WO2000047238A1 - Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures - Google Patents

Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures Download PDF

Info

Publication number
WO2000047238A1
WO2000047238A1 PCT/US2000/003534 US0003534W WO0047238A1 WO 2000047238 A1 WO2000047238 A1 WO 2000047238A1 US 0003534 W US0003534 W US 0003534W WO 0047238 A1 WO0047238 A1 WO 0047238A1
Authority
WO
WIPO (PCT)
Prior art keywords
progenitor cells
cells
neural
recipient
neural progenitor
Prior art date
Application number
PCT/US2000/003534
Other languages
English (en)
Other versions
WO2000047238A9 (fr
Inventor
Michael J. Young
Fred H. Gage
Jasodhara Ray
Simon J. Whiteley
Henry Klassen
Original Assignee
The Schepens Eye Research Institute, Inc.
The Salk Institute For Biological Studies
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Schepens Eye Research Institute, Inc., The Salk Institute For Biological Studies filed Critical The Schepens Eye Research Institute, Inc.
Priority to AU28784/00A priority Critical patent/AU2878400A/en
Priority to JP2000598189A priority patent/JP2002536423A/ja
Priority to EP00907260A priority patent/EP1154801A4/fr
Priority to CA002362476A priority patent/CA2362476A1/fr
Publication of WO2000047238A1 publication Critical patent/WO2000047238A1/fr
Publication of WO2000047238A9 publication Critical patent/WO2000047238A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • 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
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/32—Alcohol-abuse
    • 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
    • A61P25/30—Drugs for disorders of the nervous system for treating abuse or dependence
    • A61P25/36—Opioid-abuse
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • Proliferative cells present in the hippocampus of adult rodent have been isolated, cultured, and transplanted to various sites within the central nervous system (CNS) . These cells are capable of differentiating into neurons when grafted to sites where neurogenesis is known to occur (Suhonen 1996, Shihabuddin 1997, Gage 1995) .
  • CNS central nervous system
  • transplanted neurons to repopulate areas of pathological cell loss within the CNS of adult mammals have largely failed, because donor neural cells tend not to integrate with host cells. For instance, attempts to transplant neurons into the eye have not demonstrated morphological integration with the host retina (del Cerro 1992, Silverman 1992, Aramant 1994, Berson & Jacobiec 1999) .
  • the present invention is directed to methods of treating dystrophic neural tissue, particularly damaged or diseased, differentiated neural tissue, in humans and other animals. It is shown that neural progenitor cells can functionally and morphologically migrate and integrate into mature and immature neural tissue.
  • neural progenitor cells e.g., adult hippocampal progenitor cells (AHPCs)
  • AHPCs adult hippocampal progenitor cells
  • the invention encompasses methods of repairing, replacing, augmenting, or rescuing damaged or diseased, differentiated neural tissue, by introducing adult-derived neural progenitor cells into a human or other animal recipient, whether the recipient is syngeneic (of the same species and genetic strain) , allogeneic (of the same species but a different strain) , or xenogeneic (of a different species) to the donor.
  • the method comprises introducing neural progenitor cells derived from a healthy donor into dystrophic neural tissue of an animal recipient, including an adult or a young animal.
  • One embodiment of the invention encompasses repopulating or rescuing a dystrophic retina or optic nerve with neurons, by introducing neural progenitor cells, e.g., AHPCs, derived from an adult donor animal, into the dystrophic eyes of an animal recipient.
  • the neural progenitor cells may be introduced into dystrophic neural tissue by placement within a recipient's central nervous system, an eye, an optic nerve, or vitreous.
  • the recipient can be either an immature (young) or immature (adult) animal.
  • the neural progenitor cells are derived from adult brain tissue, such as the hippocampus or the ventricular zone.
  • Neural progenitor cells are preferably clonally derived.
  • the neural progenitor cells may, prior to introduction into a dystrophic neural tissue site, have been cultured in vitro in a culture medium comprising at least one trophic factor selected from the group consisting of: a neural growth factor; a neurotrophin; a mitogen; a cytokine; a growth factor; a hormone; and a combination thereof.
  • a trophic factor selected from the group consisting of: a neural growth factor; a neurotrophin; a mitogen; a cytokine; a growth factor; a hormone; and a combination thereof.
  • Figures la-f depict the localization of grafted AHPCs to specific retinal layers in recipient rats of different ages (4 weeks (a-d) ; 10 weeks (e) , and 18 weeks (f) ) ;
  • Figures 2a-i depict confocal images of expression of neuronal markers by grafted AHPCs in animal grafted at: 4 weeks, examined 4 weeks after grafting (a-c) ; at
  • Figures 3a-h depict confocal images of grafted cells treated with anti-synaptophysin/Cy3 (red) antibody in animals grafted at 4 weeks, and examined 4 weeks after grafting; and Figures 4a-c present confocal images of GFP+ neurites projecting, via the host optic fiber layer, into the optic nerve head 4 weeks after grafting (a+b) .
  • the present invention relates to successful transplantation of neural progenitor cells into dystrophic neural tissue.
  • the invention encompasses a method of treating dystrophic neural tissue, comprising introducing neural progenitor cells derived from an adult animal donor into dystrophic neural tissue in an animal recipient, e.g., by grafting or applying adult progenitor cells into tissue affected by the disorder.
  • the recipient may be an young (immature) animal or an adult (mature) animal.
  • the neural progenitor cell donor and recipient may be of different species
  • donor-recipient pairs include, but are not limited, to: a donor rat and a recipient mouse; a donor mouse and a recipient rat; a donor pig and a recipient human.
  • the donor and recipient may be of the same species (e.g., human-to-human, rat-to-rat, mouse-to-mouse) , and be allogeneic (of different strains, i.e., have different histocompatibility genes) or syngeneic (of the same strain, i.e., having identical histocompatibility genes) .
  • dystrophic neural tissue examples include the central nervous system (CNS) and neural tissue of the eye, particularly the retina or optic nerve.
  • the invention encompasses a method of repopulating or rescuing a dystrophic retina with neural cells, comprising introducing neural progenitor cells derived from an adult donor (e.g., AHPCs) into dystrophic neural tissue of an animal recipient.
  • the method is particularly useful for treating dystrophic retinal tissue caused by an optic neuropathy, e.g., glaucoma.
  • the term “dystrophic neural tissue” encompasses damaged, injured, or diseased neural tissue, which neutral tissue includes differentiated neural tissue.
  • a “neuronal disorder” or “neural disorder” is any disorder or disease that involves the nervous system.
  • One type of neuronal disorder is a neurodegenerative disorder.
  • Neurodegenerative disorders include but are not limited to: (1) diseases of central motor systems including degenerative conditions affecting the basal ganglia (e.g., Huntington's disease, Wilson's disease, Striatonigral degeneration, corticobasal ganglionic degeneration, Tourettes syndrome, Parkinson's disease, progressive supranuclear palsy, progressive bulbar palsy, familial spastic paraplegia, spinomuscular atrophy, ALS and variants thereof, dentatorubral atrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellar degeneration, cerebral angiopathy (both hereditary and sporadic)); (2) diseases affecting sensory neurons (e.g., Friedreich's ataxia, diabetes, peripheral neuropathy, retinal neuronal degeneration) ;
