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WO2000047718A1 - Isolation de cellules souches et leurs procedes d'utilisation - Google Patents

Isolation de cellules souches et leurs procedes d'utilisation Download PDF

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Publication number
WO2000047718A1
WO2000047718A1 PCT/US2000/003596 US0003596W WO0047718A1 WO 2000047718 A1 WO2000047718 A1 WO 2000047718A1 US 0003596 W US0003596 W US 0003596W WO 0047718 A1 WO0047718 A1 WO 0047718A1
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Prior art keywords
cells
acsc
tissue
cell population
acpc
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Inventor
Fred H. Gage
Theo Palmer
Francis F. Safar
Jun Takahashi
Masayo Takahashi
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Salk Institute for Biological Studies
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Salk Institute for Biological Studies
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Priority to US09/913,192 priority Critical patent/US6767738B1/en
Priority to AU35944/00A priority patent/AU3594400A/en
Publication of WO2000047718A1 publication Critical patent/WO2000047718A1/fr
Anticipated expiration legal-status Critical
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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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

  • the present invention relates to the fields of stem cells and gene therapy.
  • stem cells may be more widely distributed since cells from non-neurogenic areas repeatedly passaged in the presence of high concentrations of basic fibroblast growth factor (FGF-2) appear to begin to generate neurons in vitro.
  • FGF-2 basic fibroblast growth factor
  • This observation is consistent with the isolation of neuronal progenitors from these areas, but the protracted times in culture suggests another explanation.
  • stem cell cultures initiated from hippocampal tissues will spontaneously transform, due to accumulated genetic abnormalities. Abnormalities in chromosome number can occur in as little as 30 population doublings and, as cells become increasingly aneuploid, it is possible that glial-restricted progenitors acquire capabilities beyond those available in vivo.
  • neural tissue in particular, comprises a unique biological system that presents unique therapeutic challenges. Damaged neural tissue has proven very difficult to repair or replace. To facilitate the repair or replacement of neural tissue, scientists have focused their efforts on the identification, isolation and use of neuronal stem cells (or "progenitor cells"). With an appropriately pluripotent neural progenitor or stem cell, regeneration or augmentation of a variety of neural cell types is a possibility. Indeed, with a progenitor cell exhibiting an even more widely ranging plasticity, the regeneration or augmentation of a variety of cell types should be possible. Similarly, the stem cell would be a useful vehicle for introducing exogenous genetic material, as desired to achieve therapeutic results. .
  • Occular tissue in particular the retina, represents a highly specialized neural structure for which repair is often required.
  • the eye is frequently subjected to environmentally or genetically induced injury.
  • appropriately plastic stem cells would present a valuable vehicle for repair, replacement and/or genetic manipulation (e.g., gene therapy).
  • genetically mediated degeneration of occular tissue presents a more complex challenge.
  • stem cell-like multipotent progenitors can be isolated from adult hippocampus of rats, expanded in vitro and subsequently grafted into adult hippocampus and olfactory bulb where they demonstrate site- specific neuronal differentiation. See, for example, Palmer, T. D., Takahashi, J., Gage, F.
  • the rat hippocampus contains primordial neural stem cells, Mol. Cell. Neurosci., 8: 389 (1997); Palmer, T. D., Ray, J. & Gage, F. H. FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain, Mol. Cell Neurosci. 6: 474-486 (1995); Gage, F. H., Coates, P. W., Palmer, T. D., et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. U. S. A. 92:11879-11883 (1995); and Suhonen, J.
  • the graft-host interface is often well demarcated histologically, with ultrastructural studies revealing the presence of a dense glial scar across which few neurites are seen to cross. Ivert, L., Gouras, P., Naeser, P., and Narfstrom, K., Photoreceptor allografts in a feline model of retinal degeneration. Graefes Arch Clin Exp Ophffidmol 236, 844-52 (1998).
  • stem cells which can, in turn, be used as a therapeutic agent and/or as a vehicle for additional manipulation of gene-based therapeutics in order to treat damaged or diseased tissue, particularly neuronal tissue, and more particularly ocular tissue.
  • the present invention is directed to methods of enriching a cell population for stem cells and/or stem cell precursors.
  • Stem cells isolated by the present invention are pluripotent, however, precursor stem cells are not.
  • a method for treating precursor stem cells in a manner that produces stem cells is provided.
  • Stem cells, whether isolated by invention methods or generated by invention methods are useful in repairing damaged or diseased, specialized or differentiated tissue in mature animals, particularly neuronal tissue such as retinas.
  • One embodiment of the present invention relates to transplantation of adult, hippocampus (HC)-derived progenitor cells into a selected neural tissue site of a recipient. These cells can functionally integrate into mature and immature neural tissue.
  • the invention encompasses, in one aspect, repopulating a retina of a dystrophic animal with neurons, by injecting clonally derived, adult central nervous system derived stem cells (ACSC) derived from a healthy donor animal into an eye of the dystrophic recipient.
  • ASC central nervous system derived stem cells
  • AHPCs Fischer rat-derived adult hippocampal derived progenitor cells
  • AHPCs were also found to integrate successfully into a xenogeneic recipient (e.g., rat AHPCs into the retina of dystrophic rd-I mice).
  • a method for obtaining Adult Mammalian Stem Cells or Adult Mammalian Progenitor stem Cells from tissue comprising subjecting dissociated mammalian tissue to one or more buoyancy-based separation systems.
  • AMSC and AMPC Adult Mammalian Progenitor stem Cells from tissue
  • a method for obtaining adult mammalian CNS-derived progenitor cells (ACPC) or Adult Mammalian CNS-derived Stem Cells (ACSC) from a cell population containing adult mammalian central nervous system (CNS) tissue said method comprising subjecting dissociated mammalian CNS tissue to one or more buoyancy-based separation systems.
  • adult mammalian stem cells or adult mammalian progenitor cells isolated by invention methods.
  • adult CNS-derived progenitor cells ACPC
  • adult mammalian CNS-derived stem cells APC
  • adult mammalian CNS-derived stem cells APC isolated by subjecting dissociated mammalian CNS tissue to one or more buoyancy-based separation systems.
  • AMSC and AMPC have the ability to adapt to a heterotypic environment.
  • stem cells means cells that are self-renewing and multipotent (i.e., that are not lineage restricted). Stem cells includes AMSC and ACSC as defined herein. Stem cells are characterized as both self-renewing and able to differentiate.
  • Progenitor or “precursor” cells means an undifferentiated cell whose lineal descendants differentiate along the appropriate pathway to produce a fully differentiated phenotype (i.e., cells with a restricted lineage). For example, neural stem cells isolated from the hippocampus (HC) or the subventricular zone, are self renewing and able to generate, in vitro, multiple types of cells including neurons, glia and even hematopoetic cells.
  • neural progenitor or precursor cells are lineage restricted and while self-renewing, only generate glia in vitro.
  • Progenitor or precursor cells include AMPC and ACPC, as described herein.
  • subventricular zone or subventricular residuum means a thin lamina extending inward about 50 ⁇ m from the ependymal surface, including the hippocampus alveus but excluding ependymal cells.
  • AMSC can be characterized as self-renewing and able to generate (i.e., differentiate into) mature, differentiated cells of the tissue type from which the cells were isolated, and the like, either in vivo, or in vitro when grown in mitogen free media.
  • ASC Advanced Mammalian CNS-derived Stem Cells
  • ASC Advanced Mammalian CNS-derived Stem Cells
  • AMPC Advanced Mammalian derived Progenitor Cells
  • ACPC Advanced Mammalian CNS-derived Progenitor Cells
  • these ACPC are further characterized as being able to generate neurons, glia and hematopoetic cells in vitro when grown in the presence of mitogen, e.g., FGF, or the like.
