells (immature neurons or neuronal precursors) in the striatum of mice lesioned with ibotenic acid and treated with EGF and Epo. When administered to animals treated with EGF, Epo enhanced migration of neuronal precursors into the damaged striatum. (* p ⁇ 0.05).
  • the present invention relates to a method of enhancing or inducing migration of multipotent neural stem cells or multipotent neural stem cell progeny by utilizing erythropoietin in conjunction with at least one other growth factor.
  • multipotent neural stem cell or “neural stem cell” refers to an undifferentiated cell which is capable of self-maintenance. Thus, in essence, a stem cell is capable of dividing without limit.
  • Progenitor cells are non-stem cell progeny of a multipotent neural stem cell. A distinguishing feature of a progenitor cell is that, unlike a stem cell, it has limited proliferative ability and thus does not exhibit self-maintenance. It is committed to a particular path of differentiation and will, under appropriate conditions, eventually differentiate.
  • a neuronal progenitor cell is capable of a limited number of cell divisions before giving rise to differentiated neurons.
  • a glial progenitor cell likewise is capable of a limited number of cell divisions before giving rise to astrocytes or oligodendrocytes.
  • a neural stem cell is multipotent because its progeny include both neuronal and glial progenitor cells and thus is capable of giving rise to neurons, astrocytes, and oligodendrocytes.
  • Multipotent neural stem cell progeny include neuronal precursor cells, glial precursor cells, neurons, and glial cells.
  • a "neurosphere” is a group of cells derived from a single neural stem cell as the result of clonal expansion.
  • Primary neurospheres may be generated by plating as primary cultures brain tissue which contains neural stem cells. The method for culturing neural stem cells to form neurospheres has been described in, e.g., U.S. Patent No. 5,750,376.
  • Secondary neurospheres may be generated by dissociating primary neurospheres and allowing the individual dissociated cells to form neurospheres again.
  • growth factor is meant a substance that affects the growth of a cell or an organism, including proliferation, differentiation, and increases in cell size.
  • a growth factor is a polypeptide which shares substantial sequence identity with a native mammalian growth factor and possesses a biological activity of the native mammalian growth factor.
  • the native mammalian growth factor is a native human growth factor. Having a biological activity of a native mammalian growth factor means having at least one activity of a native mammalian growth factor, such as binding to the same receptor as a particular native mammalian growth factor binds and/or eliciting proliferation and/or differentiation and/or changes in cell size.
  • the growth factor binds to the same receptor as a particular native mammalian growth factor. This includes functional variants of the native mammalian growth factor.
  • a polypeptide which shares substantial sequence identity with a native mammalian growth factor is at least about 30% identical to the native mammalian growth factor at the amino acid level.
  • the growth factor is preferably at least about 40%, more preferably at least about 60%, and most preferably about 60% identical to the native mammalian growth factor at the amino acid level.
  • growth factor encompasses analogs which are deletional, insertional, or substitutional mutants of a native mammalian growth factor.
  • growth factor encompasses the growth factors from other species and naturally occurring and synthetic variants thereof.
  • Erythropoietin is a growth factor.
  • Other exemplary growth factors that may be used in conjunction with Epo in embodiments of the present invention include, inter alia, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulinlike growth factor- 1 and -2 (IGF-1, IGF-2), transforming growth factors a and ⁇ (TGF- ⁇ , TGF- ⁇ ), acidic and basic fibroblast growth factors (a-FGF/FGF-2, b-FGF/FGF-2), interleukins 1, 2, 6, and 8 (IL-1, IL-2, IL-6, IL-8), nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), interleukin-3, hematopoietic colony stimulating factors (CSFs), amphiregulin, interferon- ⁇ (INF- ⁇ ), thyrotropin releasing hormone (TRH), pituitary adenylate cyclase activating polypeptid
  • variants or analogs of these agents which share a substantial identity with a native mammalian growth factor listed above and are capable of bulging the receptor for a native mammalian growth factor, can be used in the present application.
