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WO1997007200A1 - Compositions cellulaires precurseurs d'oligodendrocytes adultes et procedes - Google Patents

Compositions cellulaires precurseurs d'oligodendrocytes adultes et procedes Download PDF

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Publication number
WO1997007200A1
WO1997007200A1 PCT/US1996/013279 US9613279W WO9707200A1 WO 1997007200 A1 WO1997007200 A1 WO 1997007200A1 US 9613279 W US9613279 W US 9613279W WO 9707200 A1 WO9707200 A1 WO 9707200A1
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cells
oligodendrocyte precursor
adult
precursor cells
culture
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PCT/US1996/013279
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WO1997007200A9 (fr
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Barbara A. Barres
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The Board Of Trustees Of The Leland Stanford Junior University
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Priority to AU71522/96A priority Critical patent/AU7152296A/en
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Publication of WO1997007200A9 publication Critical patent/WO1997007200A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • 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
    • C12N2501/135—Platelet-derived growth factor [PDGF]

Definitions

  • the present invention relates to substantially pure cultures of adult oligodendrocyte precursor cells and methods of isolating, purifying and using such cells.
  • Demyelinating diseases such as Multiple Sclerosis generally destroy oligodendrocytes and their myelin while leaving axons intact. The preservation of axons suggests that considerable repair might be possible if the axons could be remyelinated (i.e., if new oligodendrocytes could be generated).
  • oligodendrocyte precursor cells in recovery from demyelinating lesions. For example, after virally-induced demyelination of the mouse spinal cord, a population of cells with the antigenic profile of oligodendrocyte precursor cells incorporated 3 H-thymidine and increased in number during the recovery phase (Godfraind et al, 1989; Carroll and Jennings, 1994). Furthermore, adult precursors in mixed optic nerve cultures, in which fewer than 5% of the cells are adult precursors, have been demonstrated to generate new oligodendrocytes
  • adult oligodendrocyte precursor cells may be analogous to the stem cells that persist in adult muscle (Grounds, 1991), having the capacity to revert after injury to rapidly dividing cells in order to quickly supply new oligodendrocytes.
  • the present invention includes a culture of mammalian cells, where more that about 95% of the cells are adult oligodendrocyte precursor cells. In one embodiment, more than about 99% of the cells are adult oligodendrocyte precursor cells.
  • the cells may be purified from any of a number of central nervous system sources, e.g., rat optic nerve, human biopsy, rat or human temporal lobe, etc. Exemplary sources of the cells include rat and human CNS tissue.
  • the invention also includes an isolated population of adult oligodendrocyte precursor cells, where more than about 95% of the population are adult oligodendrocyte precursor cells. A population of cells typically consists of at least 100 cells.
  • a pharmaceutical composition containing the purified cells described above is suitable for use in the manufacture of a medicament for treatment of demyelinating diseases (e.g., multiple sclerosis) and other conditions due at least in part to loss of CNS neuron myelination, such as brain trauma (e.g., due to acute injury or infection).
  • a treatment method employing such a medicament includes obtaining a population of purified or substantially-purified adult oligodendrocyte precursor cells from the subject, and implanting or injecting the cells into the diseased or injured brain region, wherein the implanted cells remyelinate demyelinated axons near the site of injections or implantation.
  • the cells are expanded in culture prior to the implanting or injecting.
  • the composition is also useful in treatment of an individual suffering from symptoms due to a demyelinating disease or condition.
  • the treatment includes delivering the composition to a demyelinated region of the individual's central nervous system, wherein the region is responsible for causing at least a portion of the symptoms.
  • the demyelinating disease or condition is multiple sclerosis.
  • the composition i.e., cells
  • the composition is autologous to the individual (i.e., the cells are obtained from the same individualinto whom they are then delivered).
  • the invention includes a method of purifying adult oligodendrocyte precursor cells from a suspension of cells derived from adult central nervous system (CNS) tissue.
  • the method includes (i) contacting the suspension with a surface (e.g., petri plastic surface, such as the inside of a plastic petri dish), derivatized to contain an antibody specifically immunoreactive with Thy-1. A portion of the cells in the suspension becomes immobilized on this surface, and a second portion of the cells remains non-adherent. The non-adherent portion is contacted with a second petri plastic surface derivatized to contain a moiety capable of selectively binding a marker preferentially- expressed on oligodendrocyte precursor cells.
  • a surface e.g., petri plastic surface, such as the inside of a plastic petri dish
  • the invention includes a method of purifying adult oligodendrocyte precursor cells from a suspension of cells derived from adult central nervous system (CNS) tissue.
  • the method includes (i) contacting the suspension with a surface (e.g., petri plastic surface, such as the inside of a plastic petri dish), derivatized to contain an antibody specifically immunoreactive with proteolipid protein (PLP).
  • a surface e.g., petri plastic surface, such as the inside of a plastic petri dish
  • PLP proteolipid protein
  • the surface also contains an antibody specifically immunoreactive with Thy-1.
  • a portion of the cells in the suspension becomes immobilized on this surface, and a second portion of the cells remains non-adherent (i.e., doesn't stick to the surface).
  • the non- adherent portion is contacted with a second petri plastic surface derivatized to contain a moiety capable of selectively binding a marker preferentially-expressed on oligodendrocyte precursor cells.
  • a preferred moiety for this application is an antibody directed against oligodendrocyte marker 04.
  • Other suitable moieties include the A2B5 antibody, the NG-2 monoclonal antibody and peanut agglutinin.
  • Cells adhering to the second surface are substantially-purified adult oligodendrocyte precursor cells.
  • the invention includes a method of determining relative numbers of adult oligodendrocyte precursors and perinatal oligodendrocyte precursors in a mixture of cells containing both cell types. Each cell in the mixture has a cytoplasmic portion and a nuclear portion.
  • the method includes obtaining a sample of cells from the mixture, and assaying relative levels of expression of p53 in the nuclear and cytoplasmic portions of cells in the sample. Perinatal oligodendrocyte precursors have higher levels of p53 expression in their cytoplasmic portion than in their nuclear portion, and adult oligodendrocyte precursors have lower levels of p53 expression in their cytoplasmic portion than in their nuclear portion.
  • the invention includes a method of identifying a compound capable of increasing the rate of proliferation of adult oligodendrocyte precursor cells.
  • a sample of the cells is contacted with a test compound, the effect of the test compound on the rate of proliferation cells in the sample is measured, and the compound is identified as effective if its measured effect on the rate of proliferation is above a selected threshold level.
  • the threshold level may be selected, for example, to correspond to a selected number of standard deviations (s.d.) away from the mean rate of proliferation in the absence of test compounds.
  • the invention includes a method of culturing adult oligodendrocyte precursor cells.
  • the method includes incubating or growing the cells in a medium which (i) is substantially free of feeder cells or medium conditioned by feeder cells, (ii) is substantially serum-free, and (iii) contains N-Acetyl-L-cysteine, PDGF and NT-3.
  • the culturing results in maintenance of the adult oligodendrocyte precursor cells in an undifferentiated state.
