WO2001090315A2 - Production de neurones a partir de cellules souches - Google Patents

Production de neurones a partir de cellules souches Download PDF

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Publication number
WO2001090315A2
WO2001090315A2 PCT/CA2001/000756 CA0100756W WO0190315A2 WO 2001090315 A2 WO2001090315 A2 WO 2001090315A2 CA 0100756 W CA0100756 W CA 0100756W WO 0190315 A2 WO0190315 A2 WO 0190315A2
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Prior art keywords
medium
stem cell
heat
conditioned medium
treating
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PCT/CA2001/000756
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English (en)
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WO2001090315A3 (fr
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Harold A. Robertson
Cindee Leopold
Victor Rafuse
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Novaneuron Inc.
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Priority to AU2001261984A priority Critical patent/AU2001261984A1/en
Publication of WO2001090315A2 publication Critical patent/WO2001090315A2/fr
Publication of WO2001090315A3 publication Critical patent/WO2001090315A3/fr

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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
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N2501/11—Epidermal growth factor [EGF]
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system

Definitions

  • the invention relates to methods for producing neurons from stem cells.
  • the usual method of isolating neural stem cells in vitro is to dissect out a region of the fetal or adult brain known to contain dividing cells in vivo.
  • the tissue is disaggregated and the dissociated cells are exposed to a high concentration of mitogens such as fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) (B. A. Reynolds and S. Weiss, Dev. Biol. 175:1 (1996)) in either a defined or supplemented medium on a matrix as a substrate for binding.
  • mitogens such as fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) (B. A. Reynolds and S. Weiss, Dev. Biol. 175:1 (1996)
  • FGF-2 fibroblast growth factor-2
  • EGF epidermal growth factor
  • the cells proliferate, and subsequently they can be either induced to differentiate by withdrawing the mitogens or by exposing the cells to another factor that induces some of the cells to
  • the phenotypes of cells are then determined by staining with antibodies directed against antigens specific for astrocytes, oligodendrocytes, and neurons.
  • a single cell can be plated at low density and monitored to determine if a single cell can give rise to the three phenotypes (B. A. Reynolds and S. Weiss, Dev. Biol. 175:1 (1996)).
  • Stem cells were also tagged with a retro virus in vitro, after which the clones of cells derived from the original tagged cell were shown by Southern analysis to be derived from a single cell (T. D. Palmer, J. Takahashi, F. H. Gage, Mol. Cell. Neurosci. 8:389 (1997)).
  • progenitor cells differentiate into the neuronal phenotype and then differentiate into specific types of neuron (for example, dopaminergic, serotonergic, cholinergic, hippocampal pyramidal cells, striatal neurons) (S. Weiss, et al., Trends Neurosci. 19, 387 (1996)). It is important to produce cell types of a specific phenotype. For example, dopamine neurons are needed in large numbers as a potential therapy for Parkinson's disease. Parkinson's disease is caused by the loss of dopamine neurons and a large body of work suggests that replacement of these neurons by transplantation into patients will reverse the symptoms of Parkinson's disease (C.W. Olanow, et al. Trends Neurosci.
  • the present inventors have discovered a simple technique for converting stem cells into a neuronal phenotype. Following this method, it is possible to convert most or all stem cells into neurons.
  • the invention provides a method for producing a neuron comprising: (a) incubating a stem cell in a growth medium; (b) separating the growth medium from the stem cell;
  • the invention also provides a method for producing neurons comprising:
  • the invention further provides a conditioned medium for growing neurons from stem cells comprising a medium prepared by the steps of: (a) isolating a stem cell;
  • the treated medium includes the growth medium. In a further embodiment, the treated medium includes the growth medium of step (c).
  • the stem cell may be a brain stem cell, an adult stem cell, a striatal stem cell, a mammalian stem cell, a human stem cell, a mouse stem cell and/ or a stem cell from a subependymal zone.
  • the heat-treating is at a temperature of between 40°Cand
  • the heat-treating may be for a duration of between 1 minute and one week, between five minutes and one day, between 10 minutes and one hour, or about 30 minutes.