  • a method for obtaining AMSC by growing AMPC in the presence of FGF-2 in another embodiment, there are provided methods for obtaining AMSC by growing AMPC in the presence of FGF-2.
  • a method for obtaining ACSC by growing ACPC in the presence of a FGF preferably FGF-2, 4, 6, or 8; more preferably FGF-2 or 4 and most preferably FGF-2.
  • adult mammalian derived stem cells includes ACSC and “adult mammalian derived progenitor cells AMPC” includes ACPC.
  • AMSC and AMPC can be derived from any tissue, including CNS, heart, liver lung, bone marrow, and the like.
  • CNS tissue from which invention ACPC can be derived include whole brain, hippocampus, spinal cord, cortex, striatum, cerebellum, thalamus, hypothalamus, amigdyla, basal forebrain, ventral mesencephalon, optic nerve, locus ceruleus, and the like. Indeed, it is expected that any tissue can yield progenitor and stem cells if processed in the manner described herein.
  • invention ACSC/ACPC are isolated from the hippocampus, more preferably from the adult hippocampus.
  • heterotypic environments to which the cells are able to adapt include all non-source, or non-native, neural tissue such as whole brain, hippocampus, spinal cord, cortex, striatum, cerebellum, thalamus, hypothalamus, amigdyla, basal forebrain, ventral mesencephalon, optic nerve, locus ceruleus, and the like, as well as CNS associated tissues such as eye tissues, the vitreous of the eye, and the like.
  • heterotypic environments include in vitro culture systems in which the foregoing cell types and lineages derived therefrom are cultured.
  • the "ability to adapt” comprises the ability of invention AMSC to respond to temporal and/or spatial cues of the heterotypic environment, either in vivo, in vitro, or both.
  • temporal and spatial cues include very broad classes of compounds known to have regulatory effects on cells, including those that provide differentiation signals, and the like.
  • temporal cues refers to compounds and conditions provided by the heterotypic environment in a time- dependent manner, including development stage associated compounds, cell cycle associated compounds and conditions, as well as combinations of any two or more thereof.
  • spatial cues include compounds and conditions provided by the heterotypic environment in a location specific manner, including any molecule or compound found in the heterotypic environment that provides cell-differentiation signals, such as trophic factors, hormones, cognate receptors for the foregoing, and the like, as well as combinations thereof.
  • 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.
  • 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.
  • HGF-SF scatter factor
  • 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
  • EGF EGF
  • Hormones that provide spatial cues include thyroid hormone and the like.
  • Receptors include the steroid thyroid hormone superfamily of receptors, neurotrophin receptors TrkB and TrkC, and the like.
  • the temporal and spatial cues described herein may be provided to invention cells as either molecules that are supplied exogenously (i.e., extracellularly) or endogenously (e.g., through the expression of native and/or introduced nucleic acids encoding such molecules, and the like).
  • invention AMSC are self-renewing (i.e., are capable of replication to generate additional AMSC).
  • invention AMSC/AMPC due to their pluripotent character, are capable of exhibiting a wide variety of responses upon exposure to a heterotypic environment with its associated temporal and spatial bio-information (i.e., cues). Because invention AMSC/AMPC are pluripotent, in one embodiment of the present invention AMSC/AMPC response to a heterotypic environment includes differentiation into a more lineage restricted type of cell found in the tissue from which the AMSC/AMPC was isolated.
  • ACSC/ACPC are also pluripotent stem cells
  • ACSC/ACPC response to a heterotypic environment includes differentiation into neurons, and glia, including astroglia and/or oligodendroglia, and the like.
  • AMSC/AMPC containing one or more heterologous DNA sequences (e.g., transgenes, and the like).
  • the AMSC/AMPC are capable of expressing proteins encoded by the heterologous DNA sequences.
  • invention AMSC/AMPC are able to integrate and differentiate into a number of different tissue types.
  • Invention ACSC/ACPC are able to integrate and differentiate primarily into neural tissues.
  • invention AMSC/AMPC are useful as therapeutic agents for replacing or augmenting diseased or damaged tissue.
  • Invention AMSC/AMPC may, however, also carry and express heterologous DNA sequences.
  • methods of therapy comprising administering to a patient in need thereof a cell population comprising modified AMSC/AMPC , such as, for example, those described herein, in an amount sufficient to provide a desired therapeutic effect.
  • a therapeutically effective amount is an amount effective for introducing or complementing one or more missing and/or defective genes, wherein the gene(s) so introduced comprise heterologous genetic material contained and expresed within said AMSC/AMPC and their descendants.
  • the optic vesicle forms early in development and the retina becomes regionally isolated and highly specialized. Given this spatial and temporal separation, it would seem unlikely that ACSC/ACPC could be used to replace retinal neurons, yet these immature cells retain sufficient adaptability to integrate within the normal retina and provide a means to deliver gene products to the eye.
  • ACSC/ACPC adult hippocampal stem cell and progenitors
  • AHPCs hippocampal stem cell-progenitors
  • ACSC/ACPC 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. Given that they can be propagated in vitro and, following transplantation, can extensively repopulate an actively degenerating retina in visually mature animals, this invention is also useful in treating retinal diseases involving neuronal cell loss.
  • this invention is also useful in treating retinal diseases involving neuronal cell loss.
  • ACSC/ACPC will similarly be able to differentiate into the appropriate neuronal cell lineage of other neural sites into which these progenitors are transplanted in vivo. Therefore, ACSC/ACPC transplantation is also useful to treat other neurological diseases and injuries involving neuronal loss or damage.
  • AMSC/AMPC can be used to treat diseases and injuries to the type of tissue from which the cells were isolated.
  • the invention encompasses a method of treating dystrophic neural tissue, comprising introducing ACPC 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 recipient may be an young (immature) animal or an adult (mature) animal.
  • ACPC donor and recipient may be of different species (xenogeneic).
  • exemplary 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., ACPC or ACSC) 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); (3) diseases of limbic and cortical systems (e.g., s cerebral amyloidosis, Pick's atrophy, Retts syndrome; (4) neurodegenerative pathologies involving multiple neuronal
  • 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 ACPC/ACMC or ACSC/ACPC 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.
  • therapeutic methods comprising administering to a patient in need thereof an amount of ACSC/ACPC effective to repair or replace defective, damaged or dead tissue.
  • cells which are to be added to or replaced comprise optic cells, including, retinal cells, M ⁇ ller cells, amacrine cells, bipolar cells, horizontal cells, photoreceptors, astroglial cells, and the like.
  • invention AMSC/AMPC Because of the pluripotent nature of invention AMSC/AMPC , and the resulting multiplicity of loci where such cells may be introduced in order to achieve therapeutic effects, there is a broad range of tissue damage and disease states which can be treated using invention AMSC/AMPC . Many disease states (e.g., liver disease) result in damaged or necrotic tissue. These types of diseases are ideal for replacement or augmentation therapy comprising the administration of invention AMSC/AMPC .
  • the plastic and pluripotent nature of AMSC/AMPC make 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, even in the absence of exogenous genetic material.
  • invention AMSC/AMPC can be used to replace a variety of tissue types throughout the body that are encompassed within the different phenotypes that progeny of AMSC/AMPC can exhibit, upon differentiation, including glial cells, neurons, and the like.
  • therapeutic methods comprising administering to a patient in need thereof a cell population comprising AMSC/AMPC as described herein, in an amount sufficient to provide a therapeutic effect.
