WO1996004368A1 - Propagation et differenciation inductible de cellules souches du systeme nerveux central f×tal humain - Google Patents

Propagation et differenciation inductible de cellules souches du systeme nerveux central f×tal humain Download PDF

Info

Publication number
WO1996004368A1
WO1996004368A1 PCT/CA1995/000445 CA9500445W WO9604368A1 WO 1996004368 A1 WO1996004368 A1 WO 1996004368A1 CA 9500445 W CA9500445 W CA 9500445W WO 9604368 A1 WO9604368 A1 WO 9604368A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
nervous system
central nervous
cell
differentiated
Prior art date
Application number
PCT/CA1995/000445
Other languages
English (en)
Inventor
Alan Fine
Ruth M. E. Chalmers-Redman
Original Assignee
Dalhousie University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalhousie University filed Critical Dalhousie University
Priority to AU33385/95A priority Critical patent/AU3338595A/en
Publication of WO1996004368A1 publication Critical patent/WO1996004368A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • 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
    • C12N2501/105—Insulin-like growth factors [IGF]
    • 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]

Definitions

  • the present invention relates to processes and compositions for preparing human fetal central nervous system progenitor cells and differentiated neuronal and glial cells; as well as their use.
  • the mammalian central nervous system originates from cells of the embryonic neural tube. During development, these cells proliferate and differentiate into the diverse cell types of the mature central nervous system (CNS), including neurons and glia. Multipotential proliferating cells derived from fetal rat striatum, fetal rat cerebral cortex, post natal rat forebrain, chicken optic tectum and foetal and adult mouse striatum have been propagated in vitro and found capable of generating neurons and glia; long- term propagation of similar progenitor cells of human origin has not yet been described.
  • CNS central nervous system
  • epidermal growth factor exerts a mitogenic action on low-density, dissociated cultures of adult and fetal mouse CNS tissue, yielding mixed cultures of neurons and astrocytes presumably arising from a population of multipotent progenitor cells (See
  • EGF EGF-like growth factors
  • IGF-I and IGF-II Insulin-like growth factors
  • IGF-I insulin-like growth factors
  • the present invention provides that epidermal growth factor, when added to a partially defined medium, allows the survival and proliferation of progenitor cells from human forebrain, including primordia of the cortex, striatum and basal forebrain.
  • progenitors can be maintained in a proliferating state for months. At any time, they can be induced to differentiate into neurochemically defined neuron and glia consistent with the cell types found in these forebrain areas in the mature state. Differentiated neurons derived from the progenitor cells display ligand-gated conductances characteristic of normal mammalian neurons.
  • the present invention provides a process of preparing a human fetal central nervous system progenitor cell comprising the steps of:
  • the central nervous system tissue can be from the spinal cord or the brain.
  • Brain tissue can be from any specific region of the brain as exemplified by ventral forebrain tissue, cerebral cortex tissue or tissue from midbrain precursor.
  • the effective amount of epidermal growth factor is from about 1 ng/ml to about 100 ng/ml of culture medium, more preferably from about 10 to about 50 ng/ml of culture medium and, even more preferably about 30 ng/ml of culture medium.
  • the proliferaf ⁇ dn culture medium rurtnsr contains an effective amount of an insulin-like growth factor.
  • the insulin ⁇ like growth factor is preferably insulin-like growth factor I and an effective amount of insulin-like growth factor I is from about 10 to about 100 ng/ml of culture medium and, more preferably about 40 ng/ml of culture medium.
  • the proliferation culture medium can further contain transferrin, progesterone, putrescine, insulin and a selenite salt.
  • An especially preferred proliferation medium is a 3:1 mixture of DMEM and F-12, supplemented in this manner.
  • the serum in the proliferation medium is preferably normal horse serum. Serum is preferably present in a concentration of from about 1 volume percent to about 25 volumes percent, more preferably from about 2 to about 10 volumes percent and, even more preferably about 5 volumes percent.
  • the cell concentration is between about 10 2 and 10* cells per mm 2 of culture vessel surface and, more preferably about 2 x 10 3 cells per mm 2 .
  • the present invention provides a human fetal central nervous system progenitor cell produced by a process of the present invention.
  • the present invention provides a process of preparing a differentiated, human central nervous system cell comprising the steps of:
  • inducing differentiation is accomplished by dissociating progenitor cells from the cellular mass, plating the dissociated cells onto adherent culture dishes and culturing the dissociated cells in a differentiation culture medium.
  • the differentiation medium contains serum such as fetal bovine serum and a minimal essential medium such as Dulbecco's MEM.
  • a differentiated cell produced by a process of the present invention can be a glial cell or a neuron including, but not limited to, a cholinergic neuron, a peptidergic neuron, a GABAergic neuron, a glutamatergic neuron or a catecholaminergic neuron.
  • Preferred embodiments for the process of producing a differentiated cell are the same as set forth above with regard to the process of preparing a progenitor cell.
  • the present invention also provides a differentiated human central nervous system cell produced by a process of this invention.
  • That differentiated cell can be a glial cell or a neuron.
  • the present invention provides a screening assay for identifying substances that alter the function of a human central nervous system progenitor or differentiated cell, the process comprising the steps of: (a) establishing a culture of proliferating human central nervous system progenitor cells or differentiated cells; and (b) testing the ability of a substance to alter the function of those cells.
  • progenitor or differentiated cells are established in accordance with the processes set forth above.
  • testing is accomplished by exposing the cells to a substance suspected of altering function. Exposing is preferably accomplished by culturing the cells in a culture medium containing the substance suspected of altering function. In another embodiment, exposing is accomplished by transfecting the progenitor or differentiated cell with an expression vector that contains a polynucleotide at encodes the substance suspected of altering function, the expression vector driving expression of the substance in the cell.