  • the treated medium comprises about 50% the growth medium and about 50% the heat-treated medium.
  • the growth factor may be EGF, bFGF, or EGF and bFGF.
  • the growth factor may have a concentration of about 20 ng/ml.
  • the growth medium may be DMEM/F12.
  • the incubating may be carried out to a density of appoximately 12,500 cells/ml, it may be carried out at about 37 °C and/ or it may be carried out at about 5% CO 2 .
  • the incubating is carried out for about 2 weeks.
  • the separating may be by centrifugation.
  • the subsequent incubation may be carried out by plating isolated stem cells.
  • the isolated stem cells may be isolated from neurospheres.
  • the method of the invention also embodies further inducing the neurons to differentiate.
  • the invention comprises a neuron produced by any of the methods of the invention.
  • the invention is the use of the neuron to treat a neural disease.
  • the neural disease may be Parkinson's disease, Huntington's disease, brain or spinal cord injury, epilepsy, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, tumors, or stroke.
  • the present invention discloses a novel method that induces all or almost all stem cells in vitro to assume a neuronal-like phenotype.
  • the present inventors noticed that cell proliferation was significantly higher at high cell densities compared to lower cell densities. These findings suggested that the cells were releasing a factor or factors that were "conditioning" the mediums. Accordingly, the present inventors heat- treated the medium at 56°C for 30 minutes and incubated stem cells in this heat- inactivated conditioned medium. Instead of reducing the proliferation rate as expected, all of the stem cells incubated in this medium assumed a neuronal-like phenotype.
  • the method used to create the neurons is as follows. Stem cells were isolated from an adult brain. The isolated stem cells were placed in a conditioned medium and were incubated. The conditioned medium was then separated from the neurospheres. The conditioned medium was heat-treated. The stem cells were then plated in a medium which includes the heat-treated conditioned medium. By this methodology, almost all stem cells formed neurons.
  • Isolating neural stem cells in vitro may be accomplished by dissecting a region of the fetal or adult brain or fetal or adult skin cells known to contain dividing cells in vivo. This can be successfully done, for example, with the subventricular zone (SVZ) or the hippocampus from an adult brain or many structures in the developing brain.
  • SVZ subventricular zone
  • the in vitro culture of region-specific differentiated neurons may be derived from any mammalian multipotential CNS stem cell, including mouse and human.
  • stem cells are derived from the group consisting of cortex, olfactory tubercle, retina, septum, lateral ganglionic eminence, medial ganglionic eminence, amygdala, hippocampus, thalamus, hypothalamus, ventral and dorsal mesencephalon, brain stem, cerebellum, and spinal cord.
  • Stem cells as defined herein include cells able to divide to regenerate themselves and, in addition, to produce progeny which can follow other developmental patterns.
  • Cells can be obtained from donor tissue by dissociation of individual cells from the connecting extracellular matrix of the tissue. Tissue from a particular neural region is removed from the brain using a sterile procedure, and the cells are dissociated using any method known in the art including treatment with enzymes such as trypsin, collagenase and the like, or by using physical methods of dissociation such as with a blunt instrument. Dissociation of fetal cells can be carried out in tissue culture medium, while a preferable medium for dissociation of juvenile and adult cells is low Ca 2+ artificial cerebral spinal fluid (aCSF).
  • aCSF Ca 2+ artificial cerebral spinal fluid
  • Regular aCSF contains 124 mM NaCl, 5 mM KC1, 1.3 mM MgCl 2 , 2 mM CaCl 2 , 26 mM NaHCO 3 , and 10 mM D- glucose.
  • Low Ca 2+ aCSF contains the same ingredients except for MgCl 2 at a concentration of 3.2 mM and CaCl 2 at a concentration of 0.1 mM. Dissociated cells are centrifuged at low speed, between 200 and 2000 rpm, usually between 400 and
  • the neural cells can be cultured in suspension or on a fixed substrate. However, substrates tend to induce differentiation of the neural stem cell progeny. Thus, suspension cultures are preferred if large numbers of undifferentiated neural stem cell progeny are desired.
  • Cell suspensions are seeded in any receptacle capable of sustaining cells, particularly culture flasks, culture plates or roller bottles, and more particularly in small culture flasks such as 25 cm 2 culture flasks.
  • Cells cultured in suspension are resuspended at approximately 5 X 10 4 to 2 X 10 ' cells/ml, preferably 2.85 X 10 4 cells/ml.