  • FIGURES Figures IA, IB, IC, ID and IE show a schematic diagram of a panning procedure for purifying adult oligodendrocyte precursor cells.
  • Figures 2 A and 2B show an oligodendrocyte precursor cell clone (Fig. 2A) and an oligodendrocyte clone (Fig. 2B) in cultures treated with NT-3 and PDGF. Note that the precursor cells have a bipolar morphology, while oligodendrocytes have multiple interconnecting processes.
  • Figures 3 A and 3B show the proliferative capacity of perinatal (Fig. 3 A) and adult (Fig. 3B) oligodendrocyte precursor cells cultured for 12 days and 30 days, respectively, at clonal density in serum-free medium containing high insulin, PDGF, NT-3, and thyroid hormone (T3). Note that the perinatal precursor cells divided much more rapidly than the adult precursor cells and that by 12 days most of the perinatal cells had given rise to clones of oligodendrocytes. Even by 30 days, however, none of the adult precursor cells had generated oligodendrocyte clones and the average cell had divided only about 3 times during the entire culture period.
  • Figures 4A and 4B show that perinatal precursor cells express cytoplasmic p53 staining (Fig. 4A), while adult precursor cells lack cytoplasmic staining but have nuclear staining (Fig. 4B).
  • adult oligodendrocyte precursor cell or “adult oligodendrocyte progenitor cell” refers to an oligodendrocyte precursor cell derived from a mammal whose CNS has matured to the point that essentially all axons destined to become myelinated have become myelinated (e.g., a postnatal day 60 (P60) rat).
  • P60 postnatal day 60
  • adult oligodendrocyte precursor cells are similar to perinatal oligodendrocyte precursor cells but are distinguished from the perinatal cells in at least the following ways: adult oligodendrocyte precursor cells typically (i) contain more p53 antigen in their nucleus than their cytoplasm, (ii) have an endogenous cell cycle time of about 3-5 days (versus about 1 day for perinatal oligodendrocyte precursor cells), and (iii) divide about 5-10 times more slowly than perinatal oligodendrocyte precursor cells.
  • feeder cells as applied to a culture of adult oligodendrocyte precursor cells is understood to mean other types of cells present in a culture to promote the survival, differentiation, growth or viability of the cultured adult oligodendrocyte precursor cells.
  • substantially pure when used in connection with a culture of cells, refers to a culture where over about 95% of the cells are of a single defined cell type.
  • substantially purified when used in reference to a particular population of cells, indicates that the population contains 95% or more of a single cell type.
  • a suspension of cells "derived from" a tissue refers to a cell suspension prepared from a sample of that tissue (obtained using, e.g., a biopsy or surgical procedure) by, for example, mincing the tissue and subjecting the minced tissue to enzymatic dissociation.
  • a tissue e.g., adult central nervous system (CNS) tissue, optic nerve, temporal lobe, etc.
  • CNS central nervous system
  • a suspension of cells "derived from” a tissue refers to a cell suspension prepared from a sample of that tissue (obtained using, e.g., a biopsy or surgical procedure) by, for example, mincing the tissue and subjecting the minced tissue to enzymatic dissociation.
  • “Adult CNS tissue” refers to CNS tissue in which essentially all existing axons that are destined to become myelinated have been myelinated. In humans, such adult CNS tissue typically exists in individuals 3 years or more in age. “Treating” a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
  • Oligodendrocytes are relatively small cells that form myelin sheaths around axons of nerve cells of the mammalian central nervous system (CNS) by wrapping their processes concentrically around an axon in a tight spiral.
  • a single oligodendrocyte typically envelopes several different axons (average of 15). Although there may be as many as 10,000 different types of nerve cells in the CNS, all myelinated axons in the CNS are myelinated by oligodendrocytes.
  • oligodendrocytes that myelinate the diverse collection of nerve cells in the CNS are morphologically indistinguishable, i.e., they constitute a single "class" of cells.
  • oligodendrocytes isolated, e.g., from the optic nerve have the same properties as oligodendrocytes isolated, e.g., from the spinal cord.
  • Mature oligodendrocytes that form myelin sheaths are generated from oligodendrocyte precursor cells. At early stages in postnatal development, the majority of such oligodendrocyte precursor cells are "perinatal" cells.
  • these perinatal oligodendrocyte precursor cells can give rise either to type-2 astrocytes or oligodendrocytes, depending on the culture conditions. If the cells are cultured in serum- free medium, nearly all the cells differentiate into GC-positive oligodendrocytes. If, on the other hand, the cells are grown in the presence of serum, they differentiate into GFAP- positive type-2 astrocytes. In vivo, perinatal oligodendrocyte precursor cells appear to differentiate into oligodendrocytes, which then myelinate the developing axons.
  • adult oligodendrocyte precursor cells in vivo divide at rates similar to those observed in vitro About 3 % of adult oligodendrocyte precursor cells were labeled one hour after intraperitoneal injection of the thymidine analog 5-bromo-2'-deoxyuridine (BRDU; available, e.g., from Sigma Chemical Co., St. Louis, MO).
  • BRDU 5-bromo-2'-deoxyuridine
  • a similar experiment on a perinatal animal resulted in about 20% of the perinatal oligodendrocyte precursor cells being labeled.
  • the adult oligodendrocyte precursor cells express a defining characteristic of stem cells in adult animals — that is, they maintain the capacity to continually divide and differentiate.
  • these "adult" oligodendrocyte precursor cells may persist as a quiescent reservoir of stem cells in order to generate new precursor cells and oligodendrocytes after axonal injury or demyelination; alternatively or additionally, the adult oligodendrocyte precursor cells may have other functions in addition to being precursor cells.
  • oligodendrocytes form myelin sheaths around the axons of retinal ganglion cells (RGCs), which are CNS neurons that communicate signals from the retina of the eye to the brain.
  • RRCs retinal ganglion cells
  • Most adult oligodendrocyte precursors arise in the rat optic nerve after postnatal day 14 (P14), at which point they constitute as many as 10% of the oligodendrocyte precursor cells. By P21, they constitute about 50%, and by P45, essentially 100% (Wolswijk et al., 1990) of the oligodendrocyte precursor cells.
  • oligodendrocyte precursor cells employed in the methods of the presently-described experiments were purified from the optic nerves of P60 rats.
  • the time at which essentially 100% of oligodendrocyte precursors are adult oligodendrocyte precursors corresponds to the age at which essentially all existing axons that are destined to become myelinated have been myelinated, which is typically about 3 years of age.
  • Oligodendrocyte precursor cells in adult optic nerve cultures divide and differentiate more slowly in vitro than perinatal oligodendrocyte precursor cells cultured under the same conditions (Wolswijk et al, 1990; Wren et al., 1992).
  • oligodendrocyte precursor cells also migrate more slowly than perinatal precursor cells and have a different mo ⁇ hology and possibly antigenic phenotype as well (Wolswijk et al., 1990). Because all previous studies of adult precursor cells employed mixed optic nerve cultures that also contained astrocytes, oligodendrocytes, and other cell types, it has not been possible to determine whether the behavior of adult oligodendrocyte precursor cells differs from that of the perinatal cells because they are intrinsically different or because the extrinsic environment differs between adult and perinatal animals.
  • oligodendrocyte precursor cells may be derived directly from a subpopulation of perinatal precursors (Wren et al., 1992) and that perinatal and adult precursor cells could co-exist in the same culture environment, suggesting that the two cell types were intrinsically different.
  • perinatal precursors Wren et al., 1992
  • perinatal and adult precursor cells could co-exist in the same culture environment, suggesting that the two cell types were intrinsically different.
  • adult oligodendrocyte precursor cells cultured in the presence of both PDGF and bFGF behave identically to perinatal precursor cells (Wolswijk and Noble, 1992), suggesting that their behavior is not intrinsically specified as either perinatal and adult phenotypes, but rather that these are two intraconvertible phenotypes that depend on the local environment.
  • results of experiments performed in support of the present invention indicate that adult and perinatal oligodendrocyte precursor cells are intrinsically different, and that this difference is not simply due to the environment in which the cells are placed.
  • the results also suggest, however, that perinatal cells may be induced to take on the characteristics of the adult cells, as the rate of cell division of purified perinatal oligodendrocyte precursor cells cultured for several weeks slows down with age, i.e, cultured perinatal oligodendrocyte precursor cells divide progressively more slowly with increasing age.
  • the present invention provides, in one aspect, a method for the isolation and culturing of adult oligodendrocyte precursor cells.
  • the isolation of adult oligodendrocyte precursor cells facilitates screening for compounds effective to increase the proliferation of the cells. Further, such cells may be used to remyelinate axons following neuronal injury or in treatment of demyelinating diseases.
  • the present invention also enables the growing and expansion of pure or substantially pure cultures of adult oligodendrocyte precursor cells in culture in order to transplant the cells into sites of neuronal injury for therapeutic applications.
  • adult oligodendrocyte precursor cells can be purified from any suitable CNS tissue source using one of the adaptations of immunopanning procedures (Barres, et al., 1992, 1993b, 1994b, Wysocki and Sato, 1978;
  • exemplary sources of tissue include, in the case of experimental animals, as postnatal optic nerve; in the case of humans, brain biopsies or surgically-removed samples of, e.g., right temporal lobe.
  • the brain tissue is preferably isolated from an individual of a developmental age at which most of oligodendrocyte precursor cells are adult oligodendrocyte precursor cells.
  • the tissue is isolated from animals that are preferably at least about 45 days old, as it has been demonstrated that at this stage, virtually all of the oligodendrocyte precursor cells are adult oligodendrocyte precursor cells (Wolswijk, et al. , 1990).
  • biopsies of CNS tissue may be obtained from patients over the age of about 3 years who are, for example, in need of autologous oligodendrocyte cell- replacement therapy.
  • Cells are isolated from the tissue to form a tissue suspension (e.g., as described in Materials and Methods, herein).
  • adult oligodendrocyte precursor cells are isolated from rat optic nerve.
  • the cell suspension is then depleted of Thyl J -positive cells, such as macrophages, astrocytes, meningeal cells and microglia.
  • ThylJ a member of the lg superfamily, is one of the best-characterized antigens in biology. It was originally identified on thymocytes (Williams and Gagnon, 1982), and was used to differentiate T-cells from B-cells.
  • Thy-1 is a major glycoprotein found on the surface of many neurons, mature oligodendrocytes, astrocytes, microglia, macrophages and the like.
  • ThylJ A number of antibodies directed against ThylJ have been generated, and some are available from the ATTC (e.g., hybridoma cell line Tl lD7e2; Accession number TIB 103, which contains IgM antibodies against ThylJ) or through commercial sources (e.g., monoclonal antibody OX-7 is available from Serotec, Oxford, United Kingdom; another anti-Thyl.l antibody is available from Boehringer- Mannheim (Indianapolis, IN) as Cat 1199200). Any suitable anti-Thyl.l antibodies may be employed to remove ThylJ- positive cells from the suspension. For instance, in experiments described below, OX-7 monoclonal antibody was used for this pu ⁇ ose. The mouse OX-7 monoclonal was coated onto a dish that had been derivatized with a goat anti-mouse antibody, and the dish was used in a standard panning protocol as described in Example 1.
  • ThylJ-positive cells are typically accomplished in a single panning step, but may include two or more rounds of panning if a large number of ThylJ-positive cells are present in the suspension.
  • the decision as to whether or not to include additional anti-Thyl.l steps may be based in part on the relative number of ThylJ-positive cells that are present in the non-adherent cell suspension following the initial anti-Thyl. l panning step.
  • the anti-Thyl.l panning steps together typically remove over about 99% (preferably about 99.9%) of the ThylJ-positive cells that were present in the original suspension.
  • the suspension is transferred to a plate derivatized with an antibody preferentially-expressed on oligodendrocyte precursor cells.
  • An exemplary antibody useful for this pu ⁇ ose is the monoclonal antibody secreted by the A2B5 hybridoma cell line, available from the American Type Culture Collection (ATCC; Rockville, MD) under accession number CRL 1520.
  • the A2B5 antigen is expressed on both perinatal and adult oligodendrocyte precursor cells, but in not expressed on other cells found in the optic nerve, such as mature oligodendrocytes, neurons and microglia.
  • Occasional pial fibroblasts that encapsulate the nerve are Thy-1 positive and A2B5 positive. These cells are eliminated using the first (anti-Thy-1) panning plate. Because the source material for this purification contains virtually no perinatal oligodendrocyte precursor cells (i.e. , it is obtained from adults), only adult oligodendrocyte precursor cells stick to the second panning plate (the "positive-selection" plate).
  • the purified adult oligodendrocyte precursor cells are detached from the "positive selection" dish (e.g. , A2B5 dish) using trypsin.
  • the yield obtained with the procedure described in Example 1 was about 2,000 cells per adult rat (1,000 per nerve). This yield is consistent with the total yield of cells in the adult suspension: there are about 600,000 cells in an adult rat optic nerve (Barres, et al. , 1992) of which approximately 15% can be isolated enzymaticaUy (90,000).
  • the adult optic nerve contains about 8,000 oligodendrocyte precursor cells (Fulton, et al, 1992).
  • the number of adult oligodendrocyte precursor cells isolated using the methods of the present invention is proportional to the total cell yield, indicating that these methods are effective to isolate a representative sample, as opposed to a special subset, of adult oligodendrocyte precursor cells.
  • Example 5 the adult oligodendrocyte precursor cells are purified as described above, except that an antibody directed against PLP is used instead of or in addition to an anti-Thyl.l antibody in the first panning plate.
  • the second panning plate contains, as above, an antibody expressed on adult oligodendrocyte precursor cells.
  • the second (positive selection) step can employ antibodies expressed on oligodendrocytes as well as on oligodendrocyte precursor cells.
  • An exemplary antibody suitable for use with the second panning plate is one directed against oligodendrocyte antigen 04.
  • Other antibodies may be used as well, including those used in the embodiment described in Example 1.