  • diseases of central motor systems including degenerative conditions affecting the basal ganglia (e.g., Huntington's disease, Wilson's disease, Striatonigral degeneration, corticobas
  • neurodegenerative pathologies involving multiple neuronal systems and/or brainstem e.g., Alzheimer's disease, AIDS-related dementia, Leigh's disease, diffuse Lewy body disease, epilepsy, Multiple system atrophy, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscinosis, late-degenerative stages of Down's syndrome, Alper's disease, vertigo as result of CNS degeneration;
  • pathologies arising with aging and chronic alcohol or drug abuse e.g., with alcoholism the degeneration of neurons in locus oeruleus, cerebellum, cholinergic basal forebrain; with aging degeneration of cerebellar neurons and conical neurons leading to cognitive and motor impairments; and with chronic amphetamine abuse degeneration of basal ganglia neurons leading to motor impairments
  • pathological changes resulting from focal trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic-ischemic encephalopathy,
  • the presence of a neuronal or neurodegenerative disorder or injury may be indicated by subjective symptoms, such as pain, change in sensation including decreased sensation, muscle weakness, coordination problems, imbalance, neurasthenia, malaise, decreased reaction times, tremors, confusion, poor memory, uncontrollable movement, lack of affect, obsessive/compulsive behavior, aphasia, agnosia, visual neglect, etc.
  • subjective symptoms such as pain, change in sensation including decreased sensation, muscle weakness, coordination problems, imbalance, neurasthenia, malaise, decreased reaction times, tremors, confusion, poor memory, uncontrollable movement, lack of affect, obsessive/compulsive behavior, aphasia, agnosia, visual neglect, etc.
  • objective indicia or signs observable by a physician or a health care provider, overlap with subjective indicia.
  • objective indicia examples include the physician's observation of signs such as decreased reaction time, muscle fasciculations, tremors, rigidity, spasticity, muscle weakness, poor coordination, disorientation, dysphasia, dysarthria, and imbalance. Additionally, objective signs can include laboratory parameters, such as the assessment of neural tissue loss and function by Positron Emission Tomography (PET) or functional Magnetic Resonance Imaging MRI), blood tests, biopsies and electrical studies such as electromyographic data.
  • PET Positron Emission Tomography
  • MRI Magnetic Resonance Imaging
  • Treating" dystrophic neural tissue is intended to encompass repairing, replacing, augmenting, rescuing, or repopulating the diseased or damaged neural tissue, or otherwise compensating for the dystrophic condition of the neural tissue.
  • “Introduction" of neural progenitor cells into dystrophic neural tissue may be accomplished by any means known in the medical arts, including but not limited to grafting and injection. It should be understood that such means of introducing the neural progenitor cells also encompass placing, injecting or grafting them into a site separate and/or apart from the diseased or damaged neural tissue site, since the neural progenitor cells are capable of migrating to and integrating into that dystrophic site. For example, dystrophic retinal or optic nerve tissue can be treated by placing neural progenitor cells into the vitreous of the eye.
  • the neural progenitor cells used in the invention are derived from a healthy adult animal donor, and may come from brain tissue, such as the hippocampus or ventricular zone.
  • adult hippocampal progenitor cells AHPC
  • progenitor cell refers to cells which have the ability to differentiate, including stem cells and progenitor cells. In contrast to undifferentiated cells, differentiated cells have a clearly defined morphology that identifies it as a member of a defined histological type.
  • the cell can be a mammalian cell. In one embodiment, the mammalian cell is a rodent cell.
  • the cell is a primate cell, such as a human cell.
  • Progenitor cells employed herein refer to both undifferentiated cells whose lineal descendants differentiate along the appropriate pathway to produce a fully differentiated phenotype, as well as founder cells of embryonic or other cell lineage, which are undifferentiated cells displaying high proliferative potential, generating a wide variety of differentiated progeny including the principal phenotypes of the tissue, possessing the capacity for self-renewal and retaining their multi- lineage potential over time (Gage et al . (1995) Annu. Rev. Neurosci. 18:159-192, each herein incorporated by reference) . All differentiated cells have, by definition, a progenitor cell type.
  • neural progenitor cells such as neuroblasts are progenitors for neurons and germ cells for gamete cells. Additionally, it is readily appreciated that progenitor cells do not differentiate into only one type of cell. For example, neural progenitor cells give rise primarily to neurons, however, such cells can also rise to astrocytes, glial cells and oligodendrocytes . Those of skill in the art will readily recognize the associated progenitor cells for differentiated cells. Stem cells are capable of dividing to produce two daughter cell types with different fates: one is another stem cell identical to the mother cell, and the other is a lineage progenitor cell which will divide to produce more differentiated cells. In adult mammals, stem cells occur in most tissue systems. For example, the bone marrow gives rise to all blood cells and muscle.
  • the therapeutic benefit of the invention can be evaluated or assessed by any of a number of subjective or objective factors indicating a response of the condition being treated. Such indices include measures of increased neural or neuronal proliferation or more normal function of surviving brain areas. In addition, macroscopic methods of evaluating the effects of the invention can be used which may be invasive or noninvasive. Further examples of evidence of a therapeutic benefit include clinical evaluations of cognitive functions including object identification, increased performance speed of defined tasks as compared to pretreatment performance speeds, and nerve conduction velocity studies.
  • the neural progenitor cells have preferably been cultured in vitro in a culture medium comprising at least one trophic factor, or even combinations of such factors.
  • trophic factor refers to compounds with trophic actions that promote and/or control proliferation, differentiation, migration, survival and/or death (e.g., apoptosis) of their target cells.
  • Such factors include cytokines, neurotrophins, growth factors, mitogens, co-factors, and the like, including epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors, ciliary neurotrophic factor and related molecules, glial-derived growth factor and related molecules, schwanoma-derived growth factor, glial growth factor, stiatal-derived neuronotrophic factor, platelet-derived growth factor, hepatocyte growth factor, scatter factor (HGF-SF) , transforming growth factor-beta and related molecules, neurotransmitters, and hormones.
  • cytokines include epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factors, ciliary neurotrophic factor and related molecules, glial-derived growth factor and related molecules, schwanoma-derived growth factor, glial growth factor, stiatal-derived neuronotrophic factor, platelet-derived growth factor, hepatocyte growth factor, scatter factor (HGF-
  • Trophic factors have a broad range of biological activities and their activity and specificity may be achieved by cooperation with other factors. Although trophic factors are generally active at extremely low concentrations, high concentrations of mitogen together with high cell density are often required to induce proliferation of multipotent neural progenitor cell populations. For example, growth factors for early progenitors may be useful for enhancing the viability of progenitor cells as well as treating disorders by renewal of mature cells from the progenitor cell pool.