  • the present invention provides a composition for use in proliferating human fetal central nervous system progenitor cells comprising (a) a minimal essential medium; (b) serum: and (c) an effective amount of epidermal growth factor.
  • the effective amount of epidermal growth factor is from about 1 ng/ml to about 100 ng/ml of medium, more preferably from about 10 to about 50 ng/ml of culture medium and, even more preferably about 30 ng/ml of culture medium.
  • a composition of the present invention can further comprise an effective amount of an insulin-like growth factor such as insulin-like growth factor I.
  • An effective amount of insulin-like growth factor is preferably from about 10 to about 200 ng/ml of culture medium and, more preferably about 40 ng/ml of culture medium.
  • the composition can further comprise transferrin, progesterone, putrescine, insulin and a selenite salt. ⁇ . Human Fetal Central Nervous System Progenitor Cells
  • the present invention provides a process of preparing a human fetal central nervous system progenitor cell. That process comprises the steps of: (a) culturing a population of cells from human fetal central nervous system tissue in a proliferation culture medium in a non-adherent culture dish containing serum and an effective amount of epidermal growth factor; and (b) maintaining the population of cells in the culture medium for a period of time sufficient for formation of a cellular mass that contains progenitor cells.
  • a central nervous system “progenitor” cell means an omnipotent cell that is capable of asymmetric cell division and produces daughter cells capable of differentiation into neurons or glial cells.
  • Central nervous system tissue is typically obtained from the developing central nervous system of fetuses available from elective abortion.
  • Central nervous system tissue is dissociated from the fetus using standard dissociation techniques well known in the art.
  • cells are physically dissociated from specific regions of the central nervous system by blunt dissection.
  • cells are obtained by chemical means including treatment of tissue with enzymes such as trypsin, collagenase and the like.
  • central nervous system means the brain and spinal cord or fetal tissue that gives rise to those tissues in the adult.
  • Central nervous system cells once dissociated from tissue, are typically purified using standard procedures well known in the art. Exemplary purification procedures are centrifugation of cells suspended in a physiological medium and passage of suspended cells through filters (e.g., pipettes) of various pore size.
  • Preferred physiological media include isotonic solutions of NaCl, balanced salt solutions and the like.
  • a physiological medium is preferably calcium (Ca) and magnesium (Mg) free and can comprise enzymes such as trypsin and DNase to minimize clumping of cells. The existence of viable cells is confirmed by standard techniques such as ethidium bromide exclusion.
  • a population of cells for use in a process of the present invention can be obtained from any specific region of the human fetal central nervous system. Selection of a particular region will determine to a large extent the particular nature of differentiated cells obtained from formed progenitor cells. Exemplary specific regions are ventral forebrain, cerebral cortex, cerebellum, midbrain and brainstem.
  • Dissociated cells are suspended in a proliferation culture medium and plated onto a culture container having a non-adherent surface.
  • a proliferation culture medium As used herein, the term "non-adherent" or its grammatical equivalent indicates a surface coating to which cells will not adhere.
  • Non-adherent culture containers are well known in the art and are commercially available.
  • a proliferation culture medium can be any minimal essential medium (MEM)known to support cell growth.
  • a proliferation medium contains an isotonic level of balanced salts, nutrients such as amino acids, vitamins, and growth supporting factors such as transfe ⁇ in, progesterone, putrescine, insulin and selenite.
  • Exemplary proliferation media are Dulbecco's Minimal Essential Medium (DMEM) and Ham's F-12.
  • DMEM Dulbecco's Minimal Essential Medium
  • F-12 Ham's F-12
  • a proliferation medium is a mixture of DMEM and F-12 in a 3:1 ratio.
  • Media for use in a process of the present invention can be made or purchased from commercial sources.
  • a proliferation medium comprises an effective amount of epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • an effective amount means that concentration of EGF that results in proliferation of progenitor cells. Means for determimng an effective amount are well known in the art. One skilled in the art can simply alter the concentration of EGF and determine the effects of such alteration on proliferation. An effective amount of EGF depends inter alia on the concentration of cells in the proliferation medium, the nutrient level of that medium and the absence or presence of other growth supporting factors in the medium.
  • an effective amount of EGF is preferably from about 1 ng/ml of medium to about 100 ng/ml of medium. More preferably, under those conditions, an effective amount of EGF is from about 10 ng/ml of medium to about 50 ng/ml of medium and, even more preferably from about 20 ng/ml of medium to about 40 ng/ml of medium.
  • the proliferation medium can further comprise an effective amount of an insulin-like growth factor (IGF).
  • IGF insulin-like growth factor
  • a preferred IGF is IGF-I.
  • An effective amount of an IGF is determined in the same manner as set forth above with regard to EGF. Under the specific culture conditions set forth hereinafter in the examples, an effective amount of an IGF is from about 1 ng ml of medium to about 100 ⁇ g/ml of medium. Preferably, the effective amount of an IGF is from about 10 ng/ml of medium to about 100 ng/ml of medium and, more preferably about 40 ng/ml of medium.
  • the concentration of cells in the proliferation medium is selected to maximize proliferation. That concentration depends, as is well known in the art, on the nature of the proliferation medium (e.g., level of nutrients). Means for determining a cell concentration are well known in the art.
  • the concentration of cells is from about 10 2 to about 10 4 cells/mm 2 . More preferably, cell concentration is from about 10 2 to about 5 x 10 3 cells/mm 2 and, more preferably about 2 x 10 3 cells/mm 2 .
  • the proliferation medium used to prepare proliferating human fetal central nervous system cells contains normal serum.
  • Normal serum can be derived from bovine, equine, chicken and the like. Normal horse serum is preferred.
  • the concentration of serum can range from about 1 percent by volume to about 25 percent by volume. Preferably, the concentration of serum is from about 2 percent by volume to about 10 percent by volume and, more preferably about 5 percent by volume.
  • Cells are cultured in proliferation medium, under the conditions set forth above, for a period of time sufficient for formation of a cellular mass that contains progenitor cells capable of proliferation and differentiation.