  • Cells plated on a fixed substrate are plated at approximately 2-3 X 10 3 cells/cm 2 , preferably 8.0 X 10 3 cells/cm 2 .
  • Division of neural stem cells can be accomplished in numerous ways. They may undergo asymmetric division where cells are produced with different fates. Neural stem cells may also undergo proliferative divisions, which expands the stem cell population, or differentiative divisions, which reduces the stem cell population, (Doe, C.Q., Fuerstenberg, S., and Peng, CY, J Neurobiology 36:111 (1998)). Neural stem cells are also likely to produce intermediate precursors termed progenitor cells that can generate specific subpopulations.
  • the dissociated neural cells can be placed into any known culture medium capable of supporting cell growth, including HEM, DMEM, RPMI, F-12, and the like, containing supplements which are required for cellular metabolism such as glutamine and other amino acids, vitamins, minerals and useful proteins such as transferrin and the like.
  • Medium may also contain antibiotics to prevent contamination with yeast, bacteria and fungi such as penicillin, streptomycin, gentamicin and the like.
  • the medium may contain serum derived from bovine, equine, chicken and the like.
  • One embodiment for proliferation of neural stem cells is to use a defined, serum- free culture medium, as serum tends to induce differentiation and contains unknown components (i.e. is undefined).
  • a defined culture medium is also desired if the cells are to be used for transplantation purposes.
  • Conditions for culturing should be close to physiological conditions.
  • the pH of the culture medium should be close to physiological pH, preferably between pH 6- 8, more preferably between about pH 7 to 7.8, with pH 7.4 being most preferred.
  • Physiological temperatures range between about 30 °Cto 40 °C Cells are preferably cultured at temperatures between about 32 °Cto about 38 C, and more preferably between about 35 °Cto about 37C.
  • the culture medium is supplemented with at least one proliferation-inducing growth factor.
  • growth factor refers to a protein, peptide or other molecule having a growth, proliferative, differentiative, or trophic effect on neural stem cells and or neural stem cell progeny. Growth factors which may be used for inducing proliferation include any trophic factor that allows neural stem cells and precursor cells to proliferate, including any molecule which binds to a receptor on the surface of the cell to exert a trophic, or growth-inducing effect on the cell.
  • Preferred proliferation-inducing growth factors include EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor ⁇ (TGF- ⁇ ), and combinations thereof.
  • Nurrl a transcription factor of the thyroid hormone/retinoic acid nuclear receptor superfamily (Wagner, J., et al., Nature Biotechnology 17:653 (1999)), or a combination of FGF2 and epidermal growth factor (EGF) (B. A. Reynolds and S. Weiss, Science 255:1707 (1992); Morshead, CM. et al., Neuron 13:1071 (1994)).
  • EGF is particularly useful as a primary mitogen to expand the most primitive cells. Also useful for primitive cells is FGF-2 with or without EGF.
  • TGF- ⁇ Transforming growth factor ⁇
  • TGF- ⁇ another ligand for the EGF receptor
  • TGF- ⁇ has also been shown to have a proliferative effect
  • other growth factors may be added to the culture medium that influence proliferation and differentiation of the cells including NGF, platelet-derived growth factor (PDGF), thyrotropin releasing hormone (TRH), transforming growth factor ⁇ s (TGF ⁇ s), insulin-like growth factor (IGF j ) and the like.
  • PDGF platelet-derived growth factor
  • TRH thyrotropin releasing hormone
  • TGF ⁇ s transforming growth factor ⁇ s
  • IGF j insulin-like growth factor
  • Growth factors are usually added to the culture medium at concentrations ranging between about 1 fg/ml to 1 mg/ml. Concentrations between about 1 to 100 ng/ml are usually sufficient. Titration experiments can be performed to determine the optimal concentration of a particular growth factor.
  • a multipotent neural stem cell begins to divide giving rise to a cluster of undifferentiated cells referred to herein as a "neurosphere".
  • the cells all extend projections
  • neurites and these neurites form synapses with one another.
  • the extending terminals of these neurites form growth cones, the specialization that guides the extending neurites.
  • a multiplicity of mechanisms may trigger differentiation (see, for example,
  • Stem cells were isolated from the subependymal zone of the striatum of adult mouse brain according to Reynolds and Weiss (1992). Isolated stem cells were placed in a DMEM/F 12 -containing 20 ng/ml of both EGF and bFGF at a density of approximately 12,500 cells/ml and were incubated at 37°C with 5% CO 2 according to B. A. Reynolds and S. Weiss, Science 255 : 1707 (1992) or B. A. Reynolds and S.