  • Preferred trophic factors contemplated for use in the present invention are mitogenic growth factors, like fibroblast growth factor-2 (FGF-2) (Gage, F.H., et al., 1995, Proc. Natl Acad. Sci. USA 92:11879-11883) and epidermal growth factor (EGF) (Lois, C, and Alvarez- Buylla, A., 1993, Proc. Natl. Acad. Sci. USA 90(5):2074- 2077), which induce proliferation and/or propogation of progenitor cells, e.g., neural progenitor cells isolated from the brain.
  • FGF-2 fibroblast growth factor-2
  • EGF epidermal growth factor
  • the neural progenitor cells can be cultivated in the presence of a trophic factor, or combinations of trophic factors.
  • these cells can be cultivated in medium having "neurotrophins" (or “neurotrophic factor”) that promote the survival and functional activity of nerve or glial cells, including a factor that enhances neural differentiation, induces neural proliferation, influences synaptic functions, and/or promotes the survival of neurons that are normally destined to die, during different phases of the development of the central and peripheral nervous system.
  • neurotrophins include, for example, ciliary neurotrophic factor (CNF) , nerve growth factor (NGF) , fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF) , Neurotrophin-3 (NT-3) , glia derived neurotrophic factor (GDNF) , and the like.
  • CNF ciliary neurotrophic factor
  • NGF nerve growth factor
  • FGF fibroblast growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 Neurotrophin-3
  • GDNF glia derived neurotrophic factor
  • neurotropic factors that have been identified include NT-4 , NT-5 , NT-6 , NT-7 , ciliary neuronotrophic factor (CNTF) , Glial cell line-derived neurotrophic factor (GDNF) , and Purpurin.
  • Neuron-specific enolase has been found to be a neuronal survival factor.
  • FGF-2 farnesoid GF-2
  • FGF-2 alone in the adult rat hippocampus has a limited effect on the proliferation of neural stem/progenitor cells (Kuhn et al. (1997); Wagner et al. (1999) each herein incorporated by reference) .
  • the present invention employs FGF and FGF-like factors, including a-FGF, b-FGF such as FGF-2, FGF-4, FGF-6, and the like.
  • a particularly advantageous medium for culturing neural progenitor cells comprises one of the following: fibroblast growth factor (FGF) alone (particularly basic FGF or FGF-2), FGF plus epidermal growth factor (EGF) , or FGF plus EGF plus heparin, which is mitogenic.
  • the neural progenitor cells may be derived by the following steps: (a) isolating fresh neural progenitor cells from an adult donor animal;
  • step (d) cloning individual hippocampal progenitor cell lines from the cultured cells resulting from step (c) .
  • the methods of the invention can further comprise, prior to introducing the neural progenitor cells into a recipient, confirming the lineage potential of each clone of clonally derived adult hippocampal progenitor cells by inducing a sample of said clonally derived hippocampal progenitor cells to differentiate in "conditioned medium", a term of art referring to medium or supernatant removed from cultures of living cells and then filtered.
  • conditioned medium a term of art referring to medium or supernatant removed from cultures of living cells and then filtered.
  • the invention also encompasses a kit for generating neural progenitor cell lines derived from an adult donor animal, comprising the following:
  • a polyornithine/laminin-coated substrate e.g., a coated tissue culture vessel
  • a vector comprising an identifying genetic marker, for incorporation into hippocampal progenitor cells (HPC) isolated from an adult animal, upon culture of those cells (e.g., green fluorescent protein (GFP) ) ;
  • an article of manufacture comprising instructions for cloning at least one hippocampal progenitor cell line from hippocampal progenitor cells isolated from an adult donor animal.
  • Progenitor cells can be grafted into the tissue ex vivo (by cultivating the cells in vitro) or can be cultivated in vivo.
  • the progenitor cells can be native to the dystrophic tissue but propagated and/or proliferated by the administration of trophic factors in vivo or in vitro.
  • the progenitor cells can be propagated or proliferated in vitro, and incorporated or re-incorporated into the dystrophic tissue.
  • trophic factors can be administered to the dystrophic tissue to increase the level of native or transplanted progenitor cells.
  • AHPCs adult rat hippocampal progenitor cells
  • GFP + green fluorescent protein-expressing cells
  • the transplanted cells survived for at least 2 months post-grafting, without provoking a prominent immune response.
  • GFP + cells aligned themselves with the existing cytoarchitecture and exhibited extensive arborization in configurations appropriate for retinal neurons. Similar results were obtained with both immature and visually mature, recipient animals.
  • proliferative cells present in the adult rodent hippocampus can be isolated (Palmer et al . , 1997), cultured (Gage et al., 1995 and 1998), and transplanted into various sites within the CNS, where they can differentiate into neurons (Gage et al., 1995; Shihabuddin et al . , 1997; Suhonen et al . , 1996).
  • the present data indicate that transplanted, adult hippocampal progenitor cells (AHPCs) provide a more effective source of donor material for retinal transplantation. Specifically, the data show that these cells can migrate into the dystrophic retina of adult Royal College of Surgeons (RCS) rats, an extensively studied model of retinal degeneration (LaVail et al.,
  • transplanted AHPC cells can migrate into, and differentiate within, the mature retina during the active phase of neuronal degeneration.
  • Donor cell line Hippocampal progenitor cells were clonally derived from adult Fischer 344 rats, genetically modified to express the modified jellyfish (Aequorea victoria) enhanced green fluorescent protein GFP (eGFP) . In some cases, the cells were pulsed prior to transplantation with BrdU (e.g., 5 ⁇ m, 2 days, or more preferably, 50 ng/ml, 3 pulses over 3 days) .
  • BrdU e.g., 5 ⁇ m, 2 days, or more preferably, 50 ng/ml, 3 pulses over 3 days.
  • AHPCs were cultured and differentiated as follows. Primary adult hippocampal progenitor cultures were prepared from hippocampal tissues of 3-month-old female Fisher 344 rats as previously described (Gage et al. 1995a).
  • Dissociated cells were cultured on polyornithine/laminin coated dishes using a mixture of DMEM/Ham' F-12 (1:1) supplemented with N2 (Gibco) and 20 ng / ml FGF-2 (human recombinant, prepared in E. coli, kindly provided by A. Baird) .
  • Individual cells were genetically marked using replication-defective retroviral vectors expressing GFP from a tetracycline- regulatable, minimal human cytomegalovirus immediate early promoter fused to a tet-operator (NIT-GFP) .
  • Cloned cultures were derived from bulk-injected cultures.
  • AHPCs were induced to differentiate in 4-well chamber slides at a cell density of 2,500 cells per cm 2 by withdrawal of FGF-2 and treatment for 14 days in DMEM/F12 + N2, supplemented with 0.5 ⁇ M all-trans retinoic acid and 0.5% fetal bovine serum. These conditions were previously shown to favor the differentiation of neurons, astrocytes, and oligodendrocytes in a single well (Palmer et al. 1997). AHPCs were prepared for grafting in the following manner.
  • AHPCs were harvested with trypsin, washed with high glucose Dulbecco' s PBS (D-PBS, Gibco), and suspended at a density of 100,000 cells per ⁇ l in D-PBS containing 20 ng of FGF-2 per ml.
  • D-PBS high glucose Dulbecco' s PBS
  • pigmented dystrophic RCS rats received injections of AHPCs into the vitreous or subretinal space under general (Ketamine/xylazine) and topical (proparacaine) anesthesia. Injections were performed under direct observation using coaxial illumination via binocular surgical microscope (M ⁇ ller) through a dilated pupil (topical tropicamide 1%) . The injections were made via a beveled glass micropipette