  • Culture conditions are those physiological conditions necessary for cell viability and include temperature, pH, osmolality and the like.
  • the temperature is from about 30 ⁇ C to about 40°C, more preferably from about 35 ⁇ C to about 39 ⁇ C and, even more preferably about 37 ⁇ C.
  • the pH value is preferably from about a valueof about 6 to a value of about 8, more preferably from a value of about 6.5 to a value of about 7.5. and, even more preferably about 7.4.
  • Osmolality is preferably from about 250 mosmols/liter to about 320 mosmols/liter, more preferably from about 270 mosmols/liter to about 310 mosmols/liter and, even more preferably from about 280 mosmols/liter to about 300 mosmols/liter.
  • Cells in culture are fed as needed by replacing the proliferation medium with fresh medium containing the specified growth factors and serum. Typically, feeding occurs every 3-4 days.
  • Human fetal central nervous system cells cultured as described above, form cellular masses that contain progenitor cells. Those cellular masses are typically spherical in nature. The progenitor cells in those masses can be dissociated and further propagated (e.g., subcultured) to form further masses of proliferating cells. Using a process of the present invention, human fetal progenitor cells can be propagated for extended periods of time (see the Examples hereinafter showing prpagation for at least four months in culture). The propagation of progenitor cells from adult and fetal rodent central nervous system tissue has been reported (See, e.g.. PCT Publication WO 93/01275). As reported hereinafter in the examples, human fetal central nervous system progenitor cells could not be propagated using the compositions and process reported in that publication.
  • the present invention also contemplates proliferating human fetal central nervous system progenitor cells prepared in accordance with a process of this invention. Those cells can be individual cells existing in a suspension culture or cells contained in cellular masses as set forth above. Thus, the present invention provides a culture of human fetal central nervous system progenitors cells capable of propagation in culture and differentiation.
  • a progenitor cell contemplated by the present invention can be derived from any specific region of human fetal central nervous system as set forth above.
  • a human fetal central nervous system progenitor cell of the present invention can be induced to differentiate into neural and glial cells.
  • the present invention contemplates a process of preparing such differentiated cells comprising inducing differentiation of progemtor cells of the present invention.
  • a process of preparing a human central nervous system differentiated cell comprises the steps of:
  • the proliferation medium further comprises IGF and, more preferably IGF-1.
  • IGF IGF and, more preferably IGF-1.
  • Preferred concentrations of IGF are the same as set forth above.
  • Differentiation can be induced using any means well known in the art
  • differentiation is induced by dissociating progenitor cells from the cellular mass, plating the dissociated cells onto an adherent culture dish and culturing the dissociated mass in a differentiation culture medium.
  • Progenitor cells are typically dissociated from the cellular mass using trituration with a pipette, usually after prior incubation with proteolytic enzymes.
  • Culture containers having an adherent coating are well known in the art.
  • adherent coatings include polyamines such as poly-L-lysine and poly-L-ornithine and extracellular matrix substances such as collagen, laminin and fibronectin. Culture containers having such coatings are commercially available.
  • the differentiation medium is any minimal essential medium that supports growth and differentiation. Exemplary and preferred such media are the same as set forth above. A preferred differentiation medium is
  • the differentiation medium can further comprise serum and, preferably fetal serum such as bovine fetal serum.
  • the concentration of serum can range from about 1 percent by volume to about 25 percent by volume, preferably from about 5 percent by volume to about 15 percent by volume and, more preferably about 10 percent by volume.
  • Growth factors may be present in the differentiation medium.
  • a human central nervous system differentiated cell prepared by a process of the present invention can be a neuron or a glial cell. More than one type of neuron can be prepared by a process of the present invention. The particular neuron type prepared depends inter alia upon the specific region of the central nervous system from which the progenitor cells are derived. Neuron types preparable by a process of the present invention include, but are not limited to, cholinergic neurons, peptidergic neurons, GABAergic neurons, glutamatergic neurons and catecholaminergic neurons.
  • neuron prepared is determined using standard procedures well known in the art.
  • differentiated cells are screened and identified using immunohistochemical techniques employing antibodies for known cell markers.
  • immunohistochemical techniques is also used to identify differentiated glial cells.
  • Antibodies against specific neural and glial cell markers are well known in the art.
  • Exemplary cell markers are neuron-specific enolase (NSE), neurofilament
  • NF glial fibrillary associated protein
  • GalC galactocerebroside
  • acetylcholine ACh
  • dopamine DA
  • E epinephrine
  • NE norepinephrine
  • H histamine
  • SP substance P
  • SP human nerve growth factor receptor
  • NTF-R choline acetyltransferase
  • Enk enkephalin
  • GAD glutamic acid decarboxylase
  • TH tyrosine hydroxylase
  • ADC dynorphin and aromatic amino acid decarboxylase
  • the differentiation of human fetal central nervous system cells is regulated. Regulation is accomplished by adding defined growth factors and/or conditioned media to either or both of the proliferation and differentiation medium. Those growth factors can be used to direct differentiation toward specific cell types. Regulating the differentiation of specific cell types can also be accomplished as is well known in the art by
  • the present invention also contemplates differentiated human central nervous system cells as provided by a process of this invention. Those cells can exist as isolated and purified cells or as a culture of such cells. Differentiated neuronal and glial cells are contemplated by this invention.
  • Human central nervous system progenitor and differentiated cells of the present invention can be used in a variety of ways including screening assays for the identification of substances that affect the function of those cells and for ex vivo transplantation.
  • the present invention contemplates a process of screening substances for their ability to interact with a progenitor or differentiated human central nervous system cell of the present invention.