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Abstract

L'invention concerne un procédé de production de neurones consistant à incuber des cellules souches dans un milieu de croissance contenant un facteur de croissance, à séparer le milieu de croissance des cellules souches, à traiter thermiquement le milieu de croissance afin de produire un milieu traité thermiquement, puis à incuber ces cellules souches dans un milieu traité comprenant le milieu traité thermiquement. Elle concerne aussi des neurones et le milieu conditionné produits au moyen de ce procédé ainsi que leurs utilisations.
PCT/CA2001/000756 2000-05-25 2001-05-25 Production de neurones a partir de cellules souches WO2001090315A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001261984A AU2001261984A1 (en) 2000-05-25 2001-05-25 Production of neurons from stem cells

Applications Claiming Priority (2)

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US20680700P 2000-05-25 2000-05-25
US60/206,807 2000-05-25

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WO2001090315A2 true WO2001090315A2 (fr) 2001-11-29
WO2001090315A3 WO2001090315A3 (fr) 2002-08-01

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851832A (en) * 1991-07-08 1998-12-22 Neurospheres, Ltd. In vitro growth and proliferation of multipotent neural stem cells and their progeny

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62107796A (ja) * 1985-11-07 1987-05-19 Morinaga Milk Ind Co Ltd 新規なヒト顆粒球−単球系幹細胞増殖因子の製造法
JP3431268B2 (ja) * 1994-03-31 2003-07-28 雪印乳業株式会社 細胞培養に用いる培地用添加剤

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5851832A (en) * 1991-07-08 1998-12-22 Neurospheres, Ltd. In vitro growth and proliferation of multipotent neural stem cells and their progeny

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DAADI MARCEL M ET AL: "Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain." JOURNAL OF NEUROSCIENCE, vol. 19, no. 11, 1 June 1999 (1999-06-01), pages 4484-4497, XP002191901 ISSN: 0270-6474 *
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; PREV200100088076, XP002191904 & SHINGO T ET AL: "Robust induction of tyrosine hydroxylase in embryonic and adult striatal neural stem cell-derived neurons in defined media and the absence of gene transfer." SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 26, no. 1-2, 2000, pages abstract-312.18, 30th Annual Meeting of the Society of Neuroscience;New Orleans, LA, USA; November 04-09, 2000 *
DATABASE WPI Section Ch, Week 198725 Derwent Publications Ltd., London, GB; Class B04, AN 1987-175153 XP002191905 & JP 62 107796 A (MORINAGA MILK CO LTD), 19 May 1987 (1987-05-19) *
DATABASE WPI Section Ch, Week 199550 Derwent Publications Ltd., London, GB; Class B04, AN 1995-388680 XP002191906 & JP 07 265068 A (SNOW BRAND MILK PROD CO LTD), 17 October 1995 (1995-10-17) *
FERNANDES K: "Recent advances in neural stem cell technologies review" NEURAL NOTES, vol. 19, 10 November 2001 (2001-11-10), XP002191903 *
KAWASAKI HIROSHI ET AL: "Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity." NEURON, vol. 28, no. 1, October 2000 (2000-10), pages 31-40, XP002191902 ISSN: 0896-6273 *
REYNOLDS B A ET AL: "CLONAL AND POPULATION ANALYSES DEMONSTRATE THAT AN EGF-RESPONSIVE MAMMALIAN EMBRYONIC CNS PRECURSOR IS A STEM CELL" DEVELOPMENTAL BIOLOGY, ACADEMIC PRESS, NEW YORK, NY, US, vol. 175, 1996, pages 1-13, XP000916284 ISSN: 0012-1606 cited in the application *
WEISS S ET AL: "Is there a neural stem cell in the mammalian forebrain?" TRENDS IN NEUROSCIENCE, ELSEVIER, AMSTERDAM, NL, vol. 19, no. 9, 1996, pages 387-393, XP002098734 ISSN: 0166-2236 cited in the application *

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WO2001090315A3 (fr) 2002-08-01

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