  • Tissue preparation and histology Recipient animals were killed with an overdose of sodium pentobarbitol at 1, 2, 4, 8, and 16 weeks post-transplantation. The eyes were removed and immersion-fixed with 4% paraformaldehyde for 4 hours at 4 °C. The anterior segment and lens were then removed, and the posterior segment cryoprotected in 30% sucrose/PBS overnight at 4
  • Morphology Retinae containing high numbers of grafted cells were analyzed to determine their laminar localization at 4 and 8 weeks post-transplantation. Age at time of transplantation (1, 4, and 10 weeks) was also compared. A total of nine 50 ⁇ m wide regions of sectioned retina were analyzed for each animal, chosen so that both central and peripheral regions were included.
  • AHPCs Clonally derived AHPCs from adult Fischer 344 rats, which were genetically modified to express green fluorescent protein (GFP) and also labeled with BrdU in some cases, were transplanted into both immature (3 days postnatal, P3) and mature (4-36 weeks postnatal) , dystrophic eyes of RCS rats. Following transplantation, donor-derived cells were found to maintain high levels of GFP expression. GFP+ cells were clearly evident under FITC illumination and were verified to be of graft origin based upon anti-GFP immunoreactivity, anti-BrdU immunoreactivity, as well as constitutive GFP expression (data not shown) .
  • GFP green fluorescent protein
  • the GFP + cells were quite striking in appearance and were easily distinguished from autofluorescence of host photoreceptor outer segments in the recipient, based on intensity, morphology, location, and spectral specificity. Subsequent identification of donor-derived cells was therefore based on GFP fluorescence alone, obviating the need for prelabeling with BrdU or the use of anti-GFP antibodies.
  • grafted AHPCs could be seen adhering to the vitreal surface of the graft recipient or host's eye, migrating into the host retina, and taking up residence within the cellular retinal laminae of the host, including the outer nuclear layer.
  • grafted cells were seen in the host photoreceptor layer, and when examined with anti- BrdU, were found to be double labeled with GFP and BrdU, confirming the cells' derivation from the transplanted AHPCs. No evidence of viable donor cells, or host GFP expression, was seen following injection of freeze- thawed GFP + AHPCs (negative control) , confirming observations reported in Takahashi 1998, incorporated herein by reference.
  • GFP+ cells were found within the retina of 60%, 35%, 48%, and 60% of animals grafted at the ages of 1, 4, 10, and 18 weeks, respectively.
  • intra-retinal GFP+ cells were found in 80% of the recipients who were 1 week old at the time of grafting, and 50% of those initially 4 weeks old.
  • GCL/ILP ganglion cell layer and inner plexiform layer
  • INL/OPL inner nuclear layer and outer plexiform layer
  • ONL/SRS outer nuclear layer and subretinal space k N.B. At this time point clearly defined outer plexiform and outer nuclear layers are not present
  • Table 1 shows the laminar distribution of migrating AHPCs in representative 1, 4, and 10 week old recipients. The majority of grafted cells left the vitreous and entered the retina, where they migrated into the various laminae. Although grafted cells were also found in the ganglion cell and inner nuclear layers, they showed a predilection for the outer retina, particularly the outer nuclear layer, subretinal debris zone and intervening layer of photoreceptor elements
  • ONL/SRS ONL/SRS
  • the number of cells in the ONL/SRS was greater yet.
  • GFP+ cells appeared to gain access to the retina either by direct radial migration through the undamaged vitreal surface or, in greater numbers, by way of the peripheral injection tract with subsequent lateral migration. In the latter case, cells could be found migrating into as much as 60% of the longitudinal extent of the neuroretina. Regardless of the course taken by the migrating AHPCs, GFP+ cells were found in all layers of the host neuroretina, but not in the retinal pigment epithelium, choroid, or sclera.
  • Figures la-f depict the localization of grafted AHPCs to specific retinal layers in rats of different ages (4 weeks (a-d) ; 10 weeks (e) , and 18 weeks (f)).
  • Cells (green) were grafted into the vitreous of 4 (a-d) , 10 (e) , and 18 (f) week old rats, and examined 4 weeks later.
  • Retina sections were labeled with anti- synaptophysin/Cy3 antibody (red) to demarcate the synaptic and cellular layers of the host retina, and viewed under FITC and Cy3 fluorescent illumination.
  • the arrow in Fig. la indicates cell seen in Fig. lb at higher power; the arrow in Fig. lc indicates cell seen in Fig.
  • Id at higher power (vit, vitreous; gel, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; srs, subretinal debris and degenerating photoreceptor elements) .
  • Figure 2a-i depict confocal images of expression of neuronal markers by grafted AHPCs.
  • Figs. 2a-c are from animals grafted at 4 weeks of age, examined 4 weeks after grafting: constituitive GFP expression (a), anti- calbindin/Cy3 immunoreactivity (b) , and merged image (c) .
  • the arrows indicate 2 cells co-expressing these labels.
  • the arrows indicate 2 cells co- expressing these labels.
  • Fig. 1 depict confocal images of expression of neuronal markers by grafted AHPCs.
  • Figs. 2a-c are from animals grafted at 4 weeks of age, examined 4 weeks after grafting: constituitive GFP expression (a), anti
  • 2g-i are from animals grafted at 16 weeks of age, examined 1 week after grafting: constituitive GFP expression (g) , anti-NF-200 /Cy3 immunoreactivity (g) , and merged image (i) . Arrows indicate 2 cells co-expressing these labels.
  • GFP+ cells were found to co- express calbindin, a marker found on some retinal interneurons (Fig. 2a-c) , while others co-expressed the neuronal marker MAP-5 (Fig. 2 d-f) or NF-200 (Fig 2 g- I) .
  • MAP-5 a marker found on some retinal interneurons
  • NF-200 a marker found on NF-200
  • AHPCs grafted into the developing eye of normal animals failed to express neuronal markers (Takahashi 1998). While these markers are not retina-specific, they do show that hippocampal-derived progenitor cells are capable of developing mature neuronal phenotypes when grafted to a novel site such as the retina.
  • grafted AHPCs did not show any evidence of GFAP expression or astrocytic morphological development, suggesting a preference for neuronal differentiation in the microenvironment of the degenerating retina.
  • FIG. 3a-h depicts confocal images of grafted cells treated with anti-synaptophysin/Cy3 (red) antibody, which show grafted AHPCs (green) sending processes into the inner plexiform layer (a-d) , or the outer plexiform layer (e-h) (grafted at 4 weeks, examined 4 weeks after grafting) .
  • a cell is shown merged (a) , and reconstructed to show entire neuritic arbor (b) .
  • AHPCs send neurites into the inner plexiform layer (c, higher power in d) , and outer plexiform layers (e + g, higher power in f + h, respectively) . These processes intermingle with, and appear to contact synaptophysin-positive profiles of the host .
  • Figs. 3 a-d large GFP+ cells are observed to send neuritic processes into the host inner plexiform layer, while Figs. 3 e-h show cells with elaborate arbors intermingling with the host outer plexiform layer.
  • Figs. 3 a-b provide one example of the way in which the configuration of GFP+ arbors frequently reflects the orientation of the host plexiform layers.
  • One process tracks along the INL/IPL interface while another, originating from an position offset within the ILP, assumes a parallel course in the opposite direction despite the lack of a laminar interface to guide it.
  • Confocal analysis confirmed that large numbers of GFP+ processes come into direct apposition to host synaptophysin + profiles (Fig. 3e-h; 4 weeks post- grafting into 4 week old hosts) .