  • Such a screening assay comprises the steps of providing a progenitor or differentiated cell of the present invention and testing the ability of selected substances to affect the function of that cell.
  • providing a progenitor or differentiated cell is accomplished using a process set forth above.
  • Testing the ability of a selected substance to affect cell function is accomplished by exposing a culture of a progenitor or differentiated cells to a candidate substance suspected of affecting cell function, maintaining the cell under biological conditions and detecting the functional effects of that candidate substance.
  • screening assays are provided for the testing of candidate substances such as inducers of differentiation, inhibitors of differentiation, drugs that are agonists or antagonists of cell receptors or membrane channels (e.g., Na channel, K channel, Ca channel, neurotransmitter receptors), toxins, drugs that affect membrane permeability to selective ions (e.g., Na, K, Cl, Ca), second messengers or gene expression and the like.
  • a candidate substance is a substance which potentially can interact with or modulate, by binding or other intramolecular interaction, the metabolic or electrical properties of a progenitor or differentiated cell.
  • Cells of the present invention are useful because of the difficulty in obtaining sufficient human central nervous system tissue for study.
  • a screening assay of the present invention has several advantages over currently available assays.
  • a major advantage is the availability of large amounts of uniform, nontransformed human central nervous systems cells.
  • the investigator may now control the type of neuron or cell that is utilized in a screening assay. Specific neuronal cell types can be prepared expressed and their interaction with a substance can be identified.
  • Other advantages include the availability of isolated neuron types previously difficult to obtain in tissue samples, and the obviation of finding a continuous supply of human material, particularly fetal material.
  • a screening assay provides a cell under conditions suitable for the testing of a candidate substance. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant co-factors and the like.
  • the present invention provides culture conditions and compositions suitable for the prolonged culturing of both progemtor and differentiated human central nervous system cells.
  • a typical screening assay for identifying candidate substances cells are placed into a suitable culture medium (proliferation or differentiation).
  • Candidate substances are added to the medium in convenient concentrations and the interaction between the candidate substance and the cell is momtored. By comparing reactions which are carried out in the presence or absence of the candidate substance, one can then obtain information regarding the effect of candidate/receptor interaction on the cell's function.
  • this aspect of the present invention provides those of skill in the art with methodology that allows for the identification of candidate substances having the ability to modify the function of a human central nervous system progemtor or differentiated cell in one or more manners.
  • a progenitor or differentiated cell can be exposed to that substance by transfecting the cell with an expression vector that contains a polynucleotide that encodes that polypeptide.
  • the expression vector drives expression of the polypeptide in the progenitor or differentiated cell.
  • Means of transfecting central nervous system cells are well known in the art and include techniques such as calcium-phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and adenovirus infection.
  • transfection mediated by either calcium phosphate or DEAE-dextran The most widely used method is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechamsm remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells can be transfected at any one time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for experiments that require transient expression of the foreign DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.
  • protoplasts Ofettveinrom 1 Dacte ⁇ a carrying high numbers of copies of a plasmid of interest are mixed directly with cultured mammalian cells. After fusion of the cell membranes (usually with polyethylene glycol), the contents of the bacteria are delivered into the cytoplasm of the mammalian cells and the plasmid DNA is transported to the nucleus.
  • Protoplast fusion is not as efficient as transfection for many of the cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies of the plasmid DNA tandemly integrated into the host chromosome.
  • Electroporation can be extremely efficient and can be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA.
  • Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion of the liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear but transfection efficiencies can be as high as 90%.
  • Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular components such as low-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.
  • S ⁇ SSTiTUTE SHEET (RULE 26)
  • adenovirus vector-mediated cell transfection has been reported for various cells including cells of the central nervous system.
  • Expression vectors that drive expression of a polypeptide typically include (if necessary) an origin of replication, a promoter, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. Promoters specific to the cell being studied are preferred. Thus, where a neuron is transfected, a preferred promoter is a neurofilament or neuron-specific enolase promoter. Other promoters that have particular utility in neural cells are promoters associated with the production of neurotransmitters such as ACh, E, NE, 5-HT and doparnine.
  • control functions on the expression vectors are often derived from viral material.
  • promoters are derived from polyoma, Adenovirus 2, Cytomegalovirus and most frequently Simian Virus 40 (SV40).
  • SV40 Simian Virus 40
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication.
  • An origin of replication can be provided with by construction of the vector to include an exogenous origin, such as can be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV, CMV) source, or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • an exogenous origin such as can be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV, CMV) source, or can be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • a transfected progenitor or differentiated human central nervous system cell can also be used in a screening assay to test for candidate substances that affect cell function.
  • such a cell can be used for propagating human neurotropic viruses or other fastidious pathogens, for research, development of vaccines, or other purposes.
  • Human progenitor and differentiated cells of the central nervous system of the present invention provide a source of human cells for use in ex vivo transplantation into the human brain. Ex vivo transplantation is becoming a treatment of choice for certain pathological disorders of the central nervous system.
