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WO1996015226A1 - Regulation de la proliferation de cellules souches neurales - Google Patents

Regulation de la proliferation de cellules souches neurales Download PDF

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Publication number
WO1996015226A1
WO1996015226A1 PCT/CA1995/000637 CA9500637W WO9615226A1 WO 1996015226 A1 WO1996015226 A1 WO 1996015226A1 CA 9500637 W CA9500637 W CA 9500637W WO 9615226 A1 WO9615226 A1 WO 9615226A1
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stem cell
cells
proliferation
neural stem
egf
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PCT/CA1995/000637
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English (en)
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Samuel Weiss
Brent A. Reynolds
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Neurospheres Holdings Ltd.
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Priority to EP95936393A priority Critical patent/EP0792350A1/fr
Priority to CA 2204630 priority patent/CA2204630A1/fr
Priority to JP8515600A priority patent/JPH10509592A/ja
Priority to AU38367/95A priority patent/AU716811B2/en
Publication of WO1996015226A1 publication Critical patent/WO1996015226A1/fr
Priority to FI971956A priority patent/FI971956L/fi
Priority to NO972171A priority patent/NO972171L/no
Priority to MXPA/A/1997/003492A priority patent/MXPA97003492A/xx

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
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    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/475—Growth factors; Growth regulators
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    • C07K—PEPTIDES
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    • 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
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    • A01K2217/00—Genetically modified animals
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    • 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
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/11—Epidermal growth factor [EGF]
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    • 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)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Definitions

  • stem cells In actively dividing tissues, such as bone marrow which gives rise to blood cells, specialized cells, known as stem cells, are present.
  • the critical identifying feature of a stem cell is its ability to exhibit self-renewal or to generate more of itself.
  • the simplest definition of a stem cell would be a cell with the capacity for self- maintenance.
  • a more stringent (but still simplistic) definition of a stem cell is provided by Potten and Loeffler [Development, 110: 1001 (1990)] who have defined stem cells as "undifferentiated cells capable of a) proliferation, b) self- maintenance, c) the production of a large number of differentiated functional progeny, d) regenerating the tissue after injury, and e) a flexibility in the use of these options.
  • Stem cells divide, generating progeny known as precursor cells.
  • Precursor cells comprise new stem cells and progenitor cells.
  • the new stem cells are capable of dividing again, producing more stem cells, ensuring self-maintenance, and more progenitor cells.
  • the progenitor cells are capable of limited proliferation, where all of their progeny ultimately undergo irreversible differentiation into amitotic, functional cells.
  • FIG. 1 illustrates the relationship between stem cells, progenitor cells and differentiated cells.
  • the role of stem cells is to replace cells that are lost by natural cell death, injury or disease.
  • the presence of stem cells in a particular type of tissue usually correlates with tissues that have a high turnover of cells. However, this correlation may not always hold as stem cells are thought to be present in tissues, such as the liver [Travis, Science, 259: 1829 (1989)] that do not have a high turnover of cells.
  • the best characterized stem cell system is the hematopoietic stem cell.
  • Evidence suggests that a single hematopoietic stem cell, located in bone marrow, is able to give rise, via a series of progenitor cells, to all of the blood cell lineages.
  • US Patent 5,061 ,620, issued October 29, 1991 provides a means for isolating, regenerating and using the hematopoietic stem cell.
  • hematopoietic stem cells are active at many sites, including the fetal yolk sac, bone marrow, liver and spleen, Shortly before birth the bone marrow takes over as the primary site of hematopoiesis.
  • the hematopoietic stem cells in the liver and spleen become quiescent and do not resume production of blood cells unless hematopoietic stem cell activity in the bone marrow is suppressed or widespread destruction of blood cells occurs.
  • the differentiated cells of the adult mammalian CNS exhibit little or no ability to enter the mitotic cycle and generate new neural tissue — essentially all neurogenesis occurs during the prenatal and immediate post-natal p riods W hile it is believed that there is a limited and slow turnover ot astrocytes [ ⁇ rr c ⁇ al , J Co p. Neurol. , 150: 169 (1971)] and that progenitor cells which can give rise to oligodendrocytes are present [Wolsqijk and Noble, Development, 105: 386-698 (1989)], the generation of new neurons does not normally occur.
  • Rats exhibit a limited ability to generate new neurons in restricted adult brain regions such as the dentate gyrus and olfactory bulb [Kaplan, J. Comp. Neurol. , 195: 323 (1987); Bayer, S.A, NY. ⁇ cacl. Set. , 457: 163- 172 ( 1985)] but this does not apply to all mammals; and the generation of new neurons in adult primates does not occur [Rakic, P. , Science, 227: 1054 (1985)].
  • This inability to produce new neuronal cells in most mammals (and especially primates) may be advantageous for long-term memory retention; however, it is a distinct disadvantage when the need to replace lost neuronal cells arises due to injury or disease.
  • hematopoietic stem cells survival, expansion and proliferation of the hematopoietic stem cells, and stem cell systems in the liver, intestines and skin have been shown to be under the control of a number of different trophic factors.
  • growth factors such as erythropoietin and the glycoprotein CSF (colony-stimulating factor) and various interleukins have been identified as factors which regulate stem cell function [Metcalf, D. , Bioassays, 14(12): 799-805 (1992)] .