  • Grafted AHPCs are also capable of extending processes into the host optic nerve. Grafted cells residing in the ganglion cell layer extend neurites with large growth cones that approach, but do not cross, the level of the scleral outlet at 4 weeks post-grafting into 1 week old hosts (Figs. 4a-b) .
  • Figures 4a-c show confocal images of GFP+ neurites projecting, via the host optic fiber layer, into the optic nerve head 4 weeks after grafting. These fibers have large growth cones (arrows in Fig. 4a, and in higher power in Fig. 4b), which approach, but do not cross, the scleral outlet (labeled "sc”) at 4 weeks post grafting into initially 1 week-old hosts. When animals were examined
  • FIG. 4c When examined at 8 weeks post-grafting, large numbers of growing neurites were found to cross the scleral outlet, and extend long processes at least 300 ⁇ m into the optic nerve (Fig. 4c) . The apparent increased density of GFP + processes at successive time points indicate that AHPCs continue to develop along a neuronal-like pathway for at least 8 weeks post- grafting.
  • neuronal progenitor cells derived from adult, differentiated neural tissue e.g., hippocampus
  • differentiated neural tissue e.g., hippocampus
  • AHPCs Following migration, transplanted AHPCs respect the local laminar organization and exhibit a surprising ability to differentiate into neurons with morphological characteristics suggestive of native retinal cell types.
  • the cell processes extended by AHPCs within the retina tend to resemble the neuritic profiles of specific retinal neurons, including sublamina-specific ramifications within the inner plexiform layer suggestive of bipolar and horizontal cells (Dowling, 1970) .
  • the presence of distinct bands of diffuse GFP-derived fluorescence along these sublaminar zones suggests a network of fine terminals within the host neuropil.
  • neural progenitor cells such as AHPCs are capable of functional integration into the retina of animals up to 10 weeks of age, as well as limited incorporation into 18-week-old recipients, an age when the RCS retina has degenerated severely and other interventions are ineffective.
  • AHPCs At 36 weeks of age, however, AHPCs not only fail to enter the retina but show very little survival, suggesting the loss of an important trophic influence late in the course of the dystrophy.
  • grafted AHPCs disperse within the host tissue rather than remaining adherent to each other, as is typically seen with embryonic neural grafts. After taking up residence, these cells differentiate along neuronal (as opposed to glial) lines and extend processes within the host plexiform layers. Furthermore, the orientation of many of these processes is reminiscent of the arborization pattern of retinal amacrine cells. AHPCs in the ganglion cell layer frequently extend neurites into the optic fiber layer and optic nerve. More recently, neural progenitor cells have reportedly been found to differentiate into cells of the hematopoietic lineage (Bjornson 1999) , suggesting that a hippocampal to retinal fate shift should not be dismissed.
  • AHPC arborizations appear to respond to extrinsic retinal cues in preference to any intrinsic hippocampal developmental programs.
  • graft- derived neurites are intimately associated with host synaptophysin profiles does not conclusively demonstrate synapse formation
  • another laboratory using this same AHPC cell line has recently provided electron microscopic evidence of synapse formation in vitro, as well as excitatory post-synaptic potentials (Toda 1999) .
  • Neural progenitor cells such as AHPCs migrate and integrate into neonatal, nondystrophic, syngeneic Fischer rat hosts (see Takahasi 1998, incorporated herein by reference) .
  • AHPCs also readily migrate into mechanically injured retina of adult, syngeneic hosts as well as diseased retina of mature, allogeneic RCS rat hosts.
  • the data presented here provide the first definitive evidence for the survival, migration, and neuronal differentiation of a transplanted cell in diseased, mature retina.
  • This study shows that neural progenitor cells can overcome many of the obstacles to neuronal integration present in the mature mammalian central nervous system.
  • the observations here of widespread morphological integration in an allogeneic situation also argues for the importance of the specific microenvironment of the host retina in promoting migration and differentiation of grafted precursor cells. The same is expected for other specialized or differentiated neural tissue into which AHPCs are integrated.
  • the present invention will enable the attainment of the ultimate goal of restorative neuronal transplantation into the eye: introduction of new photoreceptor cells.
  • the data demonstrate that neuroprogenitor cells such as AHPCs are capable of repopulating the outer nuclear layer of the dystrophic retina with cells resembling neurons.
  • the surprising degree of plasticity exhibited by AHPCs transplanted to the diseased eye indicates that using neural progenitor cells to repopulate the eye with photoreceptor cells, seemingly impossible only a few years ago, is now a realistic objective.
  • Neuroprogenitor cells like AHPCs are capable of reaching all layers of the retina, and differentiating into cells with local phenotypic characteristics. These cells represent an exciting new tool for studying and manipulating retinal development in mammalian species. Since neural progenitor cells can be propagated in vitro and, following transplantation, can extensively repopulate an actively degenerating retina in visually mature animals, they will also be useful in treating retinal diseases involving neuronal cell loss. In view of the results discussed herein, it is reasonable to expect that AHPCs and other neural progenitor cells would similarly be able to differentiate into the appropriate neuronal cell lineage of other neural sites into which these progenitors are transplanted in vivo . Therefore, AHPC transplantation can be useful also to treat other neurological diseases and injuries involving neuronal loss or damage.
  • the survival of adult rat-derived, hippocampal neural progenitor cells transplanted into the dystrophic mouse retina was investigated. These transplanted cells were capable of integrating into the murine host retina and of maintaining expression of the green fluorescence protein (GFP) gene inserted into the progenitor cells.
  • GFP green fluorescence protein
  • Neural progenitor cells cultured from the hippocampus of adult Fischer 344 rats, were genetically modified to express GFP and a clonal cell line was isolated, as previously described. These cells were then transplanted into the vitreous of 7-day-old "rd-1" mice (50,000 cells in 1 ⁇ l) , without immunosuppression. After 2-4 weeks post-transplant, the eyes were removed and sectioned.
  • Rat, adult neural progenitor cells transplanted to a xenogeneic environment without immunosuppression are capable of surviving for at least 4 weeks and maintaining expression of a GFP marker. These cells can also migrate into the host retina, where they developed neuron-like phenotypes.
  • the use of xenogeneic, pluripotent progenitor cells as a source of donor tissue in transplantation protocols offers a viable new technology for studying and manipulating neural development and neural tissue plasticity, and repairing damaged central nervous system (CNS) tissue.
  • CNS central nervous system
  • the present technology will enable the use of xenogenic, neural tissue, such as pig- derived neural progenitor cells, to treat retinal and other neurological diseases and injuries involving neuronal loss.
  • Example III Physiological improvement in rats receiving neural progenitor cell transplants
  • Neuroprogenitor cells such as AHPCs have the capacity to restore vision in blind rodents.
  • Recent experiments have demonstrated that grafts of AHPCs into the eye of RCS rats leads to behavioral recovery, as measured by a optokinetic nystagmas (OKN) reflex test.