  • PACAP PACAP38
  • PACAP27 PACAP27
  • Any variant or analog that is capable of binding to a receptor for a native mammalian PACAP and shares a substantial sequence identity with either PACAP38 or PACAP27 is suitable for use in the present invention.
  • Particularly useful are the analogs and variants disclosed in, e.g., U.S. Patent Nos. 5,128,242; 5,198,542; 5,208,320; 5,326,860; 5,801,147; and 6,242,563.
  • EGF variants or analogs which share a substantial identity with a native mammalian EGF and are capable of binding to a receptor for the native mammalian EGF, can be used in the present application.
  • EGF variants and analogs include, but are not limited to, the recombinant modified EGF having a deletion of the two C-terminal amino acids and a neutral amino acid substitution at position 51, such as asparagine, glutamine, serine, or alanine (particularly EGF51N or EGF51Q, having asparagine (N) or glutamine (Q) at position 51, respectively; WO 03/040310); the EGF mutein (EGF-X16) in which the His residue at position 16 is replaced with a neutral or acidic amino acid (U.S.
  • Patent No. 6,191,106 the 52-amino acid deletion mutant of EGF which lacks the amino terminal residue of the native EGF (EGF-D); the EGF deletion mutant in which the amino terminal residue as well as the two C-terminal residues (Arg-Leu) are deleted (EGF-B); the EGF-D in which the Met residue at position 21 is oxidized (EGF-C); the EGF-B in which the Met residue at position 21 is oxidized (EGF-A); heparin-binding EGF-like growth factor (HB-EGF); betacellulin; amphiregulin; neuregulin; or a fusion protein comprising any of the above.
  • Other useful EGF analogs or variants are described in WO 03/040310, and U.S. Patent Nos. 6,191,106 and 5,547,935.
  • prolactins Specifically included as prolactins are the naturally occurring prolactin variants, prolactin-related protein, placental lactogens, S179D-human prolactin (Bernichtein et al., 2001), prolactins from various mammalian species, including, but not limited to, human, other primates, rat mouse, sheep, pig, and cattle, and the prolactin mutants described in U.S. Patent Nos. 6,429,186 and 5,955,346.
  • Erythropoietin refers to a polypeptide that shares substantial sequence similarity with native mammalian erythropoietin and possesses a biological activity of the native mammalian erythropoietin, including recombinant erythropoietin or epoietin. Having a biological activity of native mammalian erythropoietin means having at least one activity of a native mammalian erythropoietin, such as binding to the same receptor as the native mammalian erythropoietin binds and/or eliciting proliferation and/or differentiation, and/or changes in cell size.
  • the polypeptide binds to a native mammalian Epo receptor.
  • a native mammalian Epo receptor This includes functional variants of the native mammalian erythropoietin.
  • the native human erythropoietin is a glycoprotein of 165 or 166 amino acids (C-terminal arginine is removed in post-translational modification) and an approximate molecular weight of 30-40 kDa.
  • Erythropoietin can be generated or synthesized using genetic engineering techniques such as those found in U.S. Patent Nos. 4,703,008; 5,441,868; 5,547,993, 5,621,080, 6,618,698, and 6,376,218.
  • a polypeptide which shares "substantial sequence similarity" with the native mammalian erythropoietin is at least about 30% identical with native mammalian erythropoietin at the amino acid level.
  • the erythropoietin is preferably about 40% , more preferably about 60% , yet more preferably at least about 70% , and most preferably, at least about 80% identical with the native mammalian erythropoietin at the amino acid level.
  • erythropoietin encompasses erythropoietin analogs which are deletional, insertional, or substitutional mutants of the native mammalian erythropoietin.
  • erythropoietin encompasses erythropoietins from other species and the naturally occurring and synthetic variants thereof.
  • Percent identity refers to the percentage of amino acid sequence in a protein or polypeptide which are also found in a second sequence when the two sequences are aligned. Percent identity can be determined by any methods or algorithms established in the art, such as L ALIGN or BLAST.
  • a polypeptide possesses the "biological activity" of a growth factor, including erythropoeitin, if it is capable of exerting any of the biological activities of the native mammalian growth factor or being recognized by a polyclonal or monoclonal antibody raised against the native mammalian growth factor.