  • PDGF platelet derived growth factor
  • CNTF ciliary neurotrophic factor
  • bFGF basic f ⁇ broblast growth factor
  • EGF epidermal growth factor
  • TGF ⁇ transforming growth factor alpha
  • NGF nerve growth factor
  • 0-2A cell a type of embryonic neural progenitor cell, known as the 0-2A cell, gives rise to oligodendrocytes and type-2 astrocytes.
  • the 0-2 A cell divides and after a few divisions differentiates into oligodendrocytes.
  • CNTF and substrate factors rather than PDGF
  • bFGF produces a two-told increase in the proliferation of embryonic progenitor cells which develop into neurons [Gensberger et at. FEB Lett. , 217: 1 -5 ( 1987)].
  • Cattaneo and McKay (1990) showed that growth factors added together or in sequential fashion will elicit novel responses not seen when the factors are added individually.
  • NGF stimulated the proliferation of embryonic neuroblasts to produce neurons only after they have been previously primed with bFGF [Cattaneo, E. and McKay, R. , Nature, 347: 762-765 ( 1990)].
  • bFGF has also been shown to influence the expression of the PDGF receptor and to block the differentiation of the 0-2A progenitor cell when exposed to PDGF [McKinnon et al. , Neuron, 5: 603-614 ( 1990)].
  • EGF or TGF ⁇ show some mitogenic effects on embryonic retinal neuroepithelial cells grown in culture, resulting in progenitor cells which, in the continued presence of the growth factors, give rise to neurons but not to glial cells [Anchan et al.. Neuron, 6: 923-936 (1991)]. In the same study, neurons and Miiller cells are reported to occur in cultures derived from postnatal rat neuroepiihelium.
  • CNS disorders encompass numerous afflictions such as neurodegenerative diseases (e.g. Alzheimer's and Parkinson's), acute brain injury (e.g. stroke, head ⁇ njur ⁇ , cerebral palsy) and a large number of diseases associated ith CNS d ⁇ stun t ⁇ on (e.g. depression, epilepsy, and schizophrenia)
  • neurodegenerative diseases e.g. Alzheimer's and Parkinson's
  • acute brain injury e.g. stroke, head ⁇ njur ⁇ , cerebral palsy
  • a large number of diseases associated ith CNS d ⁇ stun t ⁇ on e.g. depression, epilepsy, and schizophrenia
  • ⁇ ears neurodegeneramc disease has become an important concern due to the expanding elderl ⁇ population which is at greatest risk for these disorders.
  • CNS dysfunction with respect to the number of affected people
  • CNS dysfunction are not characterized by a loss of neural cells but rather by an abnormal functioning of existing neural cells. This may be due to inappropriate firing of neurons, or the abnormal synthesis, release, and processing of neurotransmitters.
  • An emerging technology for treating neurological disorders entails the transplantation of cells into the CNS to replace or compensate for loss or abnormal functioning of the host's nerve cells. While embryonic CNS cells given the best results in human trials [Winder et al. , New Eni;. J. Med. , 327: 1556 ( 1992)] and are the preferred donor tissue, ethical and political considerations, as well as the availability of sufficient quantities of tissue, limit the use ⁇ i these cells. Other types of donor tissue for use in the treatment of CNS disorders are being developed. These include: genetically modified neural cell lines [Renfranz et al.
  • transplantation approaches represent a significant improvement over currently available treatments for neurological disorders, the technology has not yet been perfected.
  • some cell types fail to integrate with host CNS tissue.
  • the use of non-neuronal primary cell cultures limits the ability of the transplanted material to make connections with the host tissue.
  • Immortalized donor cells obtained from primary neural tissue could form connections but the genetic expression of the oncogenes inco ⁇ orated into these transformed cells is hard to control and could produce tumors and other complications.
  • Donor and host could result in the rejection of the implanted cells.
  • the transplanted cells can result in tumor formation or pass infectious agents from the donor tissue to the host.
  • Gage et al. in US Patent 5,082,670, disclose a method of treating defects, disease or CNS cell damage by grafting genetically modified neural cells into the appropriate CNS regions.
  • the donor cells disclosed in this patent were obtained from non-neuronal primary cultures but it was suggested that genetically transformed neural cell lines could be used. These donor cell sources are inherently problematic.
  • Gage et al. recognize the limitations imposed by the donor cells used in their technique and acknowledge that there is a ".. paucity of replicating non-transformed cell-culture systems... " They also recognize "the refractoriness of non-replicating neuronal cells to viral infection.
  • the multipotent neural stem cells which are now known to be present in the brains of mammals throughout their lives [Reynolds and Weiss, Science, 255- 1707 (1992)], provide a source of non-transformed neural cells which can be stimulated, in the presence of a growth factor such as epidermal growth factor, to become mitotically active.
  • the neural stem cells can be induced to proliferate and can provide large quantities of undifferentiated neural cells, which are capable of differentiation into the major types of neural cells and can be transplanted, genetically modified and then transplanted, or used for drug screening or other purposes.
  • gliosis In response to injury of brain or spinal cord tissue, gliosis occurs It is thought that the glial scar which results from this process may prevent neuronal axons from reestablishing connections across the injury region, thus preventing the restoration of function.
  • Astrocytes which proliferate both at the wound sue and some distance away from the immediate vicinity of the wound are the principle cellular components of a glial scar (Reier, PJ. Astrocytes vol. 3: 263-323 ( 1986)). Proliferation of neural stem cells and their progeny in response to injury may be a factor in the development of gliosis Neural repair could be enhanced if the extent of gliosis at the injury site could be reduced.
  • compositions for the in vivo regulation of the proliferation ol CNS stem cells
  • the compositions comprise specific biological lactors, such as grow th factors or combinations of such factors, which are intused into the ventricular system ot the CNS to regulate stem cell proliferation.
  • a method of regulating the in vitro proliferation ot a multipotent neural stem cell and/or the proliferation of progeny of said neural stem cell comprises the steps of dissociating mammalian neural tissue containing at least one multipotent neural stem cell capable of producing progeny that are capable of differentiating into neurons, astrocytes and oligodendrocytes, and proliferating the multipotent neural stem cell in a culture medium containing at least one proliferative factor that induces stem cell proliferation and a regulatory factor that regulates proliferation of the multipotent neural stem cell and/or proliferation of the progeny of the multipotent neural stem cell.
  • a method and compositions for regulating the in vivo proliferation of a multipotent neural stem cell and/or the proliferation of progeny of said neural stem cell are described.
  • the method comprises the steps of delivering to the ventricular regions of a mammal a therapeutic composition comprising at least one factor that has a regulatory effect on the proliferation of a multipotent neural stem cell and or the proliferation of the progeny of the multipotent neural stem cell.
  • the proliferative factor is bFGF and the regulatory factor is EGF or heparan sulfate which increase the rate of proliferation of stem cell progeny.
  • a therapeutic composition comprising a factor or combination of factors which inhibit the proliferation ⁇ i neural stem cells is administered //; V ⁇ ' VY to reduce the proliferation of the cells.
  • FIG. 1 A schematic diagram illustrating the proliferation of a multipotent neural stem cell.
  • A In the presence of a proliferative factor the stem cell divides and gives rise to a sphere of undifferentiated cells composed of more stem cells and progenitor cells.
  • B When the clonally derived sphere of undifferentiated cells is dissociated and plated as single cells, on a non-adhesive substrate and in the presence of a proliferative factor, each stem cell will generate a new sphere.
  • C If the spheres are cultured in conditions that allow differentiation, the progenitor cells differentiate into neurons, astrocytes and oligodendrocytes.
  • FIG. 1 A schematic diagram illustrating the proliferation of a multipotent neural stem cell.
  • FIG. 3 Graph showing the number of neurospheres generated from primary cells derived from the cervical, thoracic, and lumbar regions of adult mice spinal cord in the presence of 20 ng/ml EGF + 20 ng/ml FGF or 20 ng/ml FGF + 2 ⁇ g/ml heparan sulfate.
  • neural stem cell or "central nervous system (CNS) stem cell” refers to a relatively quiescent, undifferentiated stem cell from neural tissue that is capable of proliferation, giving rise to more neural stem cells (thus ensuring self-maintenance) and to progenitor cells.
  • multipotent refers to a neural stem cell that is capable ot producing progeny that give rise to each of the major types of differentiated neural cells, i.e.
  • an undifferentiated cell that gives rise to two types of differentiated cells for example, the O-2A cell, which gives rise to oligodendrocytes and astrocytes. is termed “bipotent” , and one that gives rise to only one type of differentiated cell is termed “unipotent”.
  • progenitor cell also refers to an undifferentiated cell derived from a neural stem cell but differs from a stem cell in that it has limited ability to proliferate and does not maintain itself.
  • Each of a neural progenitor cell's progeny will, under appropriate conditions, differentiate into a neuron, astrocyte (type I or type II) or oligodendrocyte. Oligodendrocytes are differentiated glial cells that form the myelin surrounding axons in the central nervous system (CNS).
  • CNS central nervous system
  • Oligodendrocytes have the phenotype galactocerebroside ( + ), myelin basic protein (+), and glial fibrillary acidic protein (-) [GalC(+), MBP(+), GFAP(-)].
  • Neurons are differentiated neuronal cells that have the phenotype neuron specific enolase (+), neurofilament (+), microtubule associated protein or Tau-1 (+) [NSE(+), NF (+), MAP-2 (+), or Tau-1 (+)].
  • Astrocytes are differentiated glial cells that have the phenotype GFAP( + ), GalC(-), and MBP(-).
  • the CNS stem cell is capable of self- aintenance and generating a large number of progeny including new stem cells and progenitor cells capable of differentiation into neurons, astrocytes and oligodendrocytes.
  • CNS stem cells can be isolated and cultured from any pre- or post-natal mammalian CNS tissue by the methods described by Rey nolds and W eiss [Saern c. 255: 1707, ( 1992)], the published PCT applications referenced e. and in Example 1 , below .
  • Multipotent CNS stem cells occur in a oi CNS regions. including the conus medullaris, cervical, thoracic and lumbar regions of the spinal cord, the brain stem, striatum and hypothalamus.
  • the neural stem cells can be obtained from tissue from each of these regions and induced to divide in vitro, exhibiting self-maintenance and generating a large number of progeny which include neurons, astrocytes and oligodendrocytes.
  • the multipotent neural stem cell is obtained from neural tissue and grown in a culture medium which is preferably serum-free and which may comprise any combination of substances known to support the survival of cells.