  • OKN is an involuntary reflex, which depends upon visual acuity level to generate a response to rotating contrast gratings. These gratings can be varied by intensity, contrast, and frequency to precisely determine visual acuity. Animals grafted with AHPCs can possess an OKN response, whereas control animals do not. Two possible mechanisms can explain this visual behavior:
  • Grafted AHPCs are actively integrated in the retinal cytoarcitecture, and are contributing in some manner to the visual pathway, either as photoreceptors, interneurons, and/or retinal ganglion cells; and/or
  • AHPCs lead to rescue of host photoreceptors. If the latter is true, AHPC grafting has great promise in the growing field of growth factor delivery, as grafted cells can integrate into the host retina in a stable manner, and can be modified in vitro to secrete specific molecules into the host retina.
  • Fibroblast growth factors are necessary for neural retina but not pigmented ipithelium differentiation in chick embryos. Development 124, 805-16.
  • FGF-2 is sufficient to isolate progenitors found in the adult mammalian spinal cord. Exp Neurol 148, 577-86.
  • AHPCs have been grafted in adult rat hippocampus where they migrate and differentiate into neurons in the dentate gyrus (Gage et al., 1995). The site specific migration and integration of these cells have been tested by grafting them in the rostral migratory pathway leading to the olfactory bulb (Suhonen et al., 1997). Cells migrated along the RMP and then laterally to granule cell and gromular cell layers. Cells migrating to the granule cell layer became calbindin/NeuN + cells, and those migrating to gromeruli became tyrosine hydroxylase + neurons (typical phenotypes of cells residing in these regions) .
  • AHPCs grafted in neonate eyes migrated to different layers assuming the morphological characteristics of cells present at those layers. However, they did not express any of the markers specific for eye cells (Takahashi et al . , 1998).
  • adult spinal cord-derived progenitor cells when grafted in the spinal cord, have been found to generate only glial cells.
  • hippocampus in the hippocampus, they migrate in a similar fashion to AHPCs and differentiate into neurons only in the dentate gyres (Shiabuddin, Horner, Ray and Gage, unpublished results) .
  • grafted cells migrate, incorporate into all major compartments of the brain, and differentiate into multiple developmentally and regionally appropriate cell types (Flax et al., 1998, Housele et al., 1998). These data indicate that xenografts of multipotent neural stem/progenitor cells not only survive, they behave like endogenous cells of the recipient species. The ultimate fate of the grafted cells is determined by the endogenous stimuli present in specific brain regions.
  • multipotent neural stem/progenitor cells derived from rat, mouse and human have made them ideal candidates for their use as a source of cells which can be used to replace or correct for cells lost in disease or injury.
  • the utility of these cells for transplantation can be tested in the following disease models.
  • Liver diseases The liver plays a central role in the pathophysiology of many inherited metabolic diseases. Despite the unusual ability of the adult liver to regenerate after injury, the liver is an important target for cell therapy. Two separate transgenic mouse models have been established wherein the animals produce a toxic by-product that damages or kills hepatocytes. In albumin-urokinase (Alb-uPA) transgenic mice, hepatocyte-targeted expression of a hepatotoxic transgene creates a functional liver deficit leading to chronic stimulus of liver growth (Rhim et al . , 1994).
  • Alb-uPA albumin-urokinase
  • tyrosinaema type I a recessive liver disease
  • FAH fumarylacetoacetate hydroxylase
  • AHPCs adult rat hippocampal-derived progenitor cells
  • AMPCs adult mouse-brain derived progenitor cells
  • fetal or adult human brain-derived progenitor cells in to these animal models to determine whether brain-derived progenitor cells can respond to local environment of the spleen, become hepatocytes, and replace the dying cells to correct the disease phenotypes .
  • IDDM autoimmune insulin-dependent diabetes mellitus
  • NID non-obese diabetic
  • TCR T-cell receptor
  • Viable islet cells isolated from pancreas have been transplanted, resulting in complete reversal of hypoglycaemia in diabetic animals (Thomas et al., 1990) .
  • AHPCs, AMPCs, or fetal or adult human brain-derived progenitor cells can be grafted in the pancreas of the NOD or transgenic mice to determine whether hypoglycemia can be corrected by the replacement of the damaged cells with the grafted cells.
  • Duchenne muscular dystrophy is characterized by slow and progressive muscle weakness affecting limb and respiratory muscles, which degenerate until fatal cardiorespiratory failure.
  • Myodystrophy of the Duchenne type results from mutations affecting the gene for dystrophin, a cytoskeletal protein.
  • Several types of mutations have been described, which encompass the complete absence of dystrophin to its presence in reduced levels or the presence of partially functional truncated forms, and which lead to severe to very mild forms of the disease (Gilis, 1996) .
  • the mdx mice that showed complete absence of dystrophin have been used as a model for DMD and have been tested for cell therapy. Normal myoblasts have also been transplanted into the muscle of patients with DMD.
  • merosin-deficient congenital muscular dystrophy A form of congenital dystrophy caused by a deficiency of the a2 subunit of the basement membrane protein laminin/merosin is termed merosin-deficient congenital muscular dystrophy (MCMD) .
  • MCMD merosin-deficient congenital muscular dystrophy
  • Most patients with MCMD are never able to walk.
  • Null mutant dyw mice have been generated (Kung et al., 1998).
  • Expression of human LAMA2 gene in the skeletal muscle of dyw mice dramatically improves the muscle disease in these animals.
  • Both mdx mice and dyw mice can be used for transplantation of AHPCs, AMPCs or fetal or adult human brain-derived progenitor cells into muscles, to determine whether these cells can become myoblasts and replace degenerating muscle cells.
  • Cardiovascular disease The regulation of cardiovascular function is complex and depends on many factors interacting in a defined and temporal fashion. Knock-out mice lacking desmin, needed to maintain the integrity of the myocardium to develop cariomyopathy (Lie et al., 1996; Milner et al., 1996, Thornell et al., 1997) . AHPCs, AMPCs, or fetal or adult human brain-derived progenitor cells can be grafted in the myocardium of these mice to determine whether the transplanted cells can replace the diseased cells and improve heart function.
  • Cystic fibrosis the most common autosomally inherited disease, is caused by the defective gene Cftr, which encodes an ion channel at the cell membrane. By homologeous recombination, several groups have disrupted the Cftr gene. All null mutation mice developed symptoms of cystic fibrosis (Dorin et al., 1992). AHPCs, AMPCs, or fetal or adult human brain-derived progenitor cells can be grafted into the lung of these mutant mice to determine whether these cells can replace the diseased cells having defective ion channels, and restore normal lung function.
  • Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I. (1996) Nature Genet. 12: 266-273.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Addiction (AREA)
  • Psychiatry (AREA)
  • Diabetes (AREA)
  • Ophthalmology & Optometry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Psychology (AREA)
  • Zoology (AREA)
  • Pain & Pain Management (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Hospice & Palliative Care (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Developmental Biology & Embryology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Obesity (AREA)
  • Cardiology (AREA)
  • Microbiology (AREA)