  • Alzheimer's Disease such as Alzheimer's Disease, Huntington's Chorea and Parkinson's Disease have been linked to the degeneration of neurons in specific locations in the brain and the inability of the brain region to synthesize and release neurotransmitters and other substances vital to neuron function.
  • drugs and other pharmacological agents has limited utility for treatment of central nervous system pathologies.
  • Drugs administered via typical parenteral routes of administration have difficulty in reaching target tissues in the brain because of transport problems across the blood-brain barrier. Administration directly into the brain or spinal cord gives rise to potential infection in those tissues. Further, tolerance to drugs is often associated with long-term use of those drugs. By way of example, despite partial restoration of dopaminergic activity seen in Parkinson's patients treated with L-dopa, those patients often become tolerant to the effects of L- dopa, and thus require increasing dosages to maintain the beneficial effects of that drug.
  • Neurological tissue grafting of tissue into the central nervous system has been used to overcome some of the problems associated with drug therapy.
  • ex vivo treatment is used to replace the source of a particular substance where the intrinsic source of that substance has been destroyed as a result of dysfunction.
  • Parkinson's Disease is associated with a loss of doparnine producing cells in the brain.
  • the transplantation of cells that produce doparnine can be used to partially restore brain function.
  • ex vivo treatment often involves the use of cells and tissue from other sources.
  • tissues and cells are adrenal medulla (adrenal chromaffin cells), neurons from the adult peripheral nervous system (PNS), chromaffin cells and fetal ventral mesencephalon from non-human species and immortalized cell lines derived either by transformation of normal cells or by culturing cells with altered growth characteristics.
  • PNS peripheral nervous system
  • chromaffin cells and fetal ventral mesencephalon from non-human species and immortalized cell lines derived either by transformation of normal cells or by culturing cells with altered growth characteristics.
  • non-human cells give rise to an immune response.
  • non-neural cells compromises the ability of the grafted cells to functionally integrate with the CNS.
  • transformed cells may form tumors in the patient's brain.
  • the present invention provides such a reliable source of human fetal central nervous system tissue or cells.
  • the present invention therefore provides a process of ex vivo treatment of human central nervous system disorders comprising administering to patients in need of such treatment, a human fetal central nervous system progenitor or differentiated cell of the present invention.
  • Human fetal progenitor cells or differentiated cells of the present invention can also, in combination with routine immunosuppressive medication, be administered to any animal with abnormal neurological or neurodegenerative symptoms obtained in any manner, including those obtained as a result of chemical or electrolytic lesions, as a result of experimental aspiration of neural areas, or as a result of aging processes.
  • Cells are delivered throughout any affected neural area. Cells are administered to a particular region using any method which maintains the integrity of surrounding areas of the brain, preferably by injection cannula.
  • cells to use in transplantation depends upon the desired purpose of the treatment. Where, for example, it is desirable to restore levels of a particular substance (e.g., a neurotransmitter such as doparnine) to a region of the brain, cells that produce that substance are selected. Means for identifying particular cell types are set forth hereinbefore.
  • a particular substance e.g., a neurotransmitter such as doparnine
  • cells administered to the particular region of the brain form neuronal or synaptic connections with neighboring neurons, and maintain contact with glial cells which may form myelin sheaths around the neuron's axon.
  • transplanted tissue survival of the transplanted tissue is determined using standard non- invasive procedures well known in the art. Such procedures include computerized axial tomography (CAT or CT scan), nuclear magnetic resonance or magnetic resonance imaging (NMR or MRI) and positron emission tomography (PET) scans. Functional integration of the cells into the host's neural network is assessed by examining the effectiveness of grafts on restoring various functions. In addition to restoring lost function (e.g., neurotransmitter production), cells of the present invention can be used to deliver drugs or other substances to specific regions of the central nervous system.
  • CAT or CT scan computerized axial tomography
  • NMR or MRI nuclear magnetic resonance or magnetic resonance imaging
  • PET positron emission tomography
  • a progenitor or differentiated cell is transfected with an expression vector containing a polynucleotide that encodes that substance, the expression vector driving expression of the substance in the transfected cell.
  • Means for transfecting cells are set forth hereinbefore. Cells expressing the desired substance are transplanted to a desired location in the brain.
  • EXAMPLE 1 Isolation of Human Fetal CNS Cells Human fetal brain tissue was obtained from routine therapeutic abortions performed at the Victoria General Hospital in Suite, Nova Scotia, with informed consent and approval of appropriate institutional ethical review boards. Evacuated tissue fragments from 6-8 week post-conception fetuses, as staged according to the developmental atlas of England (1983), were collected directly into sterile ice-cold isotonic saline with heparin (10 ⁇ g/ml).
  • Ventral forebrain was dissected under stereomicroscopic observation in a laminar flow containment hood, and transferred to a solution of 0.05% (w/v) trypsin (Type XTJJ, Sigma) in calcium- and magnesium-free Hank's Balanced Salt Solution (CMF-HBSS) for 20 minutes at 37 ⁇ C.
  • CMF-HBSS Hank's Balanced Salt Solution
  • Progenitor cell culture medium was composed of a 3:1 mixture of Dulbecco's Minimum Essential Medium (DMEM:Gibco) and Ham's F-12 (Gibco), with 5% (v/v) horse serum (Gibco), insulin (Sigma, 10 ⁇ g/ml), transferrin (Sigma, 200 ⁇ g/ml, progesterone (Sigma, 40 mM), putrescine (Sigma, 200 ⁇ M) and sodium selenite (Sigma, 60 nM).
  • DMEM Dulbecco's Minimum Essential Medium
  • Gibco Ham's F-12
  • PCM-E EGF (Upstate Biochemicals, 20ng/ml)
  • PCM-EI EGF plus IGF-I (Upstate Biochemicals, lOOng/ml)
  • PCM-EI EGF plus IGF-I