  • expression of AMSC/AMPC native genes in the cell population occurs as necessary for AMSC/AMPC to proliferate and differentiate in order to replace or add cells of a desired type.
  • 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.
  • 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.
  • 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
  • Such factors are characterized by their trophic actions, their expression patterns in the brain, and molecular aspects of their receptors and intracellular signaling pathways.
  • Neurotrophic 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.
  • FGF fibroblast growth factor
  • EGF epidermal growth factor
  • heparin which is mitogenic.
  • a therapeutically effective amount ofagent comprising one or more of a neurotrophic factor (i.e., nuerotrophin), a mitogen, a neurotrophin-like factor, or the like is administered to a subject with damaged, diseased or dystrophic tissue (e.g., neuronal tissue such as retina, or the like). It is presently preferred that the agent be directly administered to the affected tissue via injection, topical application, or the like.
  • Some disease states are characterized by one or more defective or missing genes.
  • Such diseases are ideally treated by the adminstration of invention AMSC/AMPC containing one or more transgenes.
  • therapeutic methods as described herein, wherein one or more disease associated transgenes incorporated and expressed in said invention AMSC/AMPC .
  • neuronal tissue-associated disease states and their associated genes include Huntingtons Corea (one or more of gamma amino butyric acid (GABA) decarboxyalse and ciliary neurotrophic factor (CNTF)), Alzheimer's disease (one or more of acetylcholinesterase, neuronal growth factor (NGF), brain derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF)), Parkinson's disease (one or more of tyrosine hydroxylase, DOPA decarboxylase, DMAT, GDNF, BDNF and FGF), amyotropic lateral sclerosis (CNTF), and the like.
  • GABA gamma amino butyric acid
  • CNTF ciliary neurotrophic factor
  • Alzheimer's disease one or more of acetylcholinesterase, neuronal growth factor (NGF), brain derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF)
  • Parkinson's disease one or more of tyrosine hydroxy
  • invention AMSC/AMPC are useful to introduce therapeutic genes, it may be desirable to introduce into a host or patient one or more genes that are not strictly therapeutic but which may be useful in other ways, for example, as tracking genes (i.e., markers), as genes to induce migration, as genes to induce mitosis, as survival genes, as suicide genes, and the like.
  • Marker genes contemplated for use in the practice of the present invention include genes encoding a modified green fluorescent protein (GFP) derived from jellyfish, ⁇ - Galatosidase (the LacZ gene product), neomycin phosphotransferase (neo), Luciferase, and the like.
  • GFP green fluorescent protein
  • Non-viral methods contemplated for use in the practice of the present invention include electroporation, microinjection, polyethylene glycol precipitation, high velocity ballistic penetration by small particles with the nucleic acid to be introduced contained either within the matrix of such particles, or on the surface thereof (Klein, et al. Nature 227:70, 1987), or the like.
  • Viral methods contemplated for use in the practice of the present invention include the use of retroviral vectors, and the like. It is presently preferred that retroviral vectors be employed for introducing genetic material into invention AMSC/AMPC . In one aspect of the present invention, replication deficient vectors are employed. Such vectors are well known to those of skill in the art.
  • invention AMSC/AMPC diseases that are suitable for treatment with invention AMSC/AMPC .
  • Some of these disease states are equally suited for treatment using invention AMSC/AMPC with and/or without incorporated transgenes.
  • the liver plays a central role in the pathophysiology of many inherited metabolic diseases. Although the adult liver has the unusual ability to regenerate after injury, the liver is an important target for cell therapy.
  • AMSC/AMPC are introduced into the liver where they differentiate into hepatocytes, and replace dead and dying cells, thereby correcting disease phenotypes.
  • diseases are associated with one or more missing or defective genes, such diseases are treatable with invention AMSC/AMPC wherein the missing/defective gene(s) is/are incorporated.
  • AMSC/AMPC can be grafted in the pancreas for the replacement of damaged pancreas 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.
  • 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).
  • AMSC/AMPC are grafted into muscles wherein they differentiate to become myoblasts and replace degenerating muscle cells. Cardiac disease, typified in many instances by damaged heart muscle, is another target for cell replacement.
  • AMSC/AMPC are transplanted into the heart to replace diseased cells and improve heart function.
  • Pulmonary disease i.e., Cystic fibrosis
  • Cftr which encodes an ion channel at the cell membrane.
  • Augmentation of lung tissue with AMSC/AMPC can alleviate the reduced respiratory function caused by the defective genotype.
  • AMSC/AMPC are grafted into the lung in order to replace the diseased cells having defective ion channels, and restore normal lung function.
  • this disease is also an ideal candidate for treatment using invention ACSC/ACPC with appropriately incorporated Cftr-augmenting exogenous nucleic acids.
  • therapeutic methods as described herein wherein said AMSC/AMPC have been induced to differentiate, prior to administration to the subject, by in vitro exposure to extracellular and/or intracellular factors described herein, including trophic factors, cytokines, mitogens, hormones, cognate receptors for the foregoing, and the like, as well as combinations of any two or more thereof.
  • ACSC/ACPC are used as a paradigm for the present disclosure, however, any tissue type may be employed to isolate corresponding AMSC/AMPC .
  • a whole brain or other source neuronal tissue, as described herein, all comprise ACSC/ACPC it is desirable for therapeutic purposes to provide a cell population containing primarily ACSC/ACPC and lacking a substantial amount of other cell types and/or debris. Accordingly, it is presently preferred that cell populations be enriched for ACSC/ACPC . This enrichment can be carried out by a number of methods.
  • a cell population containing adult mammalian CNS tissue for adult mammalian CNS-derived stem cells comprising subjecting dissociated mammalian CNS tissue to one or more separation systems.
  • separation systems contemplated for use in the practice of the present invention include buoyancy-based separation systems, charge-based separation systems, fluorescent activated cell sorting systems (FACS), and the like, as well as combinations thereof.
  • the bouyancy based separation system employed is density gradient centrifugation. Density gradients can be created using any suitable media, including, Percoll, Ficoll, sucrose, and the like. In an even more preferred embodiment of the present invention, a Percoll gradient is employed. Invention AMSC/AMPC will typically have a density in the range of about 1.06 up to about 1.08 g/ml; and more typically a density in the range of about 1.072 up to about 1.075 g/ml.
  • the Percoll gradient employed in the practice of the present invention is an approximately 50% Percoll gradient.
  • the gradient can be modified to take into account the bouyant density of the particular stem cells being sought (e.g., hepatic stem cells, or the like).
  • ACSC/ACPC can be isolated from whole adult brains via density gradient centrifugation.
  • Gradient Calibration In order to derive the preferred gradient conditions for isolating ACSC/ACPC , established adult hippocampal progenitor cells were harvested from culture dishes by trypsinization and a cell suspension was obtained by suspending the trypsinized cells in 65% conditioned medium, 35% Percoll. The gradients were initially prepared by mixing various ratios of Percoll to conditioned media in 14x89 mm Ultra-Clear polycarbonate centrifuge tubes (Beckman). Dilutions of 90%, 80%, 70%, 60%, and 50% were made.
  • Tissue Preparation A cell suspension was obtained by pooling the brains from 6 animals from which the cerebella had been trimmed away. The remaining brain tissue was then weighed. The brains were diced into small fragments, and then digested for 40-50 min at 37°C in Hanks' balanced salt solution containing 0.1% grade II neutral protease (Boehringer Mannheim), 0.01% papain (Worthington), and 0.01% Dnase I (Worthington). The mixture was shaken every five minutes. Every fifteen minutes during the digestion, the tissues were gently disrupted by pipetting the digestion reaction mixture through a 5-ml pipette.
  • Hanks' balanced salt solution containing 0.1% grade II neutral protease (Boehringer Mannheim), 0.01% papain (Worthington), and 0.01% Dnase I (Worthington). The mixture was shaken every five minutes. Every fifteen minutes during the digestion, the tissues were gently disrupted by pipetting the digestion reaction mixture through a 5-ml pipette
  • tissue was then rinsed two times with DMEM/F12 containing 10% fibroblast bovine serum, to inactivate digestive enzymes, and filtered through nylon mesh (Nitex, lOO ⁇ m pore size), to remove undissociated tissue.
  • the filtered cells were resuspended to a final volume of 10ml in DMEM/F12/10%FBS.
  • Percoll density gradient preparation and centrifugation 100% isotonic Percoll (Pharmacia) solutions (300mOsm/kg H 2 0 (Enerback 1980)) were prepared by dissolving 9 parts of Percoll with 1 part of 10-fold concentrated PBS. 5 ml of this 100% Percoll solution were added to each of two 14x89 mm Ultra-Clear polycarbonate centrifuge tubes (Beckman) which were pre-sterilized with ethylene dioxide gas. To each tube, 5 ml of the cell suspension was added and mixed together with the Percoll. This yields a 50% Percoll mixture. A Beckman L8-80M preparative ultracentrifuge was used for centrifugation. Continuous density gradients of Percoll were generated by centrifugation at 20,000 x g for 30 min in a Beckman SW-41 Ti swinging bucket rotor. All centrifugations were performed at 25°C.
  • the gradients were divided into four equal fractions.
  • the fractions were removed by inserting a syringe with a 22 gauge 1/2 inch needle into the side of each tube and collecting the fractions, starting from the bottom.
  • the needle was inserted near the bottom of the tube with the bevel facing upwards, just above a band of red blood cells which is consistently generated during the centrifugation.
  • the lower fractions and the upper fractions, respectively, were pooled and rinsed twice in 5 vol of PBS to remove Percoll from the cells.
  • DMEM/F12 Dulbecco's Modified Eagle's Medium Ham's F12 (DMEM/F12, 1: 1) supplemented with 10% defined fibroblast bovine serum (FBS, Hyclone) and plated into 4 polyornithine/laminin-coated 6 cm dishes (Falcon), two for the upper fractions and two for the lower fractions.
  • FBS defined fibroblast bovine serum
  • Falcon polyornithine/laminin-coated 6 cm dishes
  • the medium was removed and replaced with fresh DMEM/FI2 containing N2 supplement (Gibco) and 20 ng/ml FGF-2 (provided by A. Baird).
  • the medium removed from the initial plating was centrifuged in a tabletop clinical centrifuge in order to recover any cells that had not yet adhered to the culture dishes.
  • Example 2 Second Method for Isolation of Multipotent CNS Progenitor Cells from Adult Rodent Brain via Percoll Density Gradient Centrifugation
  • Tissue dissociation and fractionation Tissues were finely minced and digested in a solution of papain (2.5U / ml, Worthington), DNAse (250U / ml, Worthington), and neutral protease (1U / ml Dispase, Boehringer) dissolved in Hanks balanced salt solution, as described in Palmer TD, Ray J, Gage FH (1995), FGF-2-Responsive Neuronal Progenitors Reside in Proliferative and Quiescent Regions of the Adult Rodent Brain.
  • the Percoll solution was made by mixing 9 parts of Percoll (Pharmacia) with 1 part 10X phosphate buffered saline (PBS, Irvine Scientific). The cell suspension was then fractionated by centrifugation for 30 minutes, 18oC, at 20,000 x g. Cell fractions were harvested and washed free of Percoll by 3 or more rinses in DMEM, 10% FBS.
  • Bromodeoxy uridine (BrdU) labeling was first used to mark endogenously proliferating cells for identification in situ.
  • Adult rats were injected with BrdU once each day for 6 consecutive days and then brains were collected for evaluation on day 7. Two percent of all labeled nuclei within the hippocampus were found within the putative subventricular residuum. Ependymal cells proper accounted for 4% of the total labeled population and a similarly small proportion was found within the neurogenic zone of the SGZ (8%). In contrast, 52% were present in the white matter of the fimbrial ridge, and the remainder were scattered throughout the parenchyma. A similar comparison of neocortical gray and white matter showed that 15% of the BrdU-labeled cells were present in the parenchyma of the cortex. The remaining 85% were present in the subcortical white matter and associated SVZ.
  • GFAP glial fibrillary acidic protein
  • Vimentin a marker attributed to ependymal cells, immature astrocytes and radial glia, is also known to be expressed by multipotent precursors in perpetualized neural precursor cultures. Some cells in both fresh and 36-hour populations were weakly immunoreactive for 04, a marker first attributed to immature oligodendrocytes and also expressed by FGF-2 stimulated multipotent precursors in vitro. Nestin, a marker for immature precursors, was detected in 15% or 7% of the freshly isolated cells from HC or cortex, respectively, but was then transiently downregulated. At 36 hours in culture, very few cells expressed detectable nestin, yet one week later, virtually all cells in both hippocampal and cortical cultures were nestin-positive.
  • vimentin staining in the low buoyancy fraction also proved to be informative. After 36 hours in culture, vimentin staining was intense in cells with a flattened, neurepithelial-like mo ⁇ hology and weak in phase bright cells reminiscent of progenitors in long-term cultures. Upon acid pretreatment (required for the immunological detection of BrdU), only the intensely staining cells remained immunoreactive. This staining pattern was also seen in vivo where the acid-stable vimentin immunoreactivity was restricted to the ependyma proper, whereas glia in the parenchyma exhibited a weaker, acid-labile staining.
  • BrdU-labeled cells fractionated to the low buoyancy population, most of the isolated cells were unlabeled, consistent with the isolation of immature, yet relatively quiescent precursors.
  • hippocampus or cortex was fractionated and cells from the low buoyancy fraction cultured in defined medium containing 20 ng / ml FGF-2 (DMEM/F12 containing N2 supplement and 20 ng / ml FGF-2, "growth medium").
  • Cell division was monitored by counting cells and by treating replicate cultures with BrdU at different times post plating. Following a delay of several days, cells began an exponential growth pattern that reached a steady state in 7 to 10 days. After 10 days, growth rates were similar to those of the pe ⁇ etualized cultures with roughly 85% of the cells dividing in a given 24-hour period and >99% of the cells labeled following a 48 hour exposure to BrdU.
  • the freshly isolated cells also displayed a density dependent growth that was similar to that seen in pe ⁇ etualized stem cell cultures.
  • Plating densities of ⁇ 10,000 cells / cm2 or higher were required for optimum proliferation whereas cells plated at clonal densities ( ⁇ 1 cell / cm2) grew very slowly or not at all.
  • fractionating cells not only was it possible to eliminate debris and differentiated cells, but those cells remaining could be plated immediately into culture at densities that promoted the recruitment of cells into cycle.
  • the lineage potential of progenitors from cortex or hippocampus was determined by culturing low buoyancy cells (isolated per Example 2) in growth medium for 14 days and then allowing cells to differentiate under conditions known to stimulate both neuronal and glial differentiation (differentiation medium: 1 ng / ml FGF-2, 1% fibroblast bovine serum, and 100 nM all-trans retinoic acid). Culture conditions were as set forth in Example 2. At 14 days, few of the cells expressed markers for neurons or glia.
  • retroviral marking was used to evaluate the lineage potential of cells within the low buoyancy fractions.
  • Cells were first stimulated with FGF-2 for 7 days to induce proliferation (a prerequisite for retroviral infection) and then retroviruses carrying a green fluorescent protein (GFP) transgene were used to infect the proliferating population.
  • GFP green fluorescent protein
  • Individual infected cells were marked and allowed to proliferate within the non- infected bulk population for an additional 7 days. The resulting colonies were then induced to differentiate in differentiation medium. A typical population of colonies was generated using an excess of virus (multiplicity of infection, m.o.i. ⁇ 0.01).
  • Clones were grouped into five categories based on the expression of lineage-specific markers and mo ⁇ hology. Neuron-only clones were infrequent (5.3%) in the hippocampal preparations and rare ( ⁇ 0.1%) in cortex-derived cultures. Some clones were glial-restricted and contained only GFAP-positive astrocytes and/or 04-positive oligodendrocytes. A small but significant proportion of the marked cells (21% and 17% from hippocampus and cortex, respectively) produced a mixture of glia and neurons. The remaining clones were negative for all three lineage markers.
  • the marker-negative cells were further divided into two clone types; one type was very large and consisted of flattened, phase dark cells strongly immunoreactive for the acid stable vimentin epitope. The remaining marker-negative clones were small and contained large bipolar cells with simple, large caliber processes.
  • neuron-only clones When scored by size, neuron-only clones contained few cells whereas glial-restricted progenitors and multipotent progenitors generated colonies of intermediate size.
  • the largest clones were the lineage marker-negative, vimentin-positive clones. Although the large size may suggest a faster growth rate in the 7 days following infection, it was determined that this large size was an artifact caused by continued growth in differentiation medium. When observed during the first 7 days after viral infection, the clones with the flattened phase dark mo ⁇ hology typical of these large lineage negative clones actually grew more slowly than the other clones being monitored. Continued growth in differentiation medium was confirmed by repeating these experiments in the presence of BrdU during differentiation. The large marker- negative clones were uniformly labeled with BrdU whereas cells from the smaller neuron- only, glia-only, or mixed clones were unlabeled.
  • Neurogenesis is not detected in the adult cortex yet a significant number of the progenitors from cortical tissues were competent to generate neurons once removed from their in vivo environment. Activation of this neurogenic program could be triggered by several mechanisms.
  • multipotent precursors may yield progeny that are competent to differentiate into neurons but are suppressed by cell extrinsic signals in vivo. If so, the simple act of removing them from the in vivo environment may disinhibit or activate a latent neuronal differentiation program.
  • low-buoyancy cells were isolated from adult tissue and immediately plated into differentiation medium as described above. The presence of ⁇ Tubulin-positive neurons was scored after 10 days.
  • cortical and hippocampal populations generated abundant populations of glia but only the hippocampal preparations generated ⁇ Tubulin-positive neurons.
  • the absence of neurons in cortical preparations suggested that precursors from non-neurogenic tissues require signals provided in culture to acquire the competence to differentiate into neurons.
  • Neuron-competent progenitors are found in areas distant from the proliferative zones of the anterior SVZ.
  • Cortical gray matter contains a small population of endogenously dividing glial progenitors, but the underlying subcortical white matter and ventricular zone have relatively abundant populations of dividing cells.
  • many of the "cortical" progenitors were, in fact, derived from this underlying proliferative zone, it was also possible that contaminating cells from the more rostral neurogenic areas of the lateral ventricle may have been present in the cortical preparations.
  • others have shown that the adult optic nerve retains an active population of glial progenitors and the optic nerve rostral to the optic chiasma can be easily harvested without risk of contamination from the SVZ of the lateral ventricle.
  • optic nerve was harvested, dissociated and fractionated.
  • Low-buoyancy cells were cultured for 14 days in the presence of high FGF-2 and then allowed to differentiate for an additional 14 days.
  • BrdU was added during the last 72 hours of FGF-2 treatment (day 14).
  • cultures were evaluated for the presence of neurons ( ⁇ Tubulin) and those neurons present were scored for BrdU-immunoreactivity. Although many glia rapidly differentiated in the primary culture, clusters of proliferative precursor-like cells were readily detected.
  • Neuronal markers such as ⁇ Tubulin, 200 kd neurofilament, and Tau were never detected in GFAP- positive or 04-positive glia, indicating that the neuron-like cells were authentic neurons rather than glia that inappropriately expressed neuronal markers.
  • Neurons were often found in small clusters, suggesting a clonal derivation, and all cells, including neurons, were labeled with BrdU during the last 72 hours of FGF-2 treatment ( ⁇ 1 unlabeled cell per 50,000 total nuclei). This data indicates that all neurons were derived from proliferative precursors. The fact that these neurons are generated from cells isolated from the optic nerve dispels any concerns of contamination from known neurogenic zones and demonstrates that a latent neurogenic potential is retained by precursors from divergent regions of the adult brain.
  • clonal stem cells were cultured in Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, 1:1) containing N2 supplement (Gibco BRL, Gaithersburg, MD) and 20 ng/ml rhFGF-2 (provided by A. Baird, Prizm Pharmaceuticals Inc., La Jolla, CA).
  • Culture dishes (Becton Dickinsin Labware, Lincoln Park, NJ) were coated with polyornithine and mouse laminin as previously described by Ray, et al., Proliferation, differentiation and long-term culture of primary hippocampal neurons, Proc Natl Acad Sci USA 90:3602-3606 (1993).
  • cells were plated into polyornithine/laminin coated glass chamber slides (Nunc, NaperviUe, IL) at a density of 2.5 x 103 per cm2. The cells were incubated for 24 hrs in normal N2 medium containing 20 ng/ml FGF-2, and then the medium was replaced by N2 medium containing 0.5%) fibroblast bovine serum (FBS) and 0.01% DMSO (control solvent) or 0.5 mM all-trans retinoic acid (Sigma, St. Louis, MO).
  • FBS fibroblast bovine serum
  • DMSO control solvent
  • 0.5 mM all-trans retinoic acid Sigma, St. Louis, MO
  • the medium was replaced by N2 medium containing 0.5% FBS and one of the following: sterile water (control solvent), mouse 2.5S NGF (50 ng/ml, Boehringer Mannheim, Indianapolis, IN), rhBDNF (20 ng/ml, Alomone Labs, Jerusalem, Israel), rhNT-3 (40 ng/ml, Genentech, South San Francisco, CA).
  • the medium was replaced every two days.
  • K-252a 100 nM, Calbiochem, San Diego, CA
  • K-252a 100 nM, Calbiochem, San Diego, CA
  • RNA from stem cells was isolated using R easy Total RNA Kits (Qiagen, Chatsworth, CA) according to manufacturer's instructions. Fifty nanograms of total RNA was reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) with random hexamers (10 mM) as primers in a 20-ml reaction mixture. For PCR amplification, two sets of specific oligonucleotide pairs (0.5 mM) were used: one directed to the transcript of interest, and one for a constitutively expressed transcript, either ribosomal protein L27 or L27a.
  • Reactions were performed in a 100-ml reaction mixture containing 10 mM Tris-Cl (pH 8.4), 50 mM KCl, 1.5 mM MgC12, 200 mM each of the four dNTPs, 2 mCi of [a-32P]dCTP and 5 units of Taq polymerase (Promega) using a thermal cycler (Perkin-Elmer, Foster City, CA) with a regimen of 1 min at 94°C, 2 min at 60°C (55°C for trk receptors) and 2 min at 72°C for 23 cycles. Fifty microliters of each reaction was analyzed by electrophoresis on 8% polyacrylamide gels followed by autoradiography.
  • 5' primer 5'-GAACATTGATGATGGCACCTC-3' (SEQ ID NO: 1)
  • 3' primer 5'-GGGGATATCCACAGAGTACC-3' (SEQ ID NO:2), 189 bp amplified product;
  • 5' primer 5'-ATCGGTAAGCACCGCAAGCA-3' (SEQ ID NO:3), 3' primer: 5'-GGGAGCAACTCCATTCTTGT-3' (SEQ ID NO:4), 235 bp amplified product;
  • 5' primer 5'-CACTGGGTGGCAGTTCTCTT-3' (SEQ ID NO:5), 3' primer: 5'-CATGTACTCGAAGACCATGA-3' (SEQ ID NO:6), 368 bp amplified product;
  • 5' primer 5'-CTGAAGGATCCCACCTTGGC-3' (SEQ ID NO:9)
  • 3' primer 5'-CATGTATTCAAAGACCATGA-3' (SEQ ID NO: 10), 141 bp amplified product;
  • 5' primer 5'-GCATGCACGGGCTGAACGC-3' (SEQ ID NO: 13),
  • 3' primer 5 * -GGGATGCACCGGGAAGGAAG-3' (SEQ ID NO: 14), 317 bp amplified product;
  • 5' primer 5'-GCCCAAGATCTACCTGAG-3' (SEQ ID NO: 15),
  • 3' primer 5'-GTGGGCACTTCAGGGCTTTC-3' (SEQ ID NO: 16), 290 bp amplified product;
  • 5' primer 5*-GGGTTTCAACGCGGACTAC-3' (SEQ ID NO: 17),
  • 3' primer 5'-GTTGGCACTAGAGACGGA-3' (SEQ ID NO:18),166 bp amplified product.
  • BrdU Labeling and Visualization Cells plated as described above were treated with BrdU (10 mM, Sigma) for 24 hrs before fixation. Then the cells were fixed with tris- buffered 4% paraformaldehyde for 15 min, incubated with tris-buffered 0.6% H 2 0 2 for 30 min, and treated with 50% formamide/2x SSC for 2 hrs followed by a 30-min incubation in 2N HCl, and a neutralization in 0.1 M borate (Na 2 B 4 0 7 ) for 10 min.
  • Labeled cells were visualized using confocal scanning laser microscopy (Zeiss Axiovert, Thornwood, NY and Bio-Rad MRC1000, Hemel Hempstead, UK) and color images were generated using Adobe Photoshop (Adobe Systems, Mountain View, CA). Both the average percentage and the absolute number +/- SEM of immunofluorescent cells were determined by counting 10 or 50 high power fields (20x) visualized under fluorescence. The total number of cells was counted using nuclear counterstaining with 4',6-diamidino-2-phenylindole (DAPI, Sigma). Statistical analyses were carried out using ANOVA test.
  • the cells were incubated for 7 days at 4°C in the presence of the AChE substrate solution composed of the following: 4 mM acetylthiocholine iodine, 0.2 mM ethopropazine, 2 mM copper sulfate, 10 mM glycine, and 10 mg/ml gelatin in 50 mM acetate buffer (pH 5.0).
  • the AChE substrate solution composed of the following: 4 mM acetylthiocholine iodine, 0.2 mM ethopropazine, 2 mM copper sulfate, 10 mM glycine, and 10 mg/ml gelatin in 50 mM acetate buffer (pH 5.0).
  • the reaction product were washed with water, treated for 1 min with 1.25% Na 2 S, and washed again with water. Cells were then treated for 1 min with 1% AgN0 3 and washed with water.
  • Retinoic acid has been shown to be effective in promoting a variety of cell differentiation programs.
  • ACSC clonal stem cell cultures
  • RA clonal stem cell cultures
  • simple withdrawal of FGF-2 from the proliferative cultures triggered some neuronal differentiation, as evidenced by the expression of neuron-specific markers.
  • Cyclin-CDK activity can be modulated by tumor suppressors, such as pRb and p53, or CDK inhibitors, which include p21, pl6 and p27. Upregulation of p21 is known to be involved in the NGF- stimulated differentiation of PC 12 cells, and in many neuroblastoma cell lines, RA treatment alone is sufficient to induce growth arrest.
  • One set of cultures was fixed on days 2, 4, 6, 8, 10, and 12 and stained with 4,6- diamidino-2-phenylindole (DAPI) and the total number of nuclei was counted. Additional cultures were pulsed with BrdU for 72 hrs on days 0-3, 3-6, 6-9 or 9-12. The cultures were then fixed on day 12 and evaluated for the proportion of Map2ab-IR cells that had inco ⁇ orated BrdU. Less than one third of the Map2ab-IR divided in the three days immediately following the start of RA treatment. Less than 5% of the Map2ab-IR cells divided during days 3-6 of treatment and fewer than 0.5% of the Map2ab-IR neurons divided after 6 days of RA treatment.
  • DAPI 4,6- diamidino-2-phenylindole
  • NeuroD is one of the neuronal differentiation genes in vertebrates which encodes a basic- helix-loop-helix protein of the same family as atonal. It is transiently expressed in subsets of post-mitotic neurons in the CNS and peripheral nervous system (PNS) and causes premature differentiation of neurons when expressed ectopically.
  • PNS peripheral nervous system
  • RA RA influenced NeuroD expression
  • mRNA was examined after RA treatment. In controls (cells proliferating in the presence of FGF-2), low levels of NeuroD mRNA were detectable.
  • Retinoic acid also upregulates the expression of trkA, trkB, trkC and p75NGFR mRNA.
  • NT neurotrophins
  • mRNA for trkA, trkB, trkC and p75NGFR was examined by RT-PCR analysis. Primers were designed to recognize only the mRNA for full-length Trk receptors, i.e., only those receptors capable of signal transduction via an intact C-terminal catalytic domain.
  • RA treatment also results in neurotrophin-dependent induction of c-fos.
  • Transient and rapid induction of immediate early genes such as c-fos is a well-characterized response to extracellular stimuli in neural cells, c-fos is induced after neurotrophin treatment in cultured fibroblast hippocampal or cortical cells and the signaling cascade following c-fos induction is considered to be integral to subsequent steps in differentiation.
  • mRNAs for trk receptors and p75NGFR observed in the stem cell cultures results in the acquisition of functional Trk-dependent stimulation of c-fos.
  • MAP2abc c-Fos and microtubule-associated protein
  • MAP2abc c-Fos and microtubule-associated protein
  • MAP2ab MAP2ab
  • p75NGFR GFAP
  • the MAP2 protein has three isoforms that are developmentally regulated. In differentiating neurons in vivo and in vitro, MAP2c appears before MAP2a and 2b.
  • the antibodies used here recognize either all three forms (MAP2abc) and would detect MAP2 in both immature and mature neurons or recognize only the a and b forms of MAP2 (MAP2ab) expressed by more mature neurons. Very few c-Fos-positive cells were observed in proliferating cultures.
  • GABA gamma-aminobutyric acid
  • TH tyrosine hydroxylase
  • AChE acetylcholinesterase
  • MAP2ab In proliferating cultures, very few cells ( ⁇ 0.1%) were immunoreactive for MAP2ab or calbindin. No GABAergic, cholinergic, or dopaminergic cells were detected. When cultures were treated with RA for 6 days and then RA was withdrawn for an additional 6 days, the number of MAP2ab-positive cells increased 3-fold. Neurotrophin treatment had no effect on the total number of neurons. In contrast, neurotrophin treatment had a significant effect on the number of neurons expressing GABA, AChE, TH or calbindin. The most frequently encountered phenotype, GABA, was increased by BDNF and NT-3, but not NGF.
  • BDNF and NT-3 increased the number of TH-positive or calbindin- positive cells.
  • BDNF was the most effective agent for increasing the number of AChE- positive cells, although NT-3 and NGF treatment provided small increases over control cultures.
  • ACSC can be induced to differentiate in vitro by treatment with retinoic acid and one or more neurotrophins.
  • AHPCs clonally derived, adult rat hippocampal progenitor cells
  • GFP green fluorescent protein
  • 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), as more thouroughly detailed below.
  • the cells were pulsed with BrdU (5 /Am, 2 days) prior to transplantation.
  • 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 described by Gage, F. H., Ray, J. & Fisher, L. J., Isolation, characterization, and use of stem cells from the CNS, Annu. Rev. Neurosci., 18: 159-192 (1995). 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. Each AHPC clone carried a neomycin phosphofransferase gene (neo) and the enhanced green fluorescence protein (GFP) gene.
  • neo neomycin phosphofransferase gene
  • GFP enhanced green fluorescence protein
  • 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/FI2 + N2, supplemented with 0.5 ⁇ M all-trans retinoic acid and 0.5% fibroblast bovine serum. These conditions favor the differentiation of neurons, astrocytes, and oligodendrocytes in a single well.
  • AHPCs were prepared for grafting in the following manner. Cultured 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 and under direct observation using coaxial illumination via a binocular surgical microscope (Miller) through a dilated pupil
  • Tissue preparation and histology Recipient animals were sacrificed with an overdose of sodium pentobarbitol at 1, 2, 4, and 8 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° C, followed by embedding in OCT and subsequent sectioning at 7-14 ⁇ m on a cryostat.
  • Sections were processed for haematoxilin and eosin, anti- BrdU (1:200), anti-synaptophysin (1:200) and anti-GFP (1:200), followed by reaction with Cy3- conjugated secondary antibodies, thus allowing co-localization of the markers with the endogenous GFP expressed in transplanted AHPCS.
  • Tissue sections were viewed under fluorescence microscopy to identify donor cells,and were compared with adjacent sections stained with H&E to highlight the overall retinal cytoarchitecture, including the retinal laminae. Confocal microscopy was carried out on a subset of material that was of particular interest.
  • 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-fransplanted into both immature (3 days postnatal, P3) and mature (21-28 days postnatal, P21-28) dystrophic eyes of RCS rats. Following transplantation, donor-derived cells were found to maintain high levels of GFP expression.
  • AHPCs could be seen adhering to the vitreal surface of the graft recipient (i.e., host) eye, migrating into the host retina, and taking up residence within specific retinal laminae of the host.
  • 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.
  • the GFP+ cells were quite striking in appearance and were easily distinguished from host autofluorescence in the recipient. No evidence of viable donor cells, or host GFP expression, was seen following injection of freeze-thawed GFP+ AHPCs (negative control).
  • AHPCs may be due to intrinsic or extrinsic developmental factors, or may result from the restrictions imposed by the local retinal cytoarchitecture.
  • Widespread migration and mo ⁇ hological integration of AHPCs were also seen at 8 weeks post-grafting. The degree of retinal integration, however, was not entirely uniform: in some regions the host retinal cytoarchitecture was preserved, while elsewhere the laminar organization was noticeably distorted in association with high numbers of grafted cells. Similar to results observed at 4 weeks post-grafting, AHPCs developed extensive neurite-like projections, which extended throughout the host retina, including the plexiform layers.
  • the retinas of these animals When subsequently examined, the retinas of these animals exhibited widespread migration of green fluorescent protein-expressing (GFP+) donor cells into all layers of the host retina.
  • 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. These results indicate that the dystrophic retina can be substantially repopulated by using a line of adult-derived, neural progenitor or stem cells from an allogeneic donor, and that these cells can be functionally integrated, since they arborize extensively within the host neuropil.
  • the ability of transplanted AHPC cells to migrate into, and differentiate within, the mature retina during the active phase of neuronal degeneration is demonstrated.
  • AHPCs are capable of migrating in large numbers into all layers of the dystrophic neuroretina, including, in some cases, into the photoreceptor layer.
  • transplanted AHPCs exhibit a su ⁇ rising ability to differentiate into neurons with mo ⁇ hological 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.
  • the presence of distinct bands of diffuse GFP-derived fluorescence along these sublaminar zones is suggestive of a network of fine terminals within the host neuropil.
  • One of ordinary skill in the art of neuronal transplantation will appreciate how to practice the present invention and to manipulate AHPCs to account for such factors as functional capability, host immunological tolerance, and the long-term consequences of grafting (e.g., promoting graft survival and controlling undesired proliferation).
  • the demonstration here of survival in a dystrophic, allogeneic environment for at least 2 months, indicates the ultimate immunological success of progenitor cell transplantation to the diseased central nervous system.
  • 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
  • Methodology 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 8 Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina
  • AHPC culture and differentiation Primary adult hippocampal progenitor cultures were prepared from hippocampal tissues of 4- to 6-month-old female Fisher rats as previously described in Example 5. Dissociated cells were cultured on polyornithine/laminin coated dishes using a mixture of DMEM/Ham's F 12 ( 1 : 1 ) supplemented with N2 (Gibco) and 20 ng / ml FGF-2. Individual cells were genetically marked using replication-defective retroviral vectors and two previously extensively characterized multipotent normal diploid clones, PZ5 and Zn3, were used in the present studies.
  • Each AHPC clone carried a neomycin phosphofransferase gene (neo) and either a cytoplasmic ⁇ -Gal gene or a nuclear localized ⁇ - Gal gene.
  • neo neomycin phosphofransferase gene
  • 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% fibroblast bovine serum. These conditions have been demonstrated to favor the differentiation of neurons, astrocytes and oligodendrocytes in a single well.
  • AHPC or ⁇ -Gal marked normal diploid skin fibroblasts were harvested with trypsin, washed with high glucose Dulbecco's PBS (D- PBS, Gibco), and suspended at a density of 100,000 cells per ml in D-PBS containing 20 ng of FGF-2 per ml.
  • D- PBS high glucose Dulbecco's PBS
  • a 10-ml Hamilton syringe with a 30-gauge beveled needle was used to slowly inject 3.0 ml of cells into the vitreous cavity of anesthetized adult rats (2 months old) and 1.5 ml into sub retinal space or vitreous cavity of neonatal rats (P0-P2).
  • AHPC suspended in D-PBS at 100,000 cells per ml were freeze-thawed three times at -70°C prior to injection.
  • Cell phenotype was determined in vitro by double or triple immuno-staining in PBS containing 0.3% Triton X-100 and 3% horse or donkey serum (PBS/TS).
  • Antibodies and concentrations were as follows: rabbit anti- ⁇ -Gal (1:5000, Cortex); mouse anti-microtubule-associated protein 2ab (MAP2ab, 1 :500, Sigma); guinea pig anti-glial fibrillary acidic protein (GFAP, 1:500, Advanced Immunochemical); or mouse anti- galactocerebroside (GalC) diluted 1: 10 (kindly provided by O. Boegler).
  • the primary antibody was detected with flourescein isothiocyanate or Texas red-labeled Donkey secondary antibodies (1:250, Jackson Immunochemicals, West Grove, PA). Eyes were harvested 2, 4, and 8 weeks after grafting and fixed in 4% paraformaldehyde. Twenty micron sections were cut using a cryostat and subsequently stained in PBS/TS with mouse anti- ⁇ -Gal antibody (1 :5000 Promega) and/or guinea pig anti- GFAP (1 :5000).
  • the primary antibody was detected by the ABC method using a biotinylated horse anti-mouse antibody (Jackson Immunochemicals, West Grove, PA) diluted 1:80, and the immunohistochemical product was visualized using 3',3'- diaminobenzidine (DAB) as a chromagen and intensified using nickel. Detection of primary antibodies for fluorescent staining was performed as desccribed herein. For nuclear counterstains, DAB-stained sections were treated with nuclear fast red.
  • DAB 3',3'- diaminobenzidine
  • AHPC populations were isolated from fresh hippocampal tissue, and single stem cells were isolated by clonal density plating under selection for a retrovirally transferred neo gene. The clonality of each culture was confirmed by the presence of a unique proviral band by Southern blot analysis, and karyotype analysis showed that the low-passage clonal populations consisted of normal diploid cells. Two clones were chosen at random from a number of clones previously characterized in detail (See, Palmer, T. D., Takahashi, J., Gage, F. H., The rat hippocampus contains primordial neural stem cells. Mol. Cell. Neurosci. 8:389 (1997)).
  • the cell populations were also extensively evaluated for the presence of replication competent helper virus using both XC and marker- rescue assays and were free of detectable helper virus ( ⁇ 1 per 10ml). (See, Miller, A. D. & Buttimore, C, Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production, Mol. Cell Biol. 6:2895-2902 (1986)).
  • the AHPCs When proliferating in the presence of FGF-2, the AHPCs were observed to have round, bright cell bodies with short, thin processes. Following differentiation in the presence of 0.5% serum and 500 nM all-trans retinoic acid, the cells extended long processes and many cells became immunoreactive for the neuronal marker microtubule-associated protein 2ab (Map2ab), the astrocyte marker glial fibrilary acidic protein (GFAP), or the oligodendrocyte marker galactocerebroside (GalC). These results confirm that the clones used in this work were able to generate all three lineages that exist in the central nervous system.
  • Map2ab the neuronal marker microtubule-associated protein 2ab
  • GFAP astrocyte marker glial fibrilary acidic protein
  • GalC oligodendrocyte marker galactocerebroside
  • AHPCs Three hundred thousand AHPCs were injected into the vitreous space of the adult rat eye, or 150,000 were injected into the sub retinal space or vitreous space of the newborn rat eye. Rats were sacrificed and eyes were processed for histology 2, 4 or 8 weeks later. Controls to validate LacZ as an AHPC-specific marker in vivo included injection of an equivalent number of Z ⁇ cZ-labeled fibroblasts, injection of purified ⁇ -galactosidase enzyme, or injection of freeze-thawed LacZ- ⁇ abeled AHPCs (clone PZ5).
  • tissue slices were co- lableled for ⁇ -Gal and a variety of retina-specific markers including tyrosine hydroxylase, Thy- 1.1, protein kinase C (PKC), Ret PI, and SI 00 protein. None of the grafted cells expressed markers specific for retinal cells with the exception of the occasional SI 00+, and PKC+ cell. However, this finding did not necessarily suggest authentic retinal cell fates since these markers are present in the occasional cell prior to grafting.
  • PKC protein kinase C
  • Control grafts provide a considerable level of confidence that the cells observed in vivo are graft derived.
  • Control grafts of ⁇ -Gal-marked normal diploid skin fibroblasts did not integrate into the retina, but rather attached to the retinal surface. Although these cells formed an intimate and contiguous lamina with the retinal surface, no ⁇ -Gal immunoreactivity was transferred to retinal cells.
  • AHPC-derived progenitors responded to local retinal cues and adopted mo ⁇ hologically appropriate phenotypes for their final location. These results indicate that progeny from immature adult-derived stem cells can respond to local cues in several regions of the central nervous system.
  • stem cells derived from the adult hippocampus respond to a much wider range of migrational (i.e., spatial) and differentiative (i.e., temporal) environments than expected for their site of origin.
  • migrational i.e., spatial
  • differentiative i.e., temporal
  • some cells in adult-derived hippocampal cultures could travel along the rostral migratory stream to the olfactory bulb where they differentiated into olfactory bulb neurons. This suggests that some of the cells were sufficiently immature to adopt non-hippocampal cell fates.
  • Suhonen, J. O., Peterson, D. A., Ray, J. & Gage, F. H. Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo, Nature 383:624-627 (1996).
  • AHPC-derived progenitors responded to local retinal cues and adopted mo ⁇ hologically appropriate phenotypes for their final location.
  • the data further indicate that progeny from immature adult-derived stem cells can respond to local cues in several regions of the central nervous system.

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Abstract

La présente invention concerne des procédés de réparation de tissu lésé ou malade, spécialisé ou différentié, chez des animaux matures, particulièrement le tissu neuronal comme la rétine, par exemple. Plus particulièrement, cette invention concerne la transplantation de progéniteurs obtenus de l'hippocampe adulte, dans le site tissulaire neuronal sélectionné à partir d'un receveur. Ces cellules peuvent s'intégrer de manière fonctionnelle dans des tissus neuronaux matures et immatures. En outre, cette invention concerne, dans un premier aspect, la repopulation de la rétine d'un animal dystrophique avec des neurones, en lui injectant dans l'oeil des cellules souches de système nerveux adulte (ACSC) provenant d'un animal donneur sain, obtenues par clonage. En l'occurrence, cette invention constitue la première intégration stable et réussie de ACSC obtenues pas clonage dans la même espèce mais à partir de donneurs de souches différentes (par exemple, des progéniteurs de Fisher obtenus de l'hippocampe d'un rat adulte (AHPC) injectés dans des rats RCS dystrophiques). Etonnement, on a remarqué que les AHPC s'intégraient également très bien chez le receveur xénogène (par exemple, un AHPC de rat injecté dans la rétine d'une souris dystrophique rd-I).
PCT/US2000/003596 1999-02-11 2000-02-11 Isolation de cellules souches et leurs procedes d'utilisation Ceased WO2000047718A1 (fr)

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US7514259B2 (en) 2000-02-11 2009-04-07 Schepens Eye Research Institute Isolation and transplantation of retinal stem cells
US10758572B2 (en) 2012-02-17 2020-09-01 The Schepens Eye Research Institute Phenotype profile of human retinal progenitor cells
CN112626021A (zh) * 2020-12-30 2021-04-09 南通大学 一种体外纯化培养大鼠胚胎海马放射状胶质细胞的方法

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US5786357A (en) * 1991-12-02 1998-07-28 Sepracor Inc. Methods and compositions for treating sleep disorders, convulsive seizures and other disorders using optically pure (+) zopiclone
US6033906A (en) * 1993-07-26 2000-03-07 California Institute Of Technology Methods for differentiating neural stem cells to glial cells using neuregulins
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7514259B2 (en) 2000-02-11 2009-04-07 Schepens Eye Research Institute Isolation and transplantation of retinal stem cells
US10758572B2 (en) 2012-02-17 2020-09-01 The Schepens Eye Research Institute Phenotype profile of human retinal progenitor cells
US11957719B2 (en) 2012-02-17 2024-04-16 The Schepens Eye Research Institute Phenotype profile of human retinal progenitor cells
CN112626021A (zh) * 2020-12-30 2021-04-09 南通大学 一种体外纯化培养大鼠胚胎海马放射状胶质细胞的方法

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