Abstract

La présente invention concerne des méthodes de réparation des tissus nerveux différenciés dystrophiques, tels qu'une rétine endommagée ou malade ou un nerf optique endommagé ou malade, chez l'homme et autres animaux. L'invention concerne, en particulier, l'introduction de cellules progénitrices neurales dérivées de l'adulte au niveau d'un site tissulaire neural dystrophique d'un animal destinataire, y compris un animal adulte (mature), qu'il soit xénogénique, allogénique ou syngénique. Ces cellules progénitrices neurales dérivées de l'adulte peuvent s'intégrer fonctionnellement et morphologiquement dans les tissus nerveux dystrophiques matures et immatures.
PCT/US2000/003534 1999-02-11 2000-02-11 Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures WO2000047238A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU28784/00A AU2878400A (en) 1999-02-11 2000-02-11 Integration of transplanted neural progenitor cells into neural tissue of immature and mature dystrophic recipients
JP2000598189A JP2002536423A (ja) 1999-02-11 2000-02-11 未成熟及び成熟ジストロフィーレシピエントの神経組織内に移植された神経前駆細胞の統合
EP00907260A EP1154801A4 (fr) 1999-02-11 2000-02-11 Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures
CA002362476A CA2362476A1 (fr) 1999-02-11 2000-02-11 Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11964299P 1999-02-11 1999-02-11
US60/119,642 1999-02-11

Publications (2)

Publication Number Publication Date
WO2000047238A1 true WO2000047238A1 (fr) 2000-08-17
WO2000047238A9 WO2000047238A9 (fr) 2001-07-26

Family

ID=22385498

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/003534 WO2000047238A1 (fr) 1999-02-11 2000-02-11 Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures

Country Status (5)

Country Link
EP (1) EP1154801A4 (fr)
JP (1) JP2002536423A (fr)
AU (1) AU2878400A (fr)
CA (1) CA2362476A1 (fr)
WO (1) WO2000047238A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036749A2 (fr) * 2000-11-06 2002-05-10 The Salk Institute For Biological Studies Cellules souches de cadavres
WO2004050865A1 (fr) * 2002-12-02 2004-06-17 Anges Mg, Inc. Procede de culture de cellules souches neurales au moyen du facteur de croissance hepatocyte
US7312025B2 (en) 2002-07-12 2007-12-25 University Of Washington Methods and systems for extended in vitro culture of neuronal cells
US9061017B2 (en) 2006-05-03 2015-06-23 The Schepens Eye Research Institute, Inc. Isolation and therapeutic application of adult retinal stem cells collected from extra-retinal tissues
US10039790B2 (en) 2006-05-03 2018-08-07 The Schepens Eye Research Institute, Inc. Isolation and therapeutic application of adult retinal stem cells collected from extra-retinal tissues