  • Immunohistochemical staining revealed nestin-immunoreactive cells within the cellular masses when these masses were plated, undissociated, directly onto poly-L-lysine-coated plates for 48 hours. The masses attached loosely to the plate during this time period via small numbers of processes. After dissociating and replating the cells of these masses, 'secondary' cell masses formed; growth of secondary cells masses was less rapid than that of primary cell masses. After a further 30-60 DIV, secondary masses were similarly dissociated to produce 'tertiary' masses of proliferating cells. Such passage could be repeated at least 2 times.
  • spherical proliferating cell masses were dissociated as above, and the resulting cell suspension plated onto poly-L- lysine (Sigma)-coated dishes in DMEM with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • spherical masses of proliferating cells were, while still in PCM, exposed to l ⁇ g/ml bromodeoxyuridine (BrdU; Sigma), which is incorporated into the nuclear DNA of proliferating cells, for 6, 12, 18, 24, 36, 48, 60 or 72 hours. Cell masses were then dissociated, and the resulting cell suspension plated onto poly-L-lysine-coated plates in DMEM with 10% FBS.
  • Immunocytochemical staining was carried out as previously described, using well-characterized antibodies specific for BrdU (Amersham), neurofilament 200 (NF; Sigma), glial fibrillary associated protein (GFAP; Sigma and DAKO), choline acetyltransferase (ChAT; Chemcion), glutamic acid decarboxylase (GAD; Calbiochem), tyrosine hydroxylase (TH; Eugene Tech), substance P (Sub-P, Eugene Tech), the neuronal/glial lineage marker
  • Dilutions of primary antibodies or TT in 0.1 M phosphate buffer containing 1 % normal serum were as follows: rabbit anti-GFAP (1:100), rabbit anti-NF (1:200), mouse anti-BrdU (neat), rabbit anti-ChAT (1:1000), rabbit anti-GAD (1:200), rabbit anti-TH (1:1000), rabbit anti-Sub-P (1:1000), mouse anti- A2B5 (1:100), rabbit anti-nestin-129 (1:2000), and FITC-TT (200 ⁇ g/ml).
  • Tertiary cell masses collected at least 75 days after initial primary plating, were redissociated and plated as single cells onto poly-L-lysine coated tissue culture dishes. After 5 DIV, these cells were fixed and stained immunohistochemically to evaluate their cytochemical phenotype; cells immunoreactive for NF, ChAT, GFAP, Sub-P, TH, A2B5, GAD and TT were evident. When these cells were derived from cellular masses incubated with BrdU for 48 hours immediately prior to dissociation, cells of each cytochemically-identified group were found with BrdU-labelled nuclei.
  • tertiary cell masses were incubated with BrdU for various periods immediately prior to dissociation and replating; plated cells were fixed and stained immunocytochemically to detect BrdU-labelled cells.
  • the percentage of cells incorporating BrdU into their nucleus increased with increasing incubation times from 12 to 36 hours; with longer incubations, the proportion of labelled nuclei remained unchanged at approximately 80% of the total.
  • the pH of the perfusate was adjusted to 7.25 using NaOH, and osmolarity was adjusted to 350 mOsm using sucrose.
  • Drugs were diluted from concentrated stock solution into the same si at solution which, in the case of experiments involving N-methyl-D- aspartate (NMDA), was supplemented with glycine (10 ⁇ M) but lacked
  • the patch pipettes were filled with a solution containing (in mM): CsF, 120; CsCl, 10; Hepes, 10; EGTA, 10; CaCl 2 , 0.5.
  • the pH was adjusted to
  • NMDA excitatory amino acid analogs NMDA, ⁇ -amino- 3-hydroxy-5-methyl-isoxazolepropionate (AMPA) and kainate in a manner qualitatively similar to that of rat cortical neurons.
  • AMPA ⁇ -amino- 3-hydroxy-5-methyl-isoxazolepropionate
  • kainate in a manner qualitatively similar to that of rat cortical neurons.
  • EGF and IGF-I when added to a partially defined medium, permit the survival and continued proliferation in vitro of progenitor cells from the fetal human forebrain for at least 4 months.
  • progenitor cells remain in an undifferentiated state, expressing nestin, until they are stimulated to differentiate.
  • differentiated cells were induced by changing culture conditions to FBS-supplemented medium in culture dishes coated to promote attachment.
  • a wide range of differentiated cells can in this way be produced, including neurons (as indicated by expression of neurofilament) of several neurochemical classes (catecholaminergic as indicated by TH, peptidergic as indicated by Sub-P, cholinergic as indicated by ChAT and inhibitory GABAergic as indicated by GAD expression) and astrocytes (as indicated by GFAP expression).
  • These differentiated neurons can be maintained in vitro for at least 15 weeks when cultured on glial feeder layers, and display normal and medically-significant receptors by electrical recording criteria.
  • AU these differentiated cell types derived from progenitor cells of forebrain origin are also present in the normal adult forebrain. EGF and IGF-I also permitted the formation of proliferating cell masses from human fetal ventral mesencephalic cells.
  • progenitor cells were incubated with BrdU at a concentration that would be incorporated into the DNA but not affect proliferation. The cells were then induced to differentiate, and stained for both BrdU and particular neurochemical markers. Double-labelled cells, immunoreactive for BrdU and for ChAT, substance P, TH or GAD were observed, establishing that all of these cells could be produced by progenitor cells replicating in culture prior to terminal differentiation.
  • Double-labelled astrocytes (immunoreactive both for BrdU and for
  • Trophic factor support for the proliferation and differentiation of neuron cells has been demonstrated by others.
  • the ability of EGF and IGF-I to support proliferation of these undifferentiated progenitor cells supports the possibility that these mitogenic growth factors play a role in normal development of the fetal human forebrain.
  • IGF-I mRNA has been shown to be present in the human fetal CNS and similarly EGF receptors have been localized throughout the adult human brain.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Neurology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Neurosurgery (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de préparation de cellules souches ou différenciées du système nerveux central f÷tal humain prolifératives ainsi que des cellules préparées selon ce procédé. Les cellules souches prolifératives sont préparées par mise en culture de tissu du système nerveux central de f÷tus humain dans un conteneur de culture non adhérent contenant du sérum et un facteur de croissance épidermique. L'invention concerne également une méthode de criblage ainsi que des traitements ex vivo utilisant ces cellules.
PCT/CA1995/000445 1994-07-29 1995-07-31 Propagation et differenciation inductible de cellules souches du systeme nerveux central f×tal humain WO1996004368A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU33385/95A AU3338595A (en) 1994-07-29 1995-07-31 Propagation and inducible differentiation of human fetal central nervous system progenitor cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28292794A 1994-07-29 1994-07-29
US282,927 1994-07-29