  • a suitable serum-free culture medium herein after referred to as "Complete Medium”, comprises Dulbecco's modified Eagle's medium (DMEM) and F- 12 nutrient mixture (Gibco) (1 : 1), glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), HEPES (4-[2-hydroxyethyI]- l -p ⁇ peraz ⁇ neethanesulfon ⁇ c acid) buffer (5 mM) and a defined hormone mix and salt mixture (10% ; available from Sigma), used to replace serum, which comprises insulin (25 ⁇ g/ml), transferrin (100 ⁇ g/ml), progesterone (20 ⁇ M), putrescine (60 ⁇ M), and selenium chloride (30 nM).
  • At least one biological factor that induces multipotent stem cell proliferation
  • biological factor refers to a biologically active substance that is functional in CNS cells, such as a protein, peptide, nucleic acid, growth factor, steroid or other molecule, natural or man-made, that has a growth, proliferative, differentiative, trophic, or regulatory effect (either singly or in combination with other biological factors) on stem cells or stem cell progeny.
  • biological factors include growth factors such as acidic and basic fibroblast growth factors (aFGF, bFGF), epidermal grow th tactor (EGJ-) and EG1 - like ligands, transforming growth factor alpha (TGF ⁇ ), insulin-like growth factor (IGF-1), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factor betas (TGF/3); trophic factors such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF). and glial-de ⁇ ved neurotrophic factor (GDNF); regulators of intracellular pathw ay associated w ith grow th factor activity such as phorbol 12-my ⁇ state 13-acetate. staur ⁇ spormc.
  • aFGF, bFGF epidermal grow th tactor
  • TGF ⁇ transforming growth factor alpha
  • IGF-1 insulin-like growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • TGF/3
  • hormones such as a n .iU ⁇ i ⁇ ⁇ KHI OPU I le.easni - hormone (TRH); various proteins and polypeptides such as interleukins.
  • BMP-2 bone morphogemc protein
  • MlP-l ⁇ macrophage inflammatory proteins
  • MIP-1B and MIP-2 macrophage inflammatory proteins
  • oligonucleotides such as antisense strands directed, for example, against transcripts for EGF receptors, FGF receptors, and the like
  • hepa ⁇ n-like molecules such as heparan sulfate
  • TNF ⁇ J tumor necrosis factor alpha
  • proliferative factors Biological factors, such as EGF and bFGF, that individually have a proliferative effect on multipotent neural stem cells are herein referred to as "proliferative factors".
  • proliferative factors bind to a cell-surface receptor, resulting in the induction of proliferation.
  • Preferred proliferative factors include EGF, amphiregulin, aFGF, bFGF, TGF ⁇ , and combinations of these and other biological factors, such as heparan sulfate.
  • a particularly preferred combination for inducing the proliferation of neural stem cells is EGF and bFGF.
  • the proliferative factors are usually added to the culture medium at a concentration in the range of about 10 pg/ml to 500 ng/ml, preferably about 1 ng/ml to 100 ng/ml.
  • concentration in the range of about 10 pg/ml to 500 ng/ml, preferably about 1 ng/ml to 100 ng/ml.
  • concentration for EGF, aFGF and bFGF is about 20 ng/ml of each proliferative factor.
  • the stem cells may be cultured in any culture vessels, for example 96 well plates or culture flasks. In the presence of a proliferation-inducing growth factor or combination of factors a multipotent neural stem cell divides, giving rise, within 3- 4 days, to undifferentiated stem-cell progeny.
  • the stem cell progeny referred to herein as "precursor cells”, include newly generated multipotent stem cells and progenitor cells, ln vitro, the progeny of a single stem cell typically forms a cluster of precursor cells referred to herein as a "neurosphere"; however, culture conditions may be changed (e.g. by providing a treated substrate onto which the proliferating cells adhere) so that the proliferating cells do not form the characteristic neurospheres.
  • Precursor cells are not iinmunoreactive for any of the neuronal or glial cell markers, but they are immunoreactive for nestin, an intermediate filament protein found in undifferentiated CNS cells [Lehndahl et al. , Cell, 60: 585-595 ( 1990)].
  • the precursor cells within the neurosphere continue to divide resulting in an increase in the size of the neurospheres as a result of an increase in the number of undifferentiated cells [nestin( + ), NF(-), NSE (-), GFAP(-), MBP (-)]. It is possible to passage the precursor cells in the presence of the same growth factors or different growth factors that allow further proliferation to occur without promoting differentiation. Cells can be passaged 30 times or more using proliferative culture methods, resulting in an exponential increase in precursor cell numbers.
  • the culture techniques described above for the proliferation of CNS stem cells in vitro can be modified through the use of additional biological factors or combinations of factors which increase, decrease or modify in some other way the number and nature of the precursor cells obtained from the stem cells in response to EGF or other proliferative factors Changes in proliferation are observed by an increase or decrease in the number of neurospheres that form and/or an increase or decrease in the size of the neurospheres (which is a reflection of the rate of proliferation — determined by the numbers of precursor cells per neurosphere).
  • regulatory factor is used herein to refer to a biological factor that has a regulatory effect on the proliferation of stem cells and/or precursor cells
  • a biological factor would be considered a “regulatory factor” if it increases or decreases the number of stem cells that proliferate in vitro in response to a proliferation-inducing growth factor (such as EGF).
  • a proliferation-inducing growth factor such as EGF
  • the number of stem cells that respond to proliferation-inducing factors may remain the same, but addition of the regulatory factor affects the rate at which the stem cell and stem cell progeny proliferate.
  • a proliferative factor may act as a regulatory factor when used in combination with another proliferative factor
  • the neurospheres that form in the presence of a combination of bTGF and EGF are significantly larger than the neurospheres that form in the presence ot bFGF alone indicating that the rate of proliferation of stem cells and stem cell progeny is higher
  • regulatory factors include heparan sultate. transiormmg grovwh factor beta (TGF ⁇ ), activin bone morphogenic protein (BM P 2 j uhar ⁇ neurotrophic factor (CNTF), retinoic acid, tumor necrosis factor alpha (TNF ⁇ ), macrophage inflammatory proteins (MlP- l ⁇ , MIP-l ⁇ and MIP-2), nerve growth factor (NGF), platelet derived growth factor (PDGF), interleukins, and the Bcl-2 gene product.
  • TGF ⁇ transiormmg grovwh factor beta
  • BM P 2 j uhar ⁇ neurotrophic factor (CNTF) retinoic acid
  • TNF ⁇ tumor necrosis factor alpha
  • MlP- l ⁇ , MIP-l ⁇ and MIP-2 nerve growth factor
  • NGF nerve growth factor
  • PDGF platelet derived growth factor
  • interleukins interleukins
  • Antisense molecules that bind to transcripts of proliferative factors and the transcripts for their receptors also regulate stem cell proliferation
  • Other factors having a regulatory effect on stem cell proliferation include those that interfere with the activation ot the c-fos pathway (an intermediate early gene, known to be activated by EGF), including phorbol 12 my ⁇ state 13-acetate (PMA; Sigma), which up-regulates the c-fos pathway and staurospo ⁇ ne (Research
  • CGP-41251 (Ciba-Geigy), which down regulate c- fos expression and factors, such as tyrphostin [Fallon, D et al. , Mol. Cell Bioi , 11(5): 2697-2703 (1991)] and the like, which suppress tyrosine kinase activation induced by the binding of EGF to its receptor.
  • Preferred regulatory factors for increasing the rate at which neural stem cell progeny proliferate in response to FGF are heparan sulfate and EGF.
  • Preferred regulatory factors for decreasing the number of stem cells that respond to proliferative factors are members of the TGF ⁇ family, interleukins, MIPs, PDGF, BMP-2, TNF ⁇ , retinoic acid (10 ° M) and CNTF.
  • Preferred factors for decreasing the size of neurospheres generated by the proliferative factors are members of the TGF ⁇ family, retinoic acid (10 6 M) and CNTF.
  • the regulatory factors are added to the culture medium at a concentration in the range of about 10 pg/ml to 500 ng/ml, preferably about 1 ng/ml to 100 ng/ml.
  • the most preferred concentration for regulatory factors is about 10 ng/ml.
  • the regulatory factor retinoic acid is prepared from a 1 mM stock solution and used at a final concentration between about 0.01 ⁇ M and 100 ⁇ M, preferably between about 0.05 to 5 ⁇ M.
  • Preferred for reducing the proliferative effects of EGF or bFGF on neurosphere generation is a concentration of about 1 ⁇ M of retinoic acid.
  • Antisense strands can be used at concentrations from about 1 to 25 ⁇ M .
  • PMA and related molecules, used to increase proliferation may be used at a concentration of about 1 ⁇ g ml to 500 ⁇ g/ml, preferably at a concentration of about 10 ⁇ g/ml to 200 ⁇ g/ml.
  • the glycosaminoglycan, heparan sulfate is a ubiquitous component on the surface of mammalian cells known to affect a variety of cellular processes, and which binds to growth factor molecules such as FGF and amphiregulin, thereby promoting the binding of these molecules to their receptors on the surfaces of cells.
  • the precursor cells can be used for transplantation to treat various neurological disorders, as disclosed in PCT applications no. WO 93/01275, WO 94/16718, WO 94/10292, and WO 94/091 19.
  • the cells which are to be used for transplantation can be harvested from the culture medium and transplanted, using any means known in the art, to any animal with abnormal neurological or neurodegenerative symptoms, obtained in any manner, including those obtained as a result of chemical, electrical, mechanical or other lesions, as a result of experimental aspiration of neural areas or as a result of disease or aging processes.
  • the methods disclosed herein can also be used to test the proliferative or regulatory effects of biological factors on multipotent mammalian neural stem cell proliferation in vitro, prior to using the biological factors for the in vivo regulation of the proliferation of a patient's normally quiescent stem cells.
  • the neural stem cells may be obtained from a human with a neurological disorder in order to test the proliferative or regulatory effects of biological factors on dysfunctional, diseased, or injured tissue.
  • Therapeutic compositions comprising the regulatory factors can then be prepared for use in the treatment of various neurological disorders, disease, or injury.
  • the compositions comprise one or more regulatory factors at the above concentrations in a physiologically acceptable formulation.
  • the therapeutic compositions may be administered in vivo to regulate the proliferation of neural stem cells.
  • the normally quiescent neural stem cells are located throughout the CNS near ventricular regions.
  • Within the forebrain are the lateral (first and second) ventricles.
  • the third ventricle is a cav ity of the lower part of the forebrain which is connected to the fourth ventricle located in the hindbrain.
  • the central canal continuous with the aforementioned ventricular structures, is the ventricular component of the spinal cord.
  • CNS stem cells are located in the tissues lining ventricles offers several advantages for the modification and manipulation of these cells in vivo and the ultimate treatment of various neurological diseases, disorders, and injury that affect different regions of the CNS. Therapy for these can be tailored accordingly so that stem cells surrounding ventricles near the affected region would be manipulated or modified in vivo using the methods described herein.
  • the ventricular system is found in nearly all brain regions and thus allows easier access to the affected areas. If one wants to modify the stem cells by exposing them to a composition comprising a growth factor or a viral vector, it is relatively easy to implant a device that administers the composition to the ventricle. For example, a cannula attached to an osmotic pump may be used to deliver the composition.
  • the composition may be injected directly into the ventricles. This would allow the migration of the CNS stem cell progeny into regions which have been damaged as a result of injury or disease. Furthermore, the close proximity of the ventricles to many brain regions would allow for the diffusion of a secreted neurological agent by the stem cells or their progeny.
  • Gliosis which results in the formation of glial scar tissue, results from damage to CNS tissue.
  • the scar tissue is considered to have a major inhibitory effect on axonal outgrowth and the reconnection of severed elements, thus preventing functional recovery following brain or spinal cord injury.
  • astrocytes While not the only component of CNS scar tissue, astrocytes are one of the major elements involved. (Reier, PJ. Astrocytes vol. 3: 263-323 ( 1986)). It is a possibility that the gliosis results, at least in part, from the proliferation of previously quiescent stem cells.
  • a preferred inhibitory factor is BMP-2.
  • Example 1 In vitro proliferation of multipotent CNS stem cells derived from embryonic brain tissue — neurosphere proliferation in response to EGF
  • Embryonic day 14 (El 4) CD, albino mice (Charles River) were decapitated and the brain and striata removed using sterile procedure. The tissue was mechanically dissociated with a fire-polished Pasteur pipette into Complete Medium. The cells were centrifuged at 800 r.p.m. for 5 minutes, the supernatant aspirated, and the cells resuspended in Complete Medium for counting. The cells were suspended at a density of 25,000 cells/ml in Complete Medium containing 20 ng/ml EGF.
  • neurospheres When the cells were proliferated, within the first 48 hours and by 3-4 days in vitro (DIV), they formed small clusters, known as neurospheres, that lifted off the substrate between 4-6 DIV. The number of neurospheres generated per well were counted and the results were tabulated and compared with the numbers of neurospheres generated in response to EGF after passaging the cells (see example 2) and in response to other biological factors alone, or in combination with EGF (see Example 3).
  • Example 2 Passaging of proliferated neurospheres Paradigm 1 : Cells and media were prepared as outlined in Example 1 . Cells were plated at 0.2 x 10 6 cells/ml into 75 c ⁇ r tissue culture flasks (Corning) with no substrate pre-treatment and incubated as outlined in Example I
  • the neurospheres were removed, centrifuged at 400 r.p. m. for 2-5 minutes, and the pellet was mechanically dissociated into individual cells with a fire-polished glass Pasteur pipet in 2 mis of Complete Medium.
  • Paradigm 2 The methods of Example 1 and Example 2 paradigm 1 were followed except that 20 ng/ml FGF was added to the Complete Medium in place of the EGF.
  • Paradigm 3 The methods of Example 1 and Example 2 paradigm 1 were followed except that 20 ng/ml FGF was added to the Complete Medium in addition to the 20 ng/ml EGF that was added.
  • Neurospheres obtained after passaging, can be mechanically dissociated and the cells replated in 96 well plates as outlined in Example 1.
  • the effects of specific biological factors, or specific combinations of biological factors on the proliferation of neurospheres from cells derived from passaged neurospheres can be determined and compared with results obtained from cells derived from primary tissue.
  • Example 3 Assay of striatum-derived neurosphere proliferation in response to various combinations of proliferative and regulatory factors
  • Paradigm 1 Primary striatal cells prepared as outlined in Example 1 were suspended in Complete Medium, without growth factors, plated in 96 well plates
  • Activin, BMP-2, TGF-/3, IL-2, IL-6, IL-8, MlP- ld, MIP- 1 3, MIP-2 (all obtained from Chiron Corp.), TNF ⁇ , NGF (Sigma), PDGF (R&D Systems), EGF and CNTF (R. Dunn and P. Richardson, McGill University) were made up in separate flasks of compete medium to a final concentration of 0.2 ⁇ g/ml .
  • Retinoic acid (Sigma) was added at a concentration of 10 ° M. 10 ⁇ l of one of these regulatory factor-containing solutions was added to each proliferative factor-containing well of the 96 well plates. Control wells, containing only proliferative factors, were also prepared.
  • the activin, BMP-2, TGF-/3. IL-2, IL-6, IL-8, MIP- ld, MIP- 1/3, MIP-2, TNF ⁇ and EGF additions were repeated every second day, CNTF which was added each day and retinoic acid, NGF and PDGF were added only once, at the beginning of the experiment.
  • the cells were incubated for a period of 10- 12 days.
  • the number of neurospheres in each well was counted and the resulting counts tabulated using Cricket Graph III. Other relevant information regarding sphere size and shape were also noted.
  • bFGF had a greater proliferative effect than EGF on the numbers of neurospheres generated per well. In the presence of 20 ng/ml EGF, approximately 29 neurospheres per well were generated In the presence of bFGF, approximately 70 neurospheres were generated. However, in bFGF alone (FIG I B), the neurospheres were only about 20% of the size of those generated in the presence ot EGF (FIG. 1A). The combination of EGF and bFGF (FIG I C) produces significantly more neurospheres than does EGF alone, but fewer than seen w ith bFGF alone. The neurospheres are larger than those seen in bFGF alone, approximating those seen in EGF.
  • TGF ⁇ family interleukins, macrophage-inhibitory proteins, PDGF, TNF ⁇ , retinoic acid ( 10 ⁇ M) and CNTF significantly reduced the numbers of neurospheres generated in all of the proliferative factors or combinations of proliferative factors tested.
  • BMP-2 (at a dose of 10 ng/ml), completely abolished neurosphere proliferation in response to EGF.
  • EGF and heparan sulfate both greatly increased the size of the neurospheres formed in response to bFGF (about 400%).
  • TGF3 Family* -57% 57% . -34% .. -55% - -20% _
  • Numbers of neurospheres generated are given as percentages that reflect the decrease (-) or increase (+) in numbers of neurospheres per well, in response to a PROLIFERATIVE FACTOR in the presence of a REGULATORY FACTOR, compared with the number of neurospheres proliferated in the absence of the REGULATORY FACTOR
  • Antisense/sense experiments Embryonic tissue was prepared as outlined in Example 1 and plated into 96 well plates in Complete Medium. Antisense and sense experiments were carried out using the following oligodeoxynucleotides (all sequences written 5' to 3'):
  • EGF receptor Sense strand: GAGATGCGACCCTCAGGGAC
  • Antisense strand GTCCCTGAGGGTCGCATCTC
  • EGF Sense strand: TAAATAAAAGATGCCCTGG
  • Antisense strand CCAGGGCATCTTTTATTTA
  • Each oligodeoxynucleotide was brought up and diluted in ddH 2 0 and kept at -20°C. Each well of the 96 well plates received 10 / J of oligodeoxynucleotide to give a final concentration of either 1 , 2, 3, 4, 5, 10 or 25 ⁇ M. Oligodeoxynucleotides were added every 24 hours.
  • the EGF receptor (EGFr) and EGF oligodeoxynucleotides were applied to cultures grown in bFGF (20 ng/ml), and EGFr oligodeoxynucleotides were applied to cultures grown in EGF (20 ng/ml). Cells were incubated at 37°C, in a 5 % CO ?
  • FGF receptor Sense strand: GAACTGGGATGTGGGGCTGG
  • Antisense strand CCAGCCCCACATCCCAGTTC
  • FGF Sense strand: GCCAGCGGCATCACCTCG
  • FGFr FGF receptor
  • FGF oligodeoxynucleotides are applied to cultures grown in EGF
  • FGFr oligodeoxynucleotides are applied to cultures grown in bFGF.
  • Embryonic tissue is prepared as outlined in Example 1 and plated into 96 well plates. Complete Medium, containing 20 ng/ml of either EGF of bFGF is added to each well. 10 ⁇ l of diluted phorbol 12-myristate 13 acetate (PMA) is added once, at the beginning of the experiment, to each well of the 96 well plates, using an Eppendorf repeat pipetter with a 500 ⁇ l tip to give a final concentration of either 10, 20, 40, 100 or 200 ⁇ g/ml. Cells are incubated at 37°C in a 5% CO 2 100% humidity incubator. After a period of 10 to 12 days the number of neurospheres per well is counted and tabulated.
  • PMA diluted phorbol 12-myristate 13 acetate
  • Embryonic tissue is prepared as outlined in Example 1 and plated into 96 well plates. 10 ⁇ l of diluted staurosporine is added to each well of a 96 well plate, using an Eppendorf repeat pipetter with a 500 ⁇ l tip to give a final concentration of either 10, 1 , 0J , or 0.001 ⁇ M of staurosporine. Cells are incubated at 37°C, in a 5 % CO 2 100% humidity incubator. After a period of 10 to 12 days, the number of neurospheres per well is ⁇ mmed and tabulated
  • Example 4 Adult spinal cord ste cell proliferation — in vitro responses to specific biological factors or combinations of factors
  • the tissue was oxygenated, stirred and heated at 30°C tor 1 1/2 hours, then transferred to a vial for treatment with a trypsin inhibitor in media solution (DMEM/12/hormone mix).
  • media solution DMEM/12/hormone mix
  • the tissue was triturated 25-50 times with a fire narrow polished pipette.
  • the dissociated cells were centrifuged at 400 r.p.m. for 5 minutes and then resuspended in fresh media solution. Cells were plated in 35 mm dishes (Costar) and allowed to settle. Most of the media was aspirated and fresh media was added.
  • EGF alone, or EGF and bFGF were added to some of the dishes to give a final concentration of 20 ng/ml each, and bFGF (20 ng/ml) was added, together with 2 ⁇ g/ml of heparan sulfate, to the remainder of the dishes.
  • the cells were incubated in 5 % C0 2 , 100% humidity, at 37°C for 10-14 days. The numbers of neurospheres generated per well were counted and the results tabulated.
  • EGF alone resulted in the generation of no neurospheres from any of the spinal cord regions.
  • neurospheres were generated from all regions of the spinal cord, in particular the lumbar sacral region.
  • EGF -I- FGF and FGF + heparan sulfate produced similar numbers of spheres in the cervical region, whereas the combination of bFGF plus heparan sulfate resulted in fewer neurospheres from the thoracic and lumbar regions (see FIG. 3).
  • First-passage neurospheres were obtained from adult human tissue. Du ⁇ ng a routine biopsy, normal tissue was obtained from a 65 year old female patient. The biopsy site was the right frontal lobe, 6 mm from the tip of the frontal/anterior horn of the lateral ventricle. The tissue was prepared using substantially the same procedure outlined in Example 4 using aCSF. The stem cells were cultured in T25 flasks (Nunclon) in Complete Medium with 20 ng/ml EGF. 20 ng/ml bFGF, or 20 ng/ml each EGF plus bFGF. The flasks were examined every 2-3 days for neurosphere formation. More neurospheres were generated from the combination of EGF plus EGF than with either EGF or FGF alone.
  • the cannula is implanted, using a stereotaxis, into the 4th ventricle at AP -6.0 mm posterior to bregma, L -0.3 mm and DV -4.3 mm below dura, with a flat skull position between lambda and bregma.
  • the cannula is secured with dental acrylic cement.
  • the composition comprising a regulatory factor that inhibits stem cell proliferation in response to an injury stimulus is infused for 1-28 days at a flow rate of 0.5 ⁇ l/h.
  • the composition comprises 0.9% saline, 1 mg/ml mouse serum albumin (Sigma), and BMP-2 at 10 ng/ml.
  • the cannula is implanted, using a stereotaxis.
  • the cannula is secured with dental acrylic cement.
  • Factors which inhibit stem cell proliferation in response to an injury stimulus are infused for 1 -28 days at a flow rate of 0.5 ⁇ l/h.
  • the vehicle solution is 0.9% saline, containing 1 mg/ml mouse serum albumin (Sigma).
  • Inhibitory factors include those found to have an inhibitory effect on neural stem cell proliferation in vitro, such as BMP-2, CNTF, retinoic acid, members of the TGF- ⁇ and MIP family, and antisense oligodeoxynucleotides against EGF and FGF receptors.
  • the response of cells in the region of the injury is determined as outlined in Example 7.
  • mice are injected with bromodeoxyuridine (BrdU; Sigma, 120 mg/kg, i.pJ every two hours for a total ol 5 injections, to label proliferating cells in the region of the injury.
  • Animals are sacrificed 30 minutes, 2 days, 4 days, 1 week, 6 weeks or 6 months following the final injection by an anesthetic overdose and are transcardially perfused with 4% paraformaldehyde.
  • the region adjacent to and including the injury site and ventricular region adjacent to the injury site is removed and postfixed overnight at 4°C in perfusing solution, then cryoprotected. 30 ⁇ m sagittal crvostat sections are cut and directly mounted on gelled slides.
  • the tissue is processed for lmmunocytochemistry after first treating the sections w ith 1 M HCI for 30 minutes at 65°C to denature cellular DNA.
  • Rat anti-BrdU (Seralab) is utilized with donkey anti-rat-FITC for immiinocytocheinistrs
  • Antiserum to GFAP (expressed by astrocytes) is used followed by a donkey anti-mouse-FITC to visualize GFAP expression and glial scar production.
  • the effect of treatment on nestin expression is quantitated by labeling sections with an antiserum to nestin followed by a donkey anti-mouse-FITC and counting the number of nestin- immunoreactive cells near the ventricular area and the lesion site. Specificity of immunostaining is confirmed by the absence of primary antibody. Results in treated animals are compared to controls which received intraventricular treatment with the vehicle only.

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Abstract

La présente invention concerne la régulation de la prolifération in vitro et in vivo de cellules souches neurales multipotentes à l'aide de compositions composées de divers facteurs biologiques. Elle concerne plus particulièrement un procédé et des compositions thérapeutiques permettant la régulation du nombre de cellules précurseurs qui sont produites par la division de cellules souches neurales, par l'exposition desdites cellules souches à des facteurs biologiques spécifiques ou à des combinaisons de tels facteurs.
PCT/CA1995/000637 1994-11-14 1995-11-14 Regulation de la proliferation de cellules souches neurales WO1996015226A1 (fr)

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CA 2204630 CA2204630A1 (fr) 1994-11-14 1995-11-14 Regulation de la proliferation de cellules souches neurales
JP8515600A JPH10509592A (ja) 1994-11-14 1995-11-14 神経幹細胞増殖調節
AU38367/95A AU716811B2 (en) 1994-11-14 1995-11-14 Regulation of neural stem cell proliferation
FI971956A FI971956L (fi) 1994-11-14 1997-05-07 Hermosolujen lisääntymisen säännöstely
NO972171A NO972171L (no) 1994-11-14 1997-05-12 Regulering av neural stamcelleproliferasjon
MXPA/A/1997/003492A MXPA97003492A (en) 1994-11-14 1997-05-13 Regulation of proliferation of cells germinal neural

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