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JO3626B1 (ar) * 2012-02-23 2020-08-27 Merial Inc تركيبات موضعية تحتوي على فيبرونيل و بيرميثرين و طرق استخدامها

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DURING ET AL.: "Towards gene therapy for the central nervous system", MOLECULAR MEDICINE TODAY,, vol. 4, no. 11, November 1998 (1998-11-01), pages 485 - 493, XP002926800 *
MARTINEZ-SERRANO ET AL.: "CNS-derived neural progenitor cells for gene transfer of nerve growth factor to the adult rat brain: complete resceue of axotomized cholinergic neurons after transplantation into the septum", THE JOURNAL OF NEUROSCIENCE,, vol. 15, no. 8, August 1995 (1995-08-01), pages 5668 - 5680, XP002926798 *
MARTINEZ-SERRANO ET AL.: "Immortalized neural progenitor cells for CNS gene transfer and repair", TRENDS IN NEUROSCIENCE,, vol. 20, no. 11, November 1997 (1997-11-01), pages 530 - 538, XP002926796 *
See also references of EP1154801A4 *
SNYDER ET AL.: "Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain", NATURE,, vol. 374, 23 March 1995 (1995-03-23), pages 367 - 370, XP002926795 *
TAKAHASHI ET AL.: "Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina", MOLECULAR AND CELLULAR NEUROSCIENCE,, vol. 12, December 1998 (1998-12-01), pages 340 - 348, XP002926797 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002036749A2 (fr) * 2000-11-06 2002-05-10 The Salk Institute For Biological Studies Cellules souches de cadavres
WO2002036749A3 (fr) * 2000-11-06 2003-10-30 Salk Inst For Biological Studi Cellules souches de cadavres
US7312025B2 (en) 2002-07-12 2007-12-25 University Of Washington Methods and systems for extended in vitro culture of neuronal cells
WO2004050865A1 (fr) * 2002-12-02 2004-06-17 Anges Mg, Inc. Procede de culture de cellules souches neurales au moyen du facteur de croissance hepatocyte
AU2003302707B2 (en) * 2002-12-02 2008-10-02 Anges Mg, Inc. Method for culturing neural stem cells using hepatocyte growth factor
US9061017B2 (en) 2006-05-03 2015-06-23 The Schepens Eye Research Institute, Inc. Isolation and therapeutic application of adult retinal stem cells collected from extra-retinal tissues
US10039790B2 (en) 2006-05-03 2018-08-07 The Schepens Eye Research Institute, Inc. Isolation and therapeutic application of adult retinal stem cells collected from extra-retinal tissues

Also Published As

Publication number Publication date
JP2002536423A (ja) 2002-10-29
EP1154801A4 (fr) 2002-08-21
EP1154801A1 (fr) 2001-11-21
CA2362476A1 (fr) 2000-08-17
WO2000047238A9 (fr) 2001-07-26
AU2878400A (en) 2000-08-29

Similar Documents

Publication Publication Date Title
Young et al. Neuronal differentiation and morphological integration of hippocampal progenitor cells transplanted to the retina of immature and mature dystrophic rats
Klassen et al. Stem cells and retinal repair
Johnson et al. Development and characterization of an adult retinal explant organotypic tissue culture system as an in vitro intraocular stem cell transplantation model
JP5414626B2 (ja) 幹細胞の移動および増殖を促進するための修飾されたピリミジン化合物の使用
Klassen et al. Isolation of retinal progenitor cells from post‐mortem human tissue and comparison with autologous brain progenitors
Canola et al. Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate
US6767738B1 (en) Method of isolating adult mammalian CNS-derived progenitor stem cells using density gradient centrifugation
Sharp et al. A re-assessment of long distance growth and connectivity of neural stem cells after severe spinal cord injury
Gust et al. Adult donor rod photoreceptors integrate into the mature mouse retina
DE69926639T2 (de) Knochenmarkszellen als quelle von neuronen zur wiederherstellung von gehirn und knochenmark
Toda et al. Neurons generated from adult rat hippocampal stem cells form functional glutamatergic and GABAergic synapses in vitro
Dziewczapolski et al. Survival and differentiation of adult rat-derived neural progenitor cells transplanted to the striatum of hemiparkinsonian rats
Lu et al. Transplantation of EGF-responsive neurospheres from GFP transgenic mice into the eyes of rd mice
Van Den Pol et al. Olfactory ensheathing cells: time lapse imaging of cellular interactions, axonal support, rapid morphologic shifts, and mitosis
US20060003312A1 (en) Circulating stem cells and uses related thereto
DE60035191T2 (de) Materialien und methoden zur entwicklung von dopaminergen neuronen
US8691207B2 (en) Transplantation of cells expressing markers for photoreceptor cells and retinal ganglion cells induced from Müller stem cells
KR20060037346A (ko) 휘돌기교세포 전구세포 및 이를 획득하고 배양하는 방법
WO2005089420A2 (fr) Expansion de cellules souches neurales avec lif
US6214334B1 (en) Compositions and methods for producing and using homogenous neuronal cell transplants to treat neurodegenerative disorders and brain and spinal cord injuries
Shetty et al. Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor
DE69924728T2 (de) Nicht-embryonale ependymale neuronale stammzellen und methoden zu ihrer isolierung
Zhang et al. Functional and histological improvement of the injured spinal cord following transplantation of Schwann cells transfected with NRG1 gene
EP1154801A1 (fr) Integration de cellules progenitrices neurales transplantees dans les tissus nerveux de destinataires dystrophiques matures et immatures
KR20210132638A (ko) 신경계 세포 요법

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA JP MX US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C2

Designated state(s): AU BR CA JP MX US

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

COP Corrected version of pamphlet

Free format text: PAGES 1/4-4/4, DRAWINGS, REPLACED BY NEW PAGES 1/4-4/4; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

ENP Entry into the national phase

Ref document number: 2362476

Country of ref document: CA

Ref country code: CA

Ref document number: 2362476

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 598189

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 28784/00

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2000907260

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09913427

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 2000907260

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2000907260

Country of ref document: EP