Publications (1)

Publication Number Publication Date
WO1996004368A1 true WO1996004368A1 (fr) 1996-02-15

Family

ID=23083726

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1995/000445 WO1996004368A1 (fr) 1994-07-29 1995-07-31 Propagation et differenciation inductible de cellules souches du systeme nerveux central f×tal humain

Country Status (2)

Country Link
AU (1) AU3338595A (fr)
WO (1) WO1996004368A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2301114A (en) * 1995-05-24 1996-11-27 Bradley Michael John Stringer Method for controlling differentiation of precursor cells
WO2000050572A1 (fr) * 1999-02-26 2000-08-31 Stemcells, Inc. Utilisation de collagenase dans la preparation de cultures de cellules embryonnaires neuronales
WO2001030981A1 (fr) * 1999-10-25 2001-05-03 Ns Gene A/S Cultures de cellules gfap?+ nestin+¿ se differenciant en neurones
US6277820B1 (en) 1998-04-09 2001-08-21 Genentech, Inc. Method of dopaminergic and serotonergic neuron formation from neuroprogenitor cells
WO2003095629A1 (fr) * 2002-05-10 2003-11-20 Bresagen Ltd Production de cellules souches neurales
US7459152B2 (en) 2003-04-23 2008-12-02 Rush University Medical Center Erythropoietin administration to improve graft survival

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993001275A1 (fr) * 1991-07-08 1993-01-21 Neurospheres Ltd. NOUVELLES CELLULES SOUCHES REAGISSANT AU FACTEUR DE CROISSANCE ET POUVANT PROLIFERER $i(IN VITRO)
WO1994009119A1 (fr) * 1992-10-16 1994-04-28 Neurospheres Ltd. Remyelination effectue a l'aide de cellules souches neurales
WO1994010292A1 (fr) * 1992-10-28 1994-05-11 Neurospheres Ltd. Facteurs biologiques et cellules souches neurales

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993001275A1 (fr) * 1991-07-08 1993-01-21 Neurospheres Ltd. NOUVELLES CELLULES SOUCHES REAGISSANT AU FACTEUR DE CROISSANCE ET POUVANT PROLIFERER $i(IN VITRO)
WO1994009119A1 (fr) * 1992-10-16 1994-04-28 Neurospheres Ltd. Remyelination effectue a l'aide de cellules souches neurales
WO1994010292A1 (fr) * 1992-10-28 1994-05-11 Neurospheres Ltd. Facteurs biologiques et cellules souches neurales