  • the final step in the purification method detailed above employs antibodies directed against cell surface markers expressed on oligodendrocyte precursor cells, but preferably not on the other cell types typically found in association with such precursor cells (such as mature oligodendrocytes, astrocytes, neurons, microglia and the like).
  • the method detailed in Example 1, below employs an exemplary antibody — A2B5, which is immunoreactive with a ganglioside specifically expressed on oligodendrocyte precursor cells (Eisenbarth, et al. , 1979). Although this antigen is expressed in the embryonic CNS on radial glia, other precursor cells and some neurons, it is not expressed on these cells in the adult CNS.
  • peanut agglutinin Another method of achieving the final panning step is by using the lectin peanut agglutinin.
  • peanut agglutinin specifically binds to both perinatal and adult oligodendrocyte precursor cells, and that it may be used in the panning procedure described above to isolate a purified population of oligodendrocyte precursor cells.
  • Peanut agglutinin is commercially available (e.g., from Vector Labs, Burlingame, CA, as Cat. # L-1070).
  • one of skill in the art may elect to generate antibodies against antigens specifically expressed on oligodendrocyte precursor cells, and use such antibodies in the second (positive selection) panning plate.
  • Material isolated or derived from either perinatal or adult precursor cells may be used to immunize an animal for the generation of such antibodies.
  • This approach has been used to generate the NG-2 monoclonal antibody (Stallcup and Bersley, 1987), directed against the NG-2 proteoglycan, which is specific for oligodendrocyte precursor cells.
  • An antibody specifically immunoreactive with the oligodendrocyte antigen 04 may also be used in the second panning plate. Reports describing the generation of such antibodies have been published (e.g., Sommer and Schachner, 1981, 1982; Gogate, et al., 1994). Additionally, anti-04 antibodies specifically immunoreactive with human 04 are commercially available (e.g., from Boehringer-Mannheim, Indianapolis, IN).
  • the material used for immunization may include, for example, cell membrane preparations or fractions, proteins (e.g., membrane proteins) purified from the cells, glycoproteins or gangliosides or recombinantly-produced material from the precursor cells (i.e., a recombinant protein expressed using a vector containing DNA sequences encoding an antigen expressed on the precursor cells).
  • Recombinant methods of producing antigenic material may employ fusion proteins to facilitate purification of the antigenic polypeptide.
  • Hybrid, or fused, proteins may be generated using a variety of coding sequence derived from other proteins, such as glutathione-S-transferase or ⁇ -galactosidase.
  • Antibodies may be generated by immunizing a suitable animal according to methods known in the art (Harlow, et al.). Antigenic materials may be used directly for the generation of antibodies, or they may be coupled to appropriate carrier molecules. Many such carriers are known in the art and are commercially available (e.g., Pierce, Rockford IL). A recombinant antigen may also be produced as a fusion protein in tandem with a polypeptide carrier molecule.
  • a host animal such as a rabbit
  • the host serum or plasma is collected following an appropriate time interval, and the serum is tested for antibodies specific against the antigen.
  • the gamma globulin fraction or the IgG antibodies of immunized animals can be obtained, for example, by use of saturated ammonium sulfate precipitation or DEAE Sephadex chromatography, affinity chromatography, or other techniques known to those skilled in the art for producing polyclonal antibodies.
  • purified antigen or fused antigen protein may be used for producing monoclonal antibodies.
  • the spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art (e.g., Harlow, et al).
  • Antibodies secreted by the immortalized cells are screened (e.g., using enzyme linked immunesorbent assay (ELISA) or a Western blot) to determine the clones that secrete antibodies of the desired specificity (e.g., Ausubel, et al).
  • the screens may also include staining of samples (e.g., tissue culture cells or optic nerve tissue samples) to identify antibodies directed against cell-specific (e.g. , precursor cell- specific) antigens.
  • RAN-2 is an unknown protein that is specifically expressed on optic nerve type-1 astrocytes (Bartlett, et al., 1981), while GC is a glycolipid expressed specifically on oligodendrocytes.
  • the anti-RAN-2 step depleted type-1 astrocytes, meningeal cells, microglia and macrophages, while the anti-GC step depleted mature oligodendrocytes.
  • oligodendrocyte precursor cells isolated as described herein may be enhanced by maintaining the cells under conditions which support survival and expansion of the cells in the absence of substantial differentiation.
  • the fate of adult oligodendrocyte precursor cells isolated as described herein depends on the culture conditions. For example, more than 95% of the cells differentiate into GC-positive oligodendrocytes when cultured in serum-free medium lacking mitogens but containing survival factors such as insulin and CNTF. However, more than 95% of the cells differentiate into GFAP-positive type-2 astrocytes when cultured in medium containing 10% FCS. Additional experiments showed that when the adult oligodendrocyte precursor cells purified by the methods of die present invention were grown in medium containing PDGF, NT-3 and insulin, more than 95% of the cells divided. This result indicates that the purified cells are not postmitotic oligodendrocytes.
  • an exemplary medium for maintaining the cells in such an undifferentiated state is the modified Bottenstein and Sato serum-free medium minus T3 and T4 thyroid hormone, and including at least one survival factor (e.g., insulin), mitogens such as NT-3 and PDGF, and N-acetyl-L-cysteine.
  • Perinatal and adult oligodendrocyte precursor cells share a number of common cell- surface markers; which may be used to identify as well as purify oligodendrocyte precursor cells.
  • the markers that have been identified thus far as being expressed on both cell types include A2B5, 04, NG-2 and peanut agglutinin-binding carbohydrates.
  • p53 antigenic marker
  • p53 is a transcription factor that acts in the nucleus to suppress or slow the cell cycle, and that is sequestered from acting by binding to cytoplasmic proteins (Donehower and Bradley, 1993; Berns, 1994).
  • oligodendrocyte precursor cells The proliferation and differentiation behavior of purified perinatal and adult oligodendrocyte precursor cells was compared. Previous studies have shown that the proliferation and differentiation of perinatal oligodendrocyte precursor cells can be studied in vitro at clonal density under completely defined serum-free conditions in which oligodendrocyte precursor cells replicate many aspects of their normal in vivo behavior
  • Oligodendrocyte precursor cells were purified from postnatal rat optic nerve cell suspensions to greater than 99.95% purity by sequential immunopanning (Barres, et al, 1992).
  • the purified cells were cultured at clonal density in a serum-free medium that contained transferrin, progesterone, putrescine, selenium, thyroxine, triiodothyronine, albumin, and a high concentration (5 xg/ml) of insulin (modified from Bottenstein and Sato, 1979; see Materials and methods, below).
  • transferrin, progesterone, putrescine, selenium, thyroxine, triiodothyronine, albumin and a high concentration (5 xg/ml) of insulin (modified from Bottenstein and Sato, 1979; see Materials and methods, below).
  • transferrin transferrin, progesterone, putrescine, selenium, thyroxine, triiodothyr
  • NT-3 or PDGF when either NT-3 or PDGF was added at plateau concentrations (Barres, et al, 1994a) to the culture medium, all of the cells also differentiated into oligodendrocytes after 4 days of culture, although some of the cells under these conditions divided once prior to differentiating.
  • the percentage of cells that divided once in either NT-3 or PDGF was similar to the percentage that could be induced to synthesize DNA by NT-3 or PDGF (Barres, et al, 1993b).
  • oligodendrocyte precursor cells When thyroid hormone (T4 and T3) was eliminated from the serum-free medium, oligodendrocyte precursor cells were still able to divide in response to PDGF and NT-3, but they did not differentiate into oligodendrocytes (Barres, et al, 1994b). Further experiments showed that this effect was not attributable to an effect on survival or differentiation. Rather, the results indicate that thyroid hormone is required for oligodendrocyte precursor cells to stop dividing in response to mitogens.
  • adult oligodendrocyte precursor cells can be maintained in culture for extended periods (at least one month) in a proliferating, non-differentiated state suitable for expansion of the cells in vitro.
  • These "appropriate" culture conditions include the use of a serum-free culture medium containing survival factors (e.g., insulin), the mitogens NT-3 and PDGF, and N-acetyl-L-cysteine.
  • survival factors e.g., insulin
  • NT-3 and PDGF the mitogens NT-3 and PDGF
  • N-acetyl-L-cysteine Such a medium also preferably does not contain thyroid hormone.
  • adult (P60) and P8 perinatal precursor cells were purified simultaneously. The characteristics of the cells were compared when the cells were cultured at clonal density in the same serum-free culture medium containing their mitogens PDGF, NT-3, insulin, as well as thyroid hormone to activate the clock mechanism. After 12 and 30 days, the clones were counted and the identity of each cell scored as an oligodendrocyte or oligodendrocyte precursor cell, depending on mo ⁇ hology and expression of GC. As shown in Fig.
  • the average cell cycle time of the adult precursor cells is about 10 days (in the presence of T3; in the absence of T3 the adult precursor cell cycle time is about 5 days), which is about 10 times longer than that of the perinatal precursor cells.
  • This result was observed in 3/3 experiments.
  • clones of oligodendrocytes appeared and accumulated as expected in the perinatal cultures, so that after prolonged culture times the majority of the clones were oligodendrocyte clones, this was not the case with the adult cells, even after prolonged culture periods to allow them to make an equivalent number of divisions. Very few if any clones of oligodendrocytes were generated by the adult oligodendrocyte precursor cells (Fig. 3B).
  • oligodendrocyte precursor cells grown in serum-free medium differentiated into oligodendrocytes while over 95 % of the adult oligodendrocyte precursor cells grown in medium containing 10% fetal calf serum differentiated into type-2 astrocytes. Further, over 95% of the adult oligodendrocyte precursor cells cultured in vitro in serum-free medium containing mitogens (e.g., PDGF and NT-3) and at least one survival factor (e.g., insulin) divided.
  • mitogens e.g., PDGF and NT-3
  • survival factor e.g., insulin
  • Demyelinating diseases are a group of neurologic disorders significant both because of the disability that they cause and the frequency with which they occur. Demyelinating diseases are characterized by patchy or focal destruction of myelin sheaths in the CNS accompanied by an inflammatory response. The most common demyelinating disease is multiple sclerosis. Other examples include acute disseminated encephalomyelitis and acute hemorrhagic leukoencephalitis.
  • Multiple sclerosis is generally manifested by recurrent attacks of focal or multifocal neurologic dysfunction.
  • the symptoms are determined by the location of foci, or plaques, of demyelination within the CNS.
  • Classic features include impaired vision, nystagmus, dysarthria, decreased perception of vibration and position sense, ataxia and intention tremor, weakness or paralysis of one or more limbs, spasticity, and bladder problems.
  • the precise locations of these plaques can be determined using, e.g., magnetic resonance imaging (MRl).
  • MRl magnetic resonance imaging
  • Demyelination of CNS axons can also occur during acute or traumatic brain injury, such as during spinal chord injury. According to the present invention, any condition which results in CNS axons losing their myelin sheaths may be amenable to therapeutic methods described herein.
  • Oligodendrocyte precursor cells are the stem cells responsible for myelination. In the developing animal, perinatal oligodendrocyte precursor cells generate large numbers of oligodendrocytes, which myelinate the newly-developing axons. Adult oligodendrocyte precursor cells may also be responsible for the limited remyelination that occurs following certain types of neuronal injury. In many cases, however, the condition of a patient suffering from a demyelinating disease or condition could be greatly improved if the rate and/or degree of remyelination could be accelerated.
  • adult oligodendrocyte precursor cells may be isolated from patients with a neuronal injury, expanded in culture, and transplanted back into the patient to facilitate remyelination.
  • in vitro expansion/transplantation methods are routinely used in several areas of medicine, including hematopoietic cell replacement (Eaves, et al, 1993; Koller, et al, 1993; Rummel and Van Zant, 1994; Silva, et al, 1995), skin grafts in burn patients (e.g., Rheinwald and Green, 1975; Ronfard, et al, 1991; Teepe, et al, 1990), and are contemplated in other areas, e.g, bone and cartilage reconstruction (e.g., Brent, 1992; Nakahara, et al , 1991).
  • the cells are preferably isolated from a region of the brain whose removal results in little or no disruption of the individual's mental functioning and that is preferably unaffected by the demyelinating disease or condition.
  • the cells may be isolated from a region in the individual's right temporal lobe.
  • autologous human brain tissue may be isolated from a suitable region of the brain (e.g., the right temporal lobe) using a biopsy procedure, such as a computed tomography (CT)-guided needle biopsy or stereotactic biopsy (Wen, et al. , 1993).
  • CT computed tomography
  • stereotactic biopsy Wang, et al. , 1993.
  • tissue is prepared, and human adult oligodendrocyte precursor cells are purified as described, e.g., in Examples 1 or 5.
  • oligodendrocyte precursor cells purified as described above can be used immediately, or can be maintained in culture under conditions which support cell division and inhibit differentiation as described herein.
  • oligodendrocyte precursor cells purified as described above, and optionally expanded in culture can be transplanted at the site of injury using known methods, e.g., as described for transplantation of fetal cells into brains of Parkinson's patients (see, e.g., Molina, et al, 1994; Kupsch and Oertel, 1994).
  • a selected number of cells e.g., 5,000- 50,000
  • a buffer e.g., sterile, isotonic PBS
  • the suspension is delivered (e.g., via injection), to the injured area.
  • the injection does not necessarily need to be precisely at the site of the lesion, since the oligodendrocyte precursor cells are known to migrate to demyelinated axons.
  • the number of cells injected depends on several factors, such as the availability of source tissue, whether or not the cells were expanded, the size of the injured area and the like.
  • adult oligodendrocyte precursor cells purified from a portion of the brain unaffected by demyelinated plaques are optionally expanded in culture and transplanted, implanted or injected into regions of symptomatic plaques — that is, into plaques (localized using, e.g., MRl) that are in areas which correspond to the clinically- diagnosed neurological deficit.
  • the presently-described methods enable the determination of the average behavior of single oligodendrocyte precursor cells, avoiding selection or sampling errors. For instance, selection errors may have been introduced when these cells were micromanipulated if certain types of cell mo ⁇ hologies were preferentially selected.
  • the cells can be employed in screening applications (such as is described below) in which it is desirable to detect changes (due to, for example, the application of a proliferation-inducing compound) affecting the entire population of cells in a consistent way. Such screening applications would be difficult, if not impossible, to carry out using a cells purified one-at-a-time using the traditional clonal analysis technique.
  • Screen for Compounds Capable of Accelerating Proliferation An exemplary utility of the purified adult oligodendrocyte precursor cells described herein involves their use in a screen for compounds effective to treat neurological injury and demyelinating diseases. Compounds identified by such screens preferably increase the rate of remyelination at the injured/diseased sites when administered to subjects in need of treatment.
  • the screen may be carried out as follows: A sample of the cells is contacted with a test compound, the effect of the test compound on the rate of proliferation cells in the sample is measured, and the compound is identified as effective if its measured effect on the rate of proliferation is above a selected threshold level.
  • the threshold level may be selected, for example, to correspond to a selected number of standard deviations (s.d.) away from the mean rate of proliferation in the absence of test compounds.
  • the threshold level is set by the practitioner of the invention to a level corresponding to the desired potency of the test compound. For example, if the above-described screen is employed as a pre-screen to identify compounds for further detailed analyses, the threshold level may be set such that it corresponds to a relatively small change in rate of proliferation (e.g., 2-3 s.d.).
  • the threshold level may be set, for example, to 4-6 s.d. relative to the mean rate in absence of compound, or to a 2-fold or greater difference relative to the mean rate in absence of compound.
  • the cells can be screened for compounds effective to increase the rate of proliferation or cell division of the adult precursor cells.
  • the rate of proliferation may be assayed as described above.
  • Compounds identified as effective in such a screen may be used to increase the rate of cell division in cultures grown for cell transplantation therapy.
  • the compounds may be administered to individuals suffering from a neuronal injury or neurodegenerative disease which could benefit from remyelination therapy. Examples of such diseases include multiple sclerosis and other demyelinating diseases, as well as cerebral palsy and glaucoma. Further, since trauma to the CNS typically results in demyelination, such compounds may be effective at treating traumatic CNS injury, such as spinal cord injury.
  • the cells may also be screened for the ability to revert to cells having the characteristics of perinatal oligodendrocyte precursor cells.
  • exemplary indicator for such a reversion is immunostaining with anti-p53 antibody, which can differentiate between perinatal and adult oligodendrocytes.
  • Compounds effective to cause such a reversion may be employed in applications such as those described above. A variety of different compounds may be screened using the above approaches.
  • Such compounds, or molecules may be either biological, synthetic organic, or even inorganic compounds, and may be obtained from a number of sources, including pharmaceutical companies and specialty suppliers of libraries (e.g., combinatorial libraries) of compounds.
  • IGF-1 insulin-like growth factor 1
  • IGF-2 insulin-like growth factor 2
  • NT-3 mouse neurotrophin-3
  • CNTF rat ciliary neurotrophic factor
  • PDGF platelet-derived growth factor
  • Optic nerve was obtained from postnatal day 60 (P60) Sprague Dawley (S/D) rats (Simonsen Labs, Gilroy, CA). The animal was decapitated, the optic nerves and optic chiasm were dissected with micro-dissecting forceps and small scissors, collected in 35 mm petri dishes containing 2 ml of Minimal Essential Medium (MEM) supplemented with 10 mM Hepes (MEM/Hepes), and minced using small scissors.
  • MEM Minimal Essential Medium
  • the optic nerve was then dissociated enzymaticaUy to make a suspension of single cells, essentially as described by Huettner and Baughman (1986).
  • a papain solution was prepared, immediately prior to the start of the dissection, by adding 300 units of papain (Worthington Biochemical, Freehold, NJ) to 10 ml of Earle's Balanced Salt Solution (EBSS; Gibco/BRL Life Technologies, Gaithersburg, MD) in a 15 ml blue-top conical centrifuge tube, and placing the mixture in a 37°C water bath to dissolve the papain.
  • EBSS Earle's Balanced Salt Solution
  • EBSS Earle's Balanced Salt Solution
  • One hundred microliters of a 4 mg/ml DNAse (0.004%, Worthington Biochemical Co ⁇ ., Freehold, NJ) solution were added to the MEM/papain mixture after the papain had dissolved.
  • the solution was mixed with 2.4 mg of L-cysteine, adjusted to a pH of about 7.4 with IM NaOH, and passed through a 0.22 micron filter into sterilized scintillation vials.
  • the MEM bathing the tissue was removed with a sterile pasteur pipette and replaced with 2 ml of the papain solution.
  • the tissue was then decanted to a scintillation vial containing fresh papain solution, and the vial was placed in a 37 °C water bath for 30 minutes with gently swirling approximately every 10 minutes.
  • the tissue and papain solution in the scintillation vial were then decanted to a 15 ml blue-top centrifuge tube.
  • the old papain solution was removed with a sterile pipet and the tissue was gently rinsed with 3 ml of ovomucoid inhibitor solution, which contained ovomucoid (15 mg; Boehringer-Mannheim) and BSA (10 mg; Sigma catalog no. A-7638) dissolved in MEM (GIBCO/BRL).
  • ovomucoid 15 mg
  • Boehringer-Mannheim BSA
  • MEM Gibco/BRL
  • the solution was adjusted to pH 7.4 and sterilized with a 0.22 ⁇ m filter). The pieces of tissue were allowed to settle, and the rinse solution removed.
  • the tissue was then triturated sequentially with #21 and then #23 gauge needles. The following steps were repeated 6-10 times (until the tissue was completely broken up): (i) one ml of ovomucoid solution was added and the tissue was gently pulled up into the needle and expelled, (ii) the dissociate was allowed to settle by gravity for about 30 seconds, and (iii) the supernatant was collected.
  • the final cell suspension comprised of the supernatants from the 6-10 trituration cycles, contained about 50,000 cells per P8 optic nerve.
  • the cell suspension was then spun at 800 Xg for 10 minutes in a 15 ml blue-top centrifuge tube to separate the cells from the ovomucoid solution.
  • the supernatant was discarded and cells resuspended in 1 ml of MEM.
  • the cell suspension was then gently layered onto 6 ml of an MEM solution containing 60 mg of ovomucoid and 60 mg of BSA (pH adjusted to pH 7.4) and spun again at 800 Xg for 10 minutes in a 15 ml blue-top centrifuge tube.
  • the supernatant was discarded and the cells were resuspended in 12 ml of Eagle's Minimum Essential Medium (MEM) solution containing BSA (0.1 %). During this procedure the cells were never exposed to glutamate, aspartate or giutamine, or allowed to be cooled lower than room temperature.
  • MEM Eagle's Minimum Essential Medium
  • Purified cells were typically cultured in 96-well plates (Falcon) that had been coated with merosin (2 ⁇ g/ml; Telios Pharmaceuticals Inc., San Diego, CA, available from Gibco/BRL) in 100 ⁇ l of modified Bottenstein-Sato (MBS) serum-free medium. The percentage of surviving cells was assessed after 3, 7, and 14 days by the MTT ((3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (see below). All values were normalized to the percentage of surviving cells at 3 hours after plating, which represented the percentage of cells that survived the purification procedure. This initial viability was typically about 85%.
  • the MBS medium was similar to Bottenstein-Sato (B-S) medium (Bottenstein and Sato, 1979), but used "NEUROBASAL” (Gibco/BRL), instead of Dulbecco's Modified Eagle's Medium (DMEM), as the base.
  • NEUROBASAL is a recently-described basal medium that has been optimized for neuronal cell culture (Brewer, et al., 1993).
  • the serum-free components added to the "NEUROBASAL" base to make MBS medium were bovine serum albumin (BSA), selenium, putrescine, thyroxine, tri ⁇ iodothyronine, transferrin, progesterone, pyruvate, giutamine and N-Acetyl-L-cysteine.
  • BSA bovine serum albumin
  • T N-Acetyl-L-cysteine was found to potentiate the effects of all oligodendrocyte precursor cel mitogens, such as PDGF. It was included in the medium to allow the cells to continue dividing, as opposed to differentiating.
  • Various trophic factors and other additives were added as indicated in individual experiments.
  • the MBS medium was prepared with a highly purified, crystalline grade of BSA (Sigma, A4161), in order to avoid contaminating survival factors.
  • the component concentrations of the MBS medium used in the present experiments are provided in Table 1, below.
  • bovine serum albumin 100 ⁇ g/ml sodium selenite 40 ng/ml putrescine 16 ⁇ g/ml thyroxine 40 ng/ml tri-iodothyronine 30 ng/ml transferrin 100 ⁇ g/ml progesterone 60 ng/ml pyruvate I mM giutamine I mM
  • MTT survival assay was performed as described by Mosmann (1983). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) was dissolved in PBS at 5 mg/ml and sterilized by passage through a 0.22 ⁇ m Millipore filter (VWR Scientific Co ⁇ ., Westchester, PA). This stock solution was added to the culture well (1:9) and incubated at 37°C for 1 hour. Viable cells with active mitochondria cleaved the tetrazolium ring of MTT into a visible dark blue formazan reaction product. The viable and dead cells in each well were counted by bright-field microscopy.
  • mice were permeabilized by adding "TRITON” X-100 (0.4%) to the goat serum solution.
  • Cells were stained with mouse monoclonal anti-p53 antibody (pAb240, cat #OP29, Oncogene Science, Uniondale, NY) used at 5 ⁇ g/ml.
  • Anti-p53 antibodies were detected with fluorescein-conjugated goat anti ⁇ mouse IgG antibody (Jackson Labs).
  • Oligodendrocyte precursor cells were identified by A2B5 antibody staining.
  • Adult and perinatal oligodendrocyte precursor cells were distinguished based on the pattern of anti-p53 staining: nuclear for adult, cytoplasmic for perinatal.
  • Panning plates comprising the first set ("negative-selection” plates) were incubated with 5 ml of 50 mM Tris buffer (pH 9.5) containing 5 ⁇ g/ml affinity-purified goat anti- mouse IgM (mu-chain specific; Accurate Chemical & Scientific Co ⁇ ., Westbury, NY) for 12 hours at 4°C The supernatant was removed and the dishes were washed three times with 8 ml of PBS.
  • the dishes were then incubated with 5 ml of a supernatant from mouse monoclonal cell line OX-7 (Serotec, Oxford, United Kingdom), for at least one hour at room temperature. The supernatant was removed and the plate washed three times with PBS. In order to prevent nonspecific binding of cells to the panning dish, 5 ml of Minimal Essential Medium (MEM; Gibco/BRL) with 2 mg/ml BSA was placed on the plate for at least 20 minutes.
  • MEM Minimal Essential Medium
  • the second set of panning plates (“positive-selection” plates) was incubated with affinity-purified goat anti-mouse IgM (mu-chain specific, Accurate Chemical & Scientific Co ⁇ ., Westbury, NY), as above, washed, and further incubated with A2B5 monoclonal IgM ascites (ATCC, Accession # CRL 1520) at 1:2000 (Eisenbarth, et al, 1979).
  • the antibodies were diluted in Hepes-buffered Minimal Eagle's Medium (MEM/Hepes, Gibco/BRL) containing bovine serum albumin (BSA, lmg/ml; Sigma A4161), in order to block the non-specific adherence of cells to the panning plates.
  • the antibody solution was removed, the plates washed three times with PBS, and PBS left on the plates until use.
  • FIG. 1A-1E The panning procedure is summarized schematically in Figures 1A-1E.
  • An optic nerve cell suspension (20) prepared as above and containing adult oligodendrocyte precursor cells (22), macrophages (24) and various other cells, including Thyl positive cells (26) and
  • Thyl negative cells (28), was incubated on a (first set) panning plate (30; 150 mm) derivatized with goat-anti-mouse IgM (31) and OX-7 monoclonal antibody (32) at room temperature for 45 minutes (Fig. IB). The plate was gently swirled after 20 minutes to ensure access of all cells to the surface of the plate. If cells from more than 10 optic nerves were panned, the nonadherent cells were transferred to another 150-mm anti-mouse IgG/OX-7 panning plate for an additional 30 minutes.
  • Non-adherent cells were removed with the suspension, filtered through a UV- sterilized 15 micron "NITEX” mesh (Tetko, Elmsford, NY) to remove small clumps of cells, placed on a second set panning plate (34) derivatized with goat-anti-mouse IgM (36) and mouse A2B5 monoclonal IgM (38), and incubated on the plate (Fig. IC) as described above for 1 hour.
  • NITEX 15 micron
  • Non-adherent cells were discarded.
  • the plates were washed 8 times with 6 ml of PBS or MEM/Hepes with moderately vigorous agitation to remove all antigen-negative non- adherent cells.
  • solutions were removed from the panning dishes during washes, they were immediately replaced with fresh solution so that the cells did not dry out.
  • the progress of nonadherent cell removal was monitored under an inverted phase- contrast microscope, and washing was terminated when only adherent cells remained.
  • a trypsin solution (0.125%) were prepared by diluting a trypsin 20X stock (Sigma) into EBSS. Cells adhering to the panning dish (34) were incubated with this solution for 10 minutes in a 5% C0 2 incubator at 37°C The cells were dislodged by gently pipetting trypsin solution around the plate. Ten ml of a 25% fetal calf serum (FCS; Gibco/BRL) solution were added to inactivate the trypsin and the cells (Fig. IE) were spun and collected as above. To eliminate traces of FCS, the cells were resuspended and spun down again in an MEM solution containing BSA (0.5%). The cells were then resuspended in MBS culture medium for use in the experiments.
  • FCS 25% fetal calf serum
  • EXAMPLE 2 Proliferative Behavior of Adult v. Perinatal Oligodendrocyte Precursor Cells
  • the proliferative behavior of adult precursor cells is studied to determine if they divide in response to the same mitogens that elicit perinatal oligodendrocyte precursor cell clonal expansion, and how the cell-cycle time of the adult cells compares with that of the perinatal cells.
  • PDGF + bFGF + IGF- 1 to induce DNA synthesis is assessed by measuring BrdU inco ⁇ oration immunohistochemically.
  • cell division itself is followed by clonal analysis.
  • Cell-cycle time can be approximated by the rate of increase of the average number of cells per clone over time, when control experiments show that the survival in the study conditions is high.
  • the proliferation-inducing potential of trophic factors that do not induce perinatal oligodendrocyte precursor cells to divide is also assessed.
  • trophic factors that do not induce perinatal oligodendrocyte precursor cells to divide such as TGF-alpha, NGF, BDNF, IGF-1, insulin, CNTF, LIF, IL-6, GGF and SCF.
  • the effects of combinations of multiple factors on the division of the adult precursor cells are also examined.
  • the rate of proliferation of adult oligodendrocyte precursor cells in vitro and in vivo is compared.
  • adult rats are injected with BrdU intraperitoneally one hour prior to purifying the adult precursor cells.
  • the percentage of the purified adult precursor cells that were in DNA synthesis at the time of the injection is determined immunohistochemically .
  • EXAMPLE 3 Capacity of Adult Precursor Cells to Revert to Perinatal Precursor Cells The potential of adult oligodendrocyte precursor cells to promote remyelination is improved if they can be induced to revert to perinatal precursor cells, in order to rapidly generate new oligodendrocytes.
  • the experiments are performed using clonal cultures, as described above.
  • the extracellular signals that are tested include unmyelinated retinal ganglion cells and their axons, known factors that might be released by unmyelinated axons including GGF, FGF and glutamate, perinatal optic nerve extract, and co-culture with activated macrophages that would be expected to be present after injury.
  • Remyelination bv Adult Precursor Cells Purified adult and perinatal precursor cells are assayed for their ability to generate oligodendrocytes at sufficient number to support remyelination when transplanted into a developing spinal cord that lacks myelin.
  • the ability of transplanted glial cells, including purified perinatal oligodendrocyte precursor cells, to myelinate unmyelinated or demyelinated axons has been demonstrated (Utzschneider, et al, 1994).
  • oligodendrocyte precursor cells instead of mixed glial cells: Purified perinatal or adult precursor cells are transplanted into the spinal cord of postnatal day 3 md (myelin-deficient) rats, a mutant rat that lacks myelin (Duncan, et al., 1988; Utzschneider, et al. , 1994). A long-lived strain of md rats that lives up to 90 days is preferably employed, so that survival considerations are not limiting.
  • 20,000 purified cells resuspended into 1 ⁇ l PBS are injected via a glass micropipet into two or three sites along the dorsal columns of the spinal cord.
  • Each recipient rat is anesthetized with fluothane and undergoes a dorsal laminectomy at the thoracolumbar junction.
  • the transplant sites are marked with sterile charcoal before closing the incision.
  • the animals are sacrificed by inhalation anesthesia using fluothane, the spinal cords removed and immersed in glutaraldehyde.
  • the spinal cords are further processed with Epon embedding, preparation of 1 ⁇ m thick semi-thin sections and toluidine staining for viewing with a light microscope. The percentage of axons that have been myelinated after perinatal or adult oligodendrocyte precursor cell transplantation is determined and compared.
  • Human brain tissue is obtained using obtained using standard biopsy (e.g., Wen, et al., 1993) or surgical procedures (e.g., Son, et al, 1994; Shih, et al, 1994; Benbadis, 1995).
  • the tissue is preferably obtained from a region of the brain whose removal results in little or no disruption of the individual's mental functioning (e.g., the right temporal lobe).
  • the tissue is prepared, and adult oligodendrocyte precursor cells are purified as described in Example 1, above, except as follows.
  • the first "depletion" plate is derivatized using an antibody specifically immunoreactive with proteolipid protein (PLP), a major component of the myelin protein in the peripheral nervous system (PNS) and CNS (Meyer- Franke and Barres, 1994).
  • PLP proteolipid protein
  • PPS peripheral nervous system
  • CNS Central nervous system
  • antibodies prepared against any mammalian PLP typically cross-react with human PLP.
  • a number of antibodies to the protein have been generated (see, e.g., Yamamura, et al., 1991). Additionally, antibodies may be generated as described above.
  • the second panning plate (the "positive selection plate") is derivatized with an antibody specifically immunoreactive with 04.
  • antibody specifically immunoreactive with 04. Such antibodies have been generated (e.g., Sommer and Schachner, 1981, 1982; Gogate, et al., 1994), and may be generated using standard methods (e.g., the methods in the cited publications and/or as described above). Additionally, anti-04 antibodies specifically immunoreactive with human 04 are commercially available (e.g., from Boehringer-Mannheim, Indianapolis, IN, under catalog No. 1 518 925).
  • Adult oligodendrocyte precursor cells purified as described above can be used immediately, or can be maintained in culture under conditions which support cell division and inhibit differentiation as described herein.

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Abstract

L'invention se rapporte à une culture pratiquement pure de cellules (souches) précurseurs d'oligodendrocytes adultes, ainsi qu'à des procédés de purification et de mise en culture/développement de cellules précurseurs d'oligodendrocytes adultes provenant d'une suspension cellulaire, et à des procédés de différenciation des cellules précurseurs d'oligodendrocytes périnataux provenant de cellules précurseurs d'oligodendrocytes adultes.
PCT/US1996/013279 1995-08-16 1996-08-15 Compositions cellulaires precurseurs d'oligodendrocytes adultes et procedes WO1997007200A1 (fr)

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WO2001025402A1 (fr) * 1999-10-06 2001-04-12 Tigenix N.V. Isolement de cellules precurseurs et leur utilisation dans la reparation de tissus
US6245564B1 (en) 1997-01-23 2001-06-12 Cornell Research Foundation, Inc. Method for separating cells
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WO2002091820A1 (fr) 2001-05-16 2002-11-21 Japan Science And Technology Agency Modele animal non humain du trouble du developpement des oligodendrocytes
US7037493B2 (en) 2000-05-01 2006-05-02 Cornell Research Foundation, Inc. Method of inducing neuronal production in the brain and spinal cord
US7150989B2 (en) 2001-08-10 2006-12-19 Cornell Research Foundation, Inc. Telomerase immortalized neural progenitor cells
US7285415B2 (en) 2002-07-11 2007-10-23 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
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US7576065B2 (en) 2002-02-15 2009-08-18 Cornell Research Foundation, Inc. Enhancing neurotrophin-induced neurogenesis by endogenous neural progenitor cells by concurrent overexpression of brain derived neurotrophic factor and an inhibitor of a pro-gliogenic bone morphogenetic protein
US7579188B2 (en) 2002-07-11 2009-08-25 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US7785882B2 (en) 2000-01-18 2010-08-31 Cornell Research Foundation, Inc. Neuronal progenitor cells from hippocampal tissue and a method for isolating and purifying them
WO2011121158A2 (fr) 2010-03-31 2011-10-06 Fundación Hospital Nacional De Parapléjicos Para La Investigación Y La Integración (Fuhnpaiin) Méthode pour l'obtention de cellules précurseurs d'oligodendrocytes
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