  • the polypeptide is capable of specifically binding to the receptor for the native growth factor in a receptor binding assay.
  • “Hypoxic conditions” or “hypoxia” refers to a decrease in normal or optimal oxygen conditions for a cell or an organism. Normal or optimal oxygen concentration is 135 mm Hg or 95% air/5% CO 2 . Standard hypoxic conditions comprise an oxygen concentration of about 30-40 mm Hg.
  • Migration refers to the movement of a cell from one location to another.
  • a substance that "enhances" migration increases the speed, distance, or number of cells moving from one location to another over the speed, distance, or number of cells moving in the absence of the substance.
  • the Example below demonstrated that the distance traveled by multipotent neural stem cells and/or multipotent neural stem cell progeny is much greater with Epo and EGF compared to either of these alone.
  • a substance that "induces” migration elicits migration when it would not otherwise occur in the absence of the substance.
  • the present invention can be used to enhance or induce migration of neurons to damaged areas of the CNS.
  • a "neurodegenerative disease or condition” is a disease or a medical condition associated with neuron loss or dysfunction.
  • Examples of neurodegenerative diseases or conditions include neurodegenerative diseases, brain injuries or CNS dysfunctions.
  • Neurodegenerative diseases include, e.g., Alzheimer's Disease, Multiple Sclerosis, Huntington's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease.
  • Brain injuries include, e.g. , injuries to the nervous system due to surgery, stroke, and physical accidents.
  • CNS dysfunctions include, e.g., depression, epilepsy, neurosis, and psychosis.
  • Treating or ameliorating means the reduction or complete removal of the symptoms of a disease or medical condition.
  • an "effective amount” is an amount of a therapeutic agent sufficient to achieve the intended purpose.
  • an effective amount of a growth factor or erythropoietin to enhance the migration of neural stem cells is an amount sufficient, in vivo or in vitro, to result in an enhancement in migration of neural stem cells over the speed, distance, or number in the absence of the growth factor or erythropoietin.
  • An effective amount of a growth factor or erythropoietin to treat or ameliorate a neurodegenerative disease or condition is an amount of the growth factor or erythropoietin sufficient to reduce or remove the symptoms of the neurodegenerative disease or condition.
  • the effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal or subject to receive the therapeutic agent, and the purpose of administration.
  • the effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
  • Epo was able to enhance the speed, number, and distance of migration of neural stem cells and/or their progeny.
  • at least one other growth factor is also used.
  • EGF extracellular protein
  • prolactin is another preferred embodiment of the present invention.
  • the order of administration or addition can be varied.
  • Epo and the other growth factor can be administered or added sequentially or simultaneously.
  • Epo can be administered or added before or after the other growth factor.
  • the multipotent stem cells and/or their progeny can be induced to migrate or the migration to various areas of the brain can be enhanced.
  • the Example below shows the migration of cells from the SVZ to the striatum, other embodiments are also contemplated.
  • the migration of multipotent neural stem cells or their progeny can be enhanced towards other areas of the basal ganglia or any other damaged area of the brain.
  • compositions containing Epo and/or other growth factors can be delivered via any route known in the art, such as orally, or parenterally, e.g. , intravascularly, intramuscularly, transdermally, subcutaneously, or intraperitoneally.
  • the composition is administered parenterally.
  • the composition is delivered directly to the CNS. Direct administration into the CNS can he accomplished via delivery into a ventricle, such as the lateral ventricle.
  • Epo and other growth factors may be administered in vivo to treat subjects suffering from neurodegenerative diseases, brain injuries, or CNS dysfunctions.
  • Alzheimer's Disease, Huntington's Disease, and Parkinson's Disease may be treated according to various embodiments of the invention.
  • the subject may be suffering from a stroke. Because of the prevalence of neurodegenerative disease in adults, the preferred subject is an adult human. However, it is contemplated that younger subjects may also suffer from neurodegenerative disease, or more commonly, traumatic brain injury, and thus will benefit from the present invention.
  • humans are particularly preferred subjects, other species, such as those kept as pets, may also be treated according to an embodiment of the invention.
  • Subjects may be treated with Epo and/or other growth factors, or neural stem cells may be exogenously treated and then transplanted into the subject. A combination of these approaches is also possible.
  • Multipotent neural stem cells can be obtained from embryonic, juvenile, or adult mammalian neural tissue (e.g., mouse and other rodents, and humans and other primates) or from other sources as described in U.S. Patent No. 6,294,346 Bl.
  • Multipotent neural stem cells can be induced to proliferate in vitro or in vivo using the methods disclosed in published PCT application WO 93/01275 and U.S. Pat. Nos. 5,750,376 and 6,294,346 Bl. Briefly, the administration of one or more growth factors can be used to induce the proliferation and differentiation of multipotent neural stem cells.
  • Preferred proliferation-inducing growth factors include epidermal growth factor (EGF), amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor alpha (TGF- ⁇ ), and combinations thereof.
  • EGF epidermal growth factor
  • aFGF or FGF-1 acidic fibroblast growth factor
  • bFGF or FGF-2 basic fibroblast growth factor
  • TGF- ⁇ transforming growth factor alpha
  • a suitable proliferation-inducing growth factor such as EGF (20 ng/ml) is added to the culture medium to induce multipotent neural stem cell proliferation.
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TRH thyrotropin releasing hormone
  • TGF- ⁇ s transforming growth factor betas
  • IGF-1 insulin-like growth factor
  • multipotent neural stem cell proliferation can be detected by the formation of clusters of undifferentiated neural cells termed "neurospheres," which after several days in culture, lift off the floor of the culture dish and float in suspension.
  • Neurospheres results from the proliferation of a single multipotent neural stem cell and is comprised of daughter multipotent neural stem cells and neural progenitor cells.
  • the neurospheres can be dissociated to form a suspension of undifferentiated neural cells and transferred to fresh growth-factor containing medium. This re-initiates proliferation of the stem cells and the formation of new neurospheres. In this manner, an unlimited number of undifferentiated neural stem cell progeny can be produced by the continuous culturing and passaging of the cells in suitable culture conditions.
  • the ability to manipulate the fate of the differentiative pathway of the multipotent neural stem cell progeny to produce more neuronal progenitor cells and neurons is beneficial.
  • Cell cultures with an enriched neuronal-progenitor cell and/or neuron population can be used for transplantation to treat various neurological injuries, diseases or disorders.
  • the neuronal progenitor cells or neurons or a combination thereof can be harvested and transplanted into a patient needing neuronal augmentation.
  • Neuronal progenitor cells are particularly suitable for transplantation because they are still undifferentiated and, unlike differentiated neurons, there are no branched processes which can be damaged during transplantation procedures. Once transplanted, the neuronal progenitor cells can migrate to a damaged area of the brain and differentiate in situ into new, functioning neurons. Suitable transplantation methods are known in the art and are disclosed in U.S. Pat. Nos. 5,750,376 and 6,294,346 Bl.
  • a patient's endogenous multipotent neural stem cells could be induced to proliferate, migrate, and differentiate in situ by administering to the patient a composition comprising one or more growth factors, which induces the patient's neural stem cells to proliferate, and Epo, which instructs the proliferating neural stem cells to produce neuronal progenitor cells which eventually differentiate into neurons and enhances and/or induces migration to other brain regions.
  • a composition comprising one or more growth factors, which induces the patient's neural stem cells to proliferate, and Epo, which instructs the proliferating neural stem cells to produce neuronal progenitor cells which eventually differentiate into neurons and enhances and/or induces migration to other brain regions.
  • Suitable methods for administering a composition to a patient which induces the in situ proliferation of the patient's stem cells are disclosed in U.S. Pat. Nos. 5,750,376 and 6,294,346 Bl.
  • EGF has been shown to induce proliferation of neural stem cells in the subventricular zone (SVZ). Previously, it was demonstrated that after a unilateral striatal lesion, newly-generated cells from both hemispheres migrated towards the damaged area in response to EGF. Epo is able to direct neural stem cells to differentiate into neuronal precursors. (Shingo et al., 2001). A mouse model of neurodegenerative disease was used to determine the effects of EGF and Epo on neural stem cell migration. Following an injury to elicit neurodegeneration, mice were infused with epidermal growth factor (EGF) and erythropoietin to induce proliferation, differentiation, and migration of endogenous neural precursor cells.
  • EGF epidermal growth factor
  • mice were injected once every two (2) hours over a ten-hour period with bromodeoxyuridine (BrdU)(Sigma Chemical Co.), a marker for cell division.
  • bromodeoxyuridine BaxO
  • a small incision was made directly above the pump on the back, the tubing was cut, and the EGF pump was replaced with another pump containing erythropoietin (1000 IU/ml).
  • the cannula was removed from the skull and the wound was closed. The mice were sacrificed immediately following Epo delivery. A series of control mice were infused with the delivery vehicle only, mouse serum albumin.
  • mice were sacrificed via transcardial perfusion under anesthesia whereby the brain is fixed with 4% paraformaldehyde.
  • the brains were removed and subjected to a series of postfixation and cryoprotection steps before being frozen.
  • the brains were cut into 12 ⁇ m sections and immunostained with markers for migrating immature neurons (Doublecortin (Dcx) (Chemicon)) or mature neurons (NeuN (Chemicon)), and for proliferating cells (BrdU). Once brains were sectioned and stained, total BrdU and NeuN/BrdU and Dcx/BrdU cells were counted on every tenth section through the entire forebrain. The data presented below were the results of three independent experiments.
  • Newly generated cells in the striatum adopted a neuronal phenotype in the damaged striatum. Some of the newly generated cells differentiated into mature neurons
  • NeuN + /BrdU + regardless of the infusion conditions. As can be seen in Figure 2, all of the NeuN + /BrdU + mature neurons are found in the striatum, indicating that they may have migrated from the SVZ and differentiated in the striatum.
  • EGF followed by Epo infusion directs the migration of neuronal progenitors from the SVZ into the damaged striatum.
  • vehicle-only-infused mice neuronal progenitors (Dcx+) remain in the SVZ after two weeks of treatment. The same results are seen in Vehicle-Epo-infused mice.
  • EGF-infused mice neuronal progenitors moved laterally into the striatum.
  • EGF-Epo-infused mice most of the neuronal progenitors have migrated into the striatum.
  • Newly generated BrdU+ cells outside the SVZ exhibited extending processes, indicating migration laterally into the striatum (data not shown).
  • Figures 3 A and 3B show the distribution of Dcx + /BrdU + cells between the SVZ and the striatum, respectively.
  • the distribution of cells between the SVZ and the striatum indicates, surprisingly, that Epo enhanced the migration of neuronal precursors into the damaged striatum.
  • Epo when infused in combination with EGF, resulted in increased numbers of newly generated BrdU-f- cells in the ibotenate-lesioned striatum, compared to those generated using EGF alone.
  • Epo-stimulated cells infiltrate the entire striatum indicating they have migrated from their origin in the SVZ. Epo promotes increased migration and survival/differentiation of newly generated neuronal precursors and thus will be useful in therapeutic strategies aimed at enhancing functional recovery from CNS injury or disease.
  • the above example is merely illustrative of the present invention and is considered to be in no way limiting. The skilled artisan will appreciate numerous variations of the present invention.

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Abstract

L'invention concerne des procédés permettant d'accroître et/ou d'induire une migration de cellules souches neurales ou de cellules progénitrices neuronales. Des cellules souches neurales multipotentes sont exposées à l'érythropoiétine accroissant la migration desdites cellules et des cellules progénitrices neuronales. L'érythropoiétine peut être appliquée de manière exogène sur les cellules souches neurales multipotentes ou, de manière alternative, les cellules peuvent être soumises à une agression hypoxique induisant l'expression de l'érythropoiétine par les cellules. Dans un mode de réalisation préféré, des facteurs de croissance supplémentaires, tels que le facteur de croissance de l'épiderme (EGF) et la prolactine, sont utilisés.
PCT/CA2003/001181 2002-07-31 2003-07-31 Procede permettant d'accroitre et/ou d'induire une migration neuronale au moyen de l'erythropoietine Ceased WO2004011021A1 (fr)

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AU2003250704A AU2003250704A1 (en) 2002-07-31 2003-07-31 Method of enhancing and/or inducing neuronal migration using erythropoietin
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1928488A4 (fr) * 2005-09-27 2009-07-22 Stem Cell Therapeutics Corp Regulation par la prolactine de la proliferation des cellules precurseurs d'oligodendrocytes
JP2009530235A (ja) * 2006-03-17 2009-08-27 ステム セル セラピューティクス コーポレイション 神経幹細胞増殖剤および神経幹細胞分化剤の連続投与レジメン
US7846898B2 (en) 2004-02-13 2010-12-07 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
US7884072B2 (en) 2001-09-14 2011-02-08 Stem Cell Therapeutics Inc. Prolactin induced increase in neural stem cell numbers
US8895303B2 (en) 2006-11-13 2014-11-25 Charite-Universitatsmedizin Berlin Method of cell culture and method of treatment comprising a vEPO protein variant

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001028574A2 (fr) * 1999-10-20 2001-04-26 Stem Cells, Inc. Procedes d'induction de la proliferation et la migration in vivo de cellules progenitrices transplantees dans le cerveau

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001028574A2 (fr) * 1999-10-20 2001-04-26 Stem Cells, Inc. Procedes d'induction de la proliferation et la migration in vivo de cellules progenitrices transplantees dans le cerveau

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; December 2000 (2000-12-01), PARK KOOK IN: "Transplantation of neural stem cells: Cellular and gene therapy for hypoxic-ischemic brain injury", XP002259573, Database accession no. PREV200100135413 *
YONSEI MEDICAL JOURNAL, vol. 41, no. 6, December 2000 (2000-12-01), pages 825 - 835, ISSN: 0513-5796 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7884072B2 (en) 2001-09-14 2011-02-08 Stem Cell Therapeutics Inc. Prolactin induced increase in neural stem cell numbers
US7846898B2 (en) 2004-02-13 2010-12-07 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
US8217002B2 (en) 2004-02-13 2012-07-10 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
US8435949B2 (en) 2004-02-13 2013-05-07 Stem Cell Therapeutics Corp. Pheromones and the luteinizing hormone for inducing proliferation of neural stem cells and neurogenesis
EP1928488A4 (fr) * 2005-09-27 2009-07-22 Stem Cell Therapeutics Corp Regulation par la prolactine de la proliferation des cellules precurseurs d'oligodendrocytes
US7964563B2 (en) 2005-09-27 2011-06-21 Stem Cell Therapeutics Corp. Oligodendrocyte precursor cell proliferation regulated by prolactin
JP2009530235A (ja) * 2006-03-17 2009-08-27 ステム セル セラピューティクス コーポレイション 神経幹細胞増殖剤および神経幹細胞分化剤の連続投与レジメン
EP2004212A4 (fr) * 2006-03-17 2010-07-14 Stem Cell Therapeutics Corp Régimes de dosage continu pour agents de prolifération des cellules souches nerveuses et agents de différentiation desdites cellules
US8143220B2 (en) 2006-03-17 2012-03-27 Stem Cell Therapeutics Corp. Dosing regimens for neural stem cell proliferating agents and differentiating agents for the treatment of neurological disorders
US8333974B2 (en) 2006-03-17 2012-12-18 Stem Cell Therapeutics Corp. Continuous dosing regimens for neural stem cell proliferating agents and neural stem cell differentiating agents
AU2007229301B2 (en) * 2006-03-17 2013-08-01 Stem Cell Therapeutics Corp. Continuous dosing regimens for neural stem cell proliferating agents and neural stem cell differentiating agents
US8895303B2 (en) 2006-11-13 2014-11-25 Charite-Universitatsmedizin Berlin Method of cell culture and method of treatment comprising a vEPO protein variant

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