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
REYNOLDS A. ET AL.: "Generation of Astrocytes from Isolated Cells of the Adult Mammalian Central Nervous System", SCIENCE, vol. 255, 27 March 1992 (1992-03-27), LANCASTER, PA US, pages 1707 - 1710 *
VON VISGER J R ET AL: "Differentiation and maturation of astrocytes derived from neuroepithelial progenitor cells in culture.", EXPERIMENTAL NEUROLOGY 128 (1). 1994. 34-40. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2301114A (en) * 1995-05-24 1996-11-27 Bradley Michael John Stringer Method for controlling differentiation of precursor cells
US6277820B1 (en) 1998-04-09 2001-08-21 Genentech, Inc. Method of dopaminergic and serotonergic neuron formation from neuroprogenitor cells
WO2000050572A1 (fr) * 1999-02-26 2000-08-31 Stemcells, Inc. Utilisation de collagenase dans la preparation de cultures de cellules embryonnaires neuronales
US7049141B1 (en) 1999-02-26 2006-05-23 Stemcells California, Inc. Use of collagenase in the preparation of neural stem cell cultures
US6238922B1 (en) 1999-02-26 2001-05-29 Stemcells, Inc. Use of collagenase in the preparation of neural stem cell cultures
EP1533367A1 (fr) * 1999-02-26 2005-05-25 StemCells, Inc. Utilisation de collagénase pour la préparation des cellules souches neurales
US6878543B1 (en) 1999-10-25 2005-04-12 Nsgene Sa Cultures of GFAP+ nestin+ cells that differentiate to neurons
WO2001030981A1 (fr) * 1999-10-25 2001-05-03 Ns Gene A/S Cultures de cellules gfap?+ nestin+¿ se differenciant en neurones
US7303912B2 (en) 1999-10-25 2007-12-04 Nsgene A/S Cultures of GFAP nestin cells that differentiate to neurons
US7651853B2 (en) 1999-10-25 2010-01-26 Nsgene A/S Cultures of GFAP+ nestin+ cells that differentiate to neurons
US8501467B2 (en) 1999-10-25 2013-08-06 Stemcells California, Inc. Cultures of GFAP+ nestin+ cells that differentiate to neurons
WO2003095629A1 (fr) * 2002-05-10 2003-11-20 Bresagen Ltd Production de cellules souches neurales
US7459152B2 (en) 2003-04-23 2008-12-02 Rush University Medical Center Erythropoietin administration to improve graft survival

Also Published As

Publication number Publication date
AU3338595A (en) 1996-03-04

Similar Documents

Publication Publication Date Title
Hartikka et al. Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth, and expression of transmitter-specific enzymes
AU665012B2 (en) Novel growth factor-responsive progenitor cells which can be proliferated (in vitro)
US8501467B2 (en) Cultures of GFAP+ nestin+ cells that differentiate to neurons
Arias‐Carrion et al. Neurogenesis in the subventricular zone following transcranial magnetic field stimulation and nigrostriatal lesions
Chalmers-Redman et al. In vitro propagation and inducible differentiation of multipotential progenitor cells from human fetal brain
Avellana‐Adalid et al. Expansion of rat oligodendrocyte progenitors into proliferative “oligospheres” that retain differentiation potential
ES2194016T5 (es) Factores biologicos y celulas germinales neurales.
Moriarty et al. A combined cell and gene therapy approach for homotopic reconstruction of midbrain dopamine pathways using human pluripotent stem cells
Rubio et al. BDNF gene transfer to the mammalian brain using CNS-derived neural precursors
JP2007135604A (ja) ドーパミン作動性細胞のインビトロ誘導
O'malley et al. Local support cells promote survival of substantia nigra dopaminergic neurons in culture
US20090136456A1 (en) Methods of treating neurodegenerative disorders
Uchida et al. Potential functional neural repair with grafted neural stem cells of early embryonic neuroepithelial origin
WO1996004368A1 (fr) Propagation et differenciation inductible de cellules souches du systeme nerveux central f×tal humain
Mason et al. Isolation and characterization of neonatal Schwann cells from cryopreserved rat sciatic nerves
Pal-Ghosh et al. Mechanisms Regulating Mitochondrial Transfer in Human Corneal Epithelial Cells
WO2004013316A1 (fr) Procede d'isolation de cellules souches pluripotentes
EP1565549A1 (fr) Procede de preparation, de purification et de differenciation de neurospheres issues de cellules souches de mammiferes
Sen The development of media for the in vitro expansion of mammalian neural stem cells
US20040115807A1 (en) O-2a progenitors multipotent cells from neurohypophysis
MXPA97003493A (en) In vitro induction of dopaminergi cells
Colombo et al. Forebrain and midbrain astrocytes promote neuritogenesis in cultured chromaffin cells
Tereshchenko Neural stem cells from embryonic brain of mice and neural cell adhesion molecule L1 and projection aberrations to the hippocampus in NCAM deficient mice (Mus musculus L., 1758)
Yuliya Neural stem cells from embryonic brain of mice and neural cell adhesion molecule L1 and projection aberrations to the hippocampus in NCAM deficient mice (Mus musculus L., 1758)
Loo Nonsenescent serum-free mouse proastroblasts: Extended culture, growth responses in vitro, and application to the culture of human embryonic astrocytes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IS JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

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

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase