WO2001028342A1 - Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation - Google Patents

Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation Download PDF

Info

Publication number
WO2001028342A1
WO2001028342A1 PCT/US2000/041367 US0041367W WO0128342A1 WO 2001028342 A1 WO2001028342 A1 WO 2001028342A1 US 0041367 W US0041367 W US 0041367W WO 0128342 A1 WO0128342 A1 WO 0128342A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
ohgodendrocytes
culture
cell
sterile
Prior art date
Application number
PCT/US2000/041367
Other languages
English (en)
Inventor
John W. MCDONALD
Original Assignee
Washington 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 Washington University filed Critical Washington University
Priority to EP00986805A priority Critical patent/EP1221853A4/fr
Priority to JP2001530946A priority patent/JP2003511090A/ja
Priority to CA002388736A priority patent/CA2388736A1/fr
Priority to AU22979/01A priority patent/AU2297901A/en
Publication of WO2001028342A1 publication Critical patent/WO2001028342A1/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
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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]
    • 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
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • This invention relates to the field of cell culture methods and methods for treatment of diseases of the central nervous system.
  • CNS central nervous system
  • Transplantation approaches utilizing cellular bridges, fetal CNS cells, fibrob lasts expressing NT-3, hybridoma cells expressing inhibitory protein blocking antibodies, or olfactory ensheathing glial cells into the acutely injured spinal cord has produced axonal regrowth or functional benefits.
  • Transplants of rat or cat fetal spinal cord tissue into the chronically injured cord survive and integrate with the host cord, and may be associated with some functional improvements.
  • rats transplanted with fetal spinal cord cells exhibited benefits in some gait parameters, and the delayed transplantation of fetal raphe cells can enhance reflexes.
  • Neural progenitors isolated from the adult CNS differentiate into neurons and glia after transplantation into brain (Gage, et al, (1995) Proc. Natl. Acad. Sci. 92, 11879- 11883), and differentiate into ohgodendrocytes and astrocytes after transplantation into spinal cord.
  • Neural transplantation studies have been limited by ethical considerations and a lack of a reliable source for undifferentiated pluripotent cells. In vivo differentiated neural cells cultures are problematic because may contain genetically abnormal cells. Ideally, in vitro neural cell cultures would provide a better source of materials for transplantation, and other commercial and research purposes.
  • ES cells provide a partial solution to the problems encountered with in vivo derived neural cells because they are genetically normal, totipotent, capable of indefinite replication (Suda, Y et al, (1987) J. Cell. Physiol. 133, 197-201) and have been derived from several vertebrate species including mice (Evans & Kaufman, (1981) Nature 292, 154-156; Martin, (1981) Proc. Natl. Acad. Sci. USA 78, 7634- 7638) and humans (Thomson, et al., (1998) Science 282, 1145-1147; Shamblott, et al, (1998) Proc. Natl. Acad. Sci. USA 95, 13726-13731).
  • ES cells are also among the most flexible stem cell for genetic engineering. For example, the production of double gene allele knockouts in single ES cells has been accomplished (Wilder, et al., (1997) Dev. Biol. 192, 614-629; Hakem, et al, (1998) Cell 94, 339-352). In theory, ES cells can generate all cell types and preliminary work has shown that differentiation into ohgodendrocytes is possible (Fraichard, et al., (1995) J. Cell Sci. 108, 3181-3188; Dinsmore, et al., (1996) Cell Transplant. 5, 131-143; Housele, et al, (1997) Proc. Natl. Acad. Sci.
  • the present invention provides a method to treat spinal cord degeneration through the transplantation of in vitro differentiated cultures of neural cells.
  • the inventors through their superior appreciation of the problems of spinal cord transplantation techniques, have recognized the advantages of in vitro cultured cells. Furthermore, the inventors have discovered that ohgodendrocytes are the critical cells required for transplantation.
  • a method to make an enriched culture of ohgodendrocytes is provided.
  • the highly enriched ohgodendrocyte culture of the invention can be used to particular advantage in the method to treat spinal cord degeneration of the invention to increase the myelination of axons in degenerated spinal cord tissues.
  • a method to make a cell culture highly enriched in ohgodendrocytes uses retinoic acid differentiated 4-/4+ stage EB cells.
  • the method uses preconditioned oligosphere media, thereby forming an intermediate cell type referred to herein as "oligospheres".
  • Another aspect of the invention features a cell culture enriched in ohgodendrocytes.
  • the oligodendrocyte-enriched culture may comprise from about 20% to about 99% ohgodendrocytes in the population.
  • the culture is made by the inventive method described herein.
  • a method for using an in vitro differentiated culture of neural cells to treat degenerated CNS tissue.
  • This method comprises the steps of transplanting in vitro differentiated neural cells into the spinal cord of a patient in need of such treatment, and allowing the transplanted cells to replace damaged or missing tissues.
  • the neural cell culture of this method comprises the enriched ohgodendrocyte culture of the invention.
  • ES cell-derived cell transplantation improved behavioral recovery.
  • the present invention provides compositions and methods to regenerate the tissue of the central nervous system. Surprisingly, it has been discovered that the transplantation of a mixed population of in vitro differentiated neural cells into a damaged spinal cord is associated with improvements of CNS function. It has further been discovered that a preparation of a highly enriched culture of in vitro differentiated ohgodendrocytes is unexpectedly effective in restoring CNS function by transplantation.
  • a method to make a highly enriched culture of ohgodendrocytes is provided.
  • These highly enriched cultures of ohgodendrocytes can be produced from ES or other totipotent cells by generating an intermediate stage of floating cell groups termed "oligospheres".
  • Oligospheres primarily contain an early form of ohgodendrocyte progenitor, and can be produced by culturing a mixed population of in vitro differentiated neural cells in a preconditioned oligosphere medium, described in greater detail below.
  • the resulting highly enriched culture of ohgodendrocytes is a novel cell culture that will have many uses in research as well as transplantation therapy.
  • mice derived embryonic stem (ES) cells induced to differentiate into a mixed population of neural cells by retinoic acid were transplanted into physically and chemically injured spinal cords of rats.
  • the transplanted mouse cells differentiated into primarily ohgodendrocytes in the spinal tissue and the rats had improved hind-limb functionality as compared to the control group.
  • the use of ES cultures was surprisingly superior to the use of in vivo derived cells in the speed at which the transplanted cells migrated into the damaged spinal cord tissue and degree of remyelination.
  • a method to culture a highly enriched population of ohgodendrocytes from mixed population of neural cells was developed and optimized, as described in Examples 2 and 3.
  • the method utilizes a novel culture medium that selectively encouraged the growth of an intermediate class of cells termed "oligospheres", containing primarily immature and mature ohgodendrocytes.
  • This enrichment method is based on the unexpected simple concept of using the preconditioned media from existing ohgodendrocyte cultures to selectively induce the division of ohgodendrocytes in a mixed population to neural cells.
  • this enriched ohgodendrocyte culture was transplanted into the spinal cord of shiverer mice, which lack myelin basic protein, the introduced cells oriented with native ohgodendrocytes and myelinated the native axons.
  • the enriched ohgodendrocyte culture was surprisingly more effective than the retinoic acid differentiated ES cells in the rate of cell migration and myelination in the spinal cord.
  • the present invention provides a method of therapy for degenerated CNS tissues.
  • the method comprises the step of transplanting in vitro differentiated neural cells into the site of injury and allowing sufficient time for the introduced cells to replace missing or defective cell types. This method is particularly effective when a highly enriched population of ohgodendrocytes is used for the transplantation.
  • transplanted ohgodendrocytes act by remyelinating damaged axons. In many injuries to the spinal cord, the axons remain intact but are rendered useless due to the loss of their myelin sheath.
  • Ohgodendrocytes in the CNS wrap the axon of a nerve in a myelin sheath and prevent current from leaking across the axonal membrane.
  • the insulating function of the myelin allows the action potential to move faster along the axon and to use less metabolic energy.
  • the myelin sheath is absent from the majority of axons in a region due to injury, genetic mutation or disease, signals cannot be carried up and down the spinal cord, resulting in loss of muscular control and/or sensory signals. Remyelination may therefore be able to restore nervous system function in some situations.
  • in vitro differentiated ohgodendrocytes is particularly useful in situations involving loss of myelination.
  • this method of therapy is contemplated to be useful for treating multiple sclerosis,
  • Alzheimer's Disease leukodystrophies, cerebrial palsey, stroke, cardiac arrest and CNS trauma, among others.
  • the inventors have determined how to manipulate cultured pluripotent cells to produce a culture of differentiated neural cells that is highly enriched in ohgodendrocytes.
  • an oligodendrocyte-enriched cell culture is featured in accordance with the present invention. It has been discovered that such an oligodendrocyte-enrichced culture comprising as few as about 20% ohgodendrocytes in the population is suitable for use in the therapeutic methods of the invention. However, the inventors have devised methods for producing a culture comprising up to 99% ohgodendrocytes. Therefore, the present invention includes oligodendrocyte-enriched cultures comprising at least about 20%, preferably at least about 30%, more preferably at least about 40%, yet more preferably at least about 50%, and even more preferably at least about 60%, 70%, 80% or 90%, ohgodendrocytes in the population.
  • Oligodendrocyte-enriched cultures derived from ES cells are exemplified in the present invention.
  • other pluripotent cell types can be differentiated into neural cells; therefore, oligodendrocyte-enriched cultures derived from any pleuripotent cell type are included in the invention.
  • These include, but are not limited to, progenitor cells from the developing nervous system, cells derived from naturally occuring carcinomas and embryonal carcinoma cells, such as PI 9 (Bain et al, 1998), among others. Since basic features of pleuripotent cells are consistent across species, oligodendrocyte-enriched cell cultures from such cells of any vertebrate are included in the invention.
  • the ohgodendrocytes are produced from mammalian cells; particularly preferred are primate cells, and most preferred are human cells.
  • Other vertebrate cells from which oligodendrocyte-enriched cultures can be obtained include, but are not limited to, laboratory rodents such as mice (exemplified herein), rats (also exemplified herein) and hamsters, and domesticated animals such as cats, dogs and horses.
  • Presented with the present invention is a method to make a highly purified culture of ohgodendrocytes. This method comprises the several steps of:
  • Example 3 sets forth a detailed set of instructions on how to prepare an oligodendrocyte-enriched culture from ES cells.
  • This last step in which the cells are further cultured in a medium that contains both fresh SATO medium and "old" medium that is oligsphere- conditioned,is key to culturing this highly enriched culture. While not limiting the operation of this culture method to any one explanation, it is likely that differentiated ohgodendrocytes condition the medium in which they grow with a factor(s) that selectively promotes the survival and proliferation of ohgodendrocytes.
  • the culture method produces oligospheres, which are primarily composed of immature and mature ohgodendrocytes and nestin positive progenitor cells.
  • This novel ohgodendrocyte culture method has several advantages over previous methods.
  • the few astrocytes that are generated in this method adhere to the side of the culture flask and are easily removed from the culture. Additionally, the free-floating spherical cell clusters (oligospheres) can be easily concentrated or moved to a different medium for use.
  • the method to produce an enriched ohgodendrocyte culture may be used with EB cells from other species.
  • Examples 1 and 2 teach the use of the method with Mus musculus and Rattus EB cells, illustrating the flexibility of the method. Due to the great similarity between EB cells from all mammalian species, it is contemplated that the culture method may be used with equal efficacy with EB cells of other species. Species of particular interest include, but are not limited to, human and monkey.
  • Bioengineered stem cell cultures may also be used to advantage with the culture method.
  • Autologous ES cells are particularly advantageous when the cells are to be used for transplantation. Autologous ES cells may be created by transferring a ES cell nucleus into a denucleated cell from the patient. These autologous ES cells will have the totipotent characteristics of the ES cell but will not be rejected when transplanted into the patient.
  • Embryonic stem cell cultures from the same species may also be genetically engineered to remove markers that will cause rejection
  • Also presented with the invention is a method to regenerate the central nervous system in patients that are deficient in axon myelination, which encompasses introducing ohgodendrocyte cells cultured in vitro from embryonic stem cells.
  • the use of /w vitro cultures is superior to previous methods of transplanting in vivo differentiated cells because it is known that the ES cells are genetically normal while in vivo differentiated populations may be harboring tumor cells.
  • the use of ES- derived ohgodendrocytes is also less invasive than previous methods where precursor cerebellar cells were used.
  • Embryonic stem cells are also advantageous over in vitro differentiated cells because they can be easily genetically modified.
  • the ES cells may be engineered to produce factors that will aid spinal cord regeneration and/or make the cell more likely to survive transplantation, i.e. tolerance of low oxygen conditions.
  • Factors of particular interest for spinal cord reneration include, but are not limited to, neurotrophin 3 (NT-3), ⁇ -FGF and LI.
  • NT-3 neurotrophin 3
  • ⁇ -FGF ⁇ -FGF
  • LI neurotrophin 3
  • the use of embryonic stem cells to culture an oligodendrocyte culture is particularly advantageous because it allows cells that are genetically identical to the patient to be treated.
  • ohgodendrocyte culture of the invention may be used to study disorders of ohgodendrocyte cells found in humans, the mechanisms that regulate production of ohgodendrocytes and the factors that the cells produce to regulate the development of other ohgodendrocytes. This culture may also be used to develop an in vitro model system of neural tissue that will be invaluable for understanding how such tissues work and can be regenerated.
  • D3 or ROSA26 mouse ES cells were maintained and differentiated in culture according to the 4-/4+ protocol of Bain et al. (Dev. Biol. 168, 342-357 (1995)). Undifferentiated ES cells were propagated in the presence of leukemia inhibitory factor (LIF, Gibco). Cells were cultured as embryoid bodies in the absence of LIF for 4 days, then treated for 4 days with retinoic acid (a ⁇ -trans-RA, 500 nM, Sigma). On the 9 th day, embryoid bodies were partially trypsinized (5 min. at 37°C, 0.25% trypsin with EDTA) and resuspended in ES cell media (Bain, et al., Dev. Biol. 168, 342-357 (1995)) prior to transplantation.
  • LIF leukemia inhibitory factor
  • BBB scores were obtained the day before transplantation (day 8 post-injury) and control and experimental groups were matched and randomly assigned to ensure that initial locomotor scores were equalized between groups.
  • the weight-drop injury level was chosen based on previous experience with the NYU impact model, to produce spontaneous recovery at a BBB score 8, the most sensitive portion of the scale corresponding to absent weight supported walking.
  • rats received transplants of neural differentiated ES cells (approximately 1 million), vehicle medium, or 1 million adult mouse neocortical cells using a spinal stereotaxic frame, a 100 ⁇ m diameter tip glass pipette configured to a 5 ⁇ l Hamilton syringe, and a Kopf microstereotaxic injection system (Kopf Model 5000 & 900). Five ⁇ l of the ES cell or mouse neocortical cell suspension or vehicle medium was injected into the center of the syrinx at the T9 level over a 5 minute period. Three independent experiments with time matched controls were completed in total.
  • Behavioral testing was performed weekly by two individuals blinded to treatment using the BBB Locomotor Rating Scale (Basso, et al., J. Neurotrauma 12,1-21 (1995)). Behavioral outcomes and examples of specific BBB locomotor scores were recorded using digital video.
  • Immunohistochemistry Primary antibodies used were directed against the following antigens: astrocytes, GFAP rabbit polyclonal, 1 :4 (Incstar); ohgodendrocytes, APC CC-1 mlgG ! 1:400 (Calbiochem Oncogene Sciences); neurons, NeuN mlgG,, 1 :500 (Chemicon); anti-mouse EMA rat hybridoma, 1 : 1 (Baumrind, et al., Dev. Dyn. 194, 311-325 (1992)); anti-mouse M2 rat hybridoma (Lagenaur & Schachner J. Supramol. Struct. Cell Biochem.
  • ES cells Neural differentiated mouse embryonic stem (ES) cells were transplanted into a rat spinal cord 9 days after traumatic injury. Histological analysis 2-5 weeks later revealed that transplant-derived cells survived and differentiated into astrocytes, ohgodendrocytes and neurons, and migrated up to 8 mm away from the lesion edge. Furthermore, gait analysis revealed that transplanted rats exhibited hindlimb weight support and partial hindlimb coordination not found in sham- operated controls or controls transplanted with adult mouse neocortical cells.
  • rats received the same transplantation procedure but using ROSA26 ES cells [a lacZ transgene containing, ⁇ -galactosidase ( ⁇ -gal)-expressing mouse ES cell line] and animals were sacrificed 2 weeks after transplantation for histology and quantitative cell counting.
  • ROSA26 ES cells a lacZ transgene containing, ⁇ -galactosidase ( ⁇ -gal)-expressing mouse ES cell line
  • Mouse ES cell-derived cells marked genetically (using the ROSA26 ⁇ - gal-expressing line) and pre-labeled in vitro with BrdU (24 hr pulse, 10 ⁇ M) could be identified in situ 14-33 d after transplantation; alternatively identification could be achieved with the mouse specific antibodies M2 (Lagenaur & Schachner J. Supramol. Struct. Cell Biochem. 15, 335-346 (1981)), EMA (Baumrind, et al., Dev. Dyn. 194, 311-325 (1992)) or Thy 1.1/1.2 (data not shown for EMA and Thy 1.1/1.2).
  • ES cell-derived cells When examined 2-5 weeks after transplantation, ES cell-derived cells were found in aggregates or dispersed singly throughout the injury site; furthermore single cells could be found as far as 8 mm away from the syrinx edge in either the rostral or caudal direction (Fig. 1). In the majority of the transplanted rats, by 2 weeks after transplantation ES cell-derived cells filled the space normally occupied by a syrinx in medium-treated rats. By 5 weeks, the density of ES cell-derived cells in this area was reduced and replaced with an extracellular matrix containing fibers positive for Thy 1.1/1.2 labeling.
  • mouse-specific markers M2 and EMA, offered advantages over the genetic and DNA markers (which only mark cell bodies) in that they also labeled ES cell-derived processes, which were abundant in ES cell transplanted rats, but not present in sham injected rats.
  • ES cell-derived cells labeled with antibodies against mouse- specific markers or BrdU, also labeled with antibodies against markers specific for ohgodendrocytes (adenomatous polyposis coli gene product, APC CC-1), astrocytes (glial fibrillary acidic protein, GFAP), and neurons (neuron-specific nuclear protein, NeuN); nuclei could be clearly identified with Hoechst 33342 staining.
  • ohgodendrocytes adenomatous polyposis coli gene product, APC CC-1
  • astrocytes glial fibrillary acidic protein, GFAP
  • neurons neuroon-specific nuclear protein, NeuN
  • the ES cell transplant group demonstrated partial weight-supported ambulation.
  • a statistical difference in BBB scores was achieved by two weeks following transplantation (Fig. 2a). After one month, a difference of two points on the BBB scale was observed between groups: 7.9 ⁇ 0.6 for sham vehicle transplantation group, 10.0 ⁇ 0.4 for ES cell transplant group.
  • the former score signifies a gait characterized by no hindlimb weight bearing and no coordinated hindlimb movements, whereas the latter score signifies a gait characterized by partial hindlimb weight bearing and partial hindlimb coordination.
  • a third experimental series examined the possibility that a rat versus mouse immune response could contribute to the observed behavioral benefit.
  • mouse ES cell-derived cells when transplanted into the spinal cord 9 days after weight drop injury: (1) survive for at least 5 weeks; (2) migrate at least 8 mm away from the site of transplantation; (3) differentiate into astrocytes, ohgodendrocytes, neurons without forming tumors; and (4) produce improved locomotor function. Behavioral recovery similar in magnitude to that shown here has previously only been shown in acute injury models (Bernstein, et al., Exp. Neurol. 98, 633-644 (1987); Bregman, et al., Exp. Neurol. 123, 3-16
  • Factors possibly responsible for the benefits observed include enhancement of myelination, reduction of delayed ohgodendrocyte death, or enhancement of host axonal regeneration, for example by providing a favorable substrate for regrowth, or by producing growth factors.
  • a method for producing enriched cultures of ES cell-derived ohgodendrocytes has been developed and the resulting ohgodendrocytes are capable of myelinating axons in vitro and in vivo after transplantation in the injured and dysmyelinated spinal cord.
  • the first model localized chemical demyelination injury, without damaging passing axons, was induced in the dorsal column white matter of rats.
  • the second model utilized myelin-deficient shiverer (shi/shi) mutant mice that lack myelin basic protein (MBP).
  • mice and Care Homozygous (shi/shi) shiverer mice and female Long-Evans rats were obtained from Jackson (Bar Harbor, ME) and Simonsen (Gilroy, CA) labs, respectively. Interventions were in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals (NTH, EHEW Pub. No. 78-23, Revised, 1978) and the Guidelines and Policies for Rodent Survival Surgery provided by the Animal Studies Committee of Washington University School of Medicine. Anesthesia was induced by ketamine/medetomidine (75:0.5 mg/kg for rats, 75: 1 mg/kg for mice; i.p.) and reversed with atipamezole (1.0 mg/kg, s.q.).
  • D3 (lacZ-) or ROSA26 (lacZ+) mouse ES cells were differentiated using the 4-/4+ protocol (Bain, et al., (1995) Dev. Biol. 168, 342- 357; Example 1). After 8 days in vitro, the 4-/4+ stage floating embryoid bodies (EBs), were partially trypsinized (5 min. at 37°C, 0.25% trypsin with EDTA) and resuspended in ES cell media (ESLM) (Bain, et al., (1995) Dev. Biol.
  • ESLM ES cell media
  • Demyelination Demyelination of dorsal column white matter was induced chemically in rats using characterized methods (Hall, (1972) J. Cell Sci. 10, 535-546; Blakemore, (1976) Neuropathol. Appl. Neurobiol. 2, 21-39; Waxman, et al., (1979) J. Neurol. Sci. 44, 45-53; Blakemore, & Crang, (1985) J. Neurol. Sci. 70, 207- 223).
  • ethidium bromide (1 ⁇ l of 0.1 % ethidium bromide in 0.9% saline) or lysophosphatidyl choline (LPC - lysolecithin; 2 ⁇ l of 1.0% LPC in 0.9% saline) was injected at a depth of 0.5 mm in the dorsal column over a 10 min period using a stereotaxic microinjector (Stoelting) and a 30 ⁇ m tip glass pipet attached to a 5 ⁇ l Hamilton syringe. Three days later, the demyelinated areas were transplanted with partially dissociated EBs.
  • Preparation of Cells for Transplantation Pre-labeled (see Cell Tracking methods below) 4-/4+ stage EBs or oligospheres were prepared for transplantation using methods described previously (Example 1) to produce suspensions of small clusters of cells. Cell density was calculated using a hemocytometer and adjusted to 50,000 viable cells per ⁇ l.
  • Demyelination injury rats received transplants of approximately 125,000 cells from partially dissociated 4-/4+ EBs or media vehicle.
  • a 50-100 ⁇ m tip diameter glass pipet was stereotaxically advanced 0.5 mm into the dorsal column white matter.
  • 2.5 ⁇ l of the ES cell suspension or vehicle media was injected at a rate of 0.25 ⁇ l/min. The needle was left in place for 5 more min., slowly withdrawn, and the laminectomy site was covered with artificial dura.
  • shiverer (shi/shi) mice were transplanted with 100,000 oligosphere cells or vehicle medium (n > 6 each) at the T8 & T10 level (1 ⁇ l at 2 sites 0.35 ⁇ m below the dura).
  • partially trypsin-dissociated 4-/4+ EBs were incubated with stable fluorescent marker cell tracker orange (Molecular Probes, Eugene, OR) for 20 min, washed, incubated for another 20 min, and then washed prior to transplantation.
  • Cell tracker orange diffuses into cells and is transformed into a fixable, membrane-impermeant form in the cytoplasm.
  • Mouse-specific antibodies were used to detect the mouse ES cells in the rat demyelination experiments: anti-M2 (labels mouse glia > neurons), and anti- EMA (labels mouse neurons > glia).
  • Antibodies used to identify the ohgodendrocyte lineage included: anti-NG2 for ohgodendrocyte progenitors (Chemicon), anti-O4 for immature ohgodendrocytes (hybridoma), anti-Ol for mature ohgodendrocytes (hybridoma), anti-MBP for terminally mature ohgodendrocytes (Boehringer- Mannheim).
  • Homozygous shiverer (shi/shi) mice are devoid of MBP and the presence of MBP(+) myelin in these animals following transplantation provided a useful marker for identification of transplanted ohgodendrocytes.
  • Electron Microscopy EBs and cultures were processed using standard methods (Mulvey, et al., (1998) Science 282, 1494-1497). Samples were viewed with a Hitachi S-450 Scanning Electron Microscope operated at 20 KV accelerating voltage and JEOL 100CX Transmission Electron Microscope.
  • ES cells differentiated using the 4-/4+ retinoic acid protocol (Bain, et al., (1995) Dev. Biol. 168, 342-357) produce ohgodendrocytes when transplanted into injured spinal cord (Example 1). Based on this protocol, methods were developed for reliable generation of mixed cultures of ohgodendrocytes, neurons and astrocytes from 4-/4+ stage EBs. EBs floated in cell clusters, many of which contained internal cysts. Ultrastructural SEM examination revealed that the cells on the surface were covered with extensive microprocesses.
  • ES-derived type-I astrocytes formed a confluent layer adherent to the bottom of the dish, other cells grew on top, and neurons grew in small clumps with large bundles of axons radiating outward.
  • Mixed cultures grew best in SATO defined media supplemented with serum and could be maintained for at least one month. Ohgodendrocyte longevity was enhanced by the presence of neurons.
  • ⁇ FGF 10 ng/ml
  • ohgodendrocyte production and inhibition of cell division 10 "5 M cytosine arabinoside), as employed in previous studies of cultured ES derived neurons (Bain, et al., (1995) Dev. Biol. 168, 342-357), limited ohgodendrocyte viability.
  • ES cell-derived ohgodendrocytes produced myelin.
  • OI myelin
  • axonal myelin profiles with 2-3 loosely wrapped layers were common. It is known that development of compact mature myelin profiles in vitro typically takes 3-6 weeks, and limited survival of neurons past 14
  • ohgodendrocytes formed sheets of myelin, similar to cultures of primary ohgodendrocytes.
  • oligospheres Enriched cultures of ohgodendrocytes were produced by development of an intermediate in vitro stage termed "oligospheres".
  • 4- /4+ EBs were trypsinized and triturated, then placed in T25 flasks containing 5 ml of pre-conditioned "oligosphere media" consisting of SATO defined media, ⁇ -FGF (10 ng/ml) and PDGF (2 ng/ml). This media promoted survival and proliferation of ohgodendrocytes.
  • non-adherent cells primarily ohgodendrocyte precursors
  • oligospheres contained a small number of neurons, few astrocytes, a large number of immature and mature ohgodendrocytes, and substantial numbers of nestin positive progenitor cells (Table 1).
  • One-week survival periods could be readily attained and longer-term growth was possible if cultures were fed with media conditioned by oligospheres.
  • CNS partially dissociated 4-/4+ EBs were transplanted into the dorsal column of rat spinal cord 3 days after chemical demyelination.
  • Successful engraftment in the demyelinated region was evident in 9/10 rats when examined 1 week after transplantation, as indicated by immunostaining with anti-mouse specific antibodies, lacZ expression, and by an increased cell density demonstrated by Hoechst 33342 labeling in transplanted animals.
  • rats that received a sham vehicle transplant axons of passage were largely spared and a paucity of Hoechst nuclear labeling was present at the demyelination site.
  • ES cells differentiated primarily into ohgodendrocytes (anti-APC CC-1), but not astrocytes.
  • Enhanced GFAP reactivity was consistently observed at the lesion borders in both ES cell and vehicle medium transplanted rats, indicating the association with host reactive astrocytes.
  • Little evidence of ES cell-derived neurons (anti-NeuN or anti-neuron specific enolase) was found in the zone of demyelination or in host tissues.
  • Nine of the ten rats that received transplants exhibited this pattern of ES cell differentiation. Histologic evidence of acute graft rejection was present in 1/10 rats.
  • ES ohgodendrocytes were transplanted into the thoracic spinal cord of shiverer (shi/shi) mice (>2 months old), which lack MBP, an essential component of functional myelin.
  • Transplanted cells were tracked by pre-labeling the oligospheres with the fluorescence marker cell tracker orange or by detecting MBP expressed by transplanted cells, but absent in the host shiverer (shi/shi) mice.
  • ES cell- derived cell tracker orange(+) and MBP(+) ohgodendrocytes predominated in white matter.
  • ES ohgodendrocytes conformed to the organization that ohgodendrocytes normally respect in white matter: ES ohgodendrocytes would align with host intrafascicular ohgodendrocytes and myelinate axons. Since homozygous shiverer mice do not exhibit substantial MBP immunoreactivity, MBP immunoreactivity could be attributed to the transplanted ES cell-derived ohgodendrocytes. In oligosphere- transplanted mice, but not sham-transplanted mice, widespread MBP immunoreactivity was evident in regions surrounding the sites of transplantation and the pattern of MBP expression was similar to that found in the normal spinal cord of mice. The longitudinal parallel arrays of MBP immunoreactivity separated by spaces occupied by axons in white matter is characteristic of axonal myelination.
  • ES cells can be used to reliably generate mixed and enriched cultures of ohgodendrocytes and that these ohgodendrocytes are capable of producing myelin and myelinating axons in vitro.
  • transplanted ES cells can: 1) preferentially differentiate into ohgodendrocytes in areas of demyelination, suggesting that environmental cues in the injury site can direct ES cell differentiation, and 2) myelinate host axons in the dysmyelinated spinal cord.
  • Remyelination is an enticing mechanism potentially underlying the rapid recovery of locomotor function observed when dissociated 4-/4+ stage ES cells were transplanted 9 days after moderate contusion injury in rats (Example 1). Significant recovery of locomotion was first evident 11 days after transplantation and ohgodendrocytes represented the largest differentiated population of ES cell derived cells in that study. The above results also suggest that local conditions in the lesioned
  • CNS can select for differentiation or survival of particular types of ES- derived neural cells.
  • ES cells When ES cells are transplanted into a contusion- injured spinal cord, they differentiate into substantial numbers of astrocytes and ohgodendrocytes arranged in specific patterns relative to one another (Example 1).
  • primary demyelinated lesions, sparing passing axons preferentially induce ES cells to differentiate into ohgodendrocytes.
  • ES cells Formation of teratomas or other tumor types remains a concern in any transplantation study.
  • a heartening feature of ES cells is that they are the only stem cells that can be proven genetically normal by generating a normal animal after implantation into blastocysts.
  • Oligospheres from ES Cells This example sets forth detailed instructions on the preparation of an oligodendrocyte-enriched cell culture from ES cells. To the extent specific media, reagents and materials are mentioned, they are intended to be illustrative, not limiting, of the invention.
  • the process of culturing oligospheres is outlined in this example from the point where Embryoid Bodies (EBs) have been successfully created (Example 1; Bain et al, 1998). There are many stock solutions used as components of this recipe that must be created prior to beginning a particular step. Some of these must be made fresh, others can be stored and used many times.
  • EBs Embryoid Bodies
  • SATO 100X stock solution is the medium supplement basis for SATO-no-serum medium and is the first series of steps in the process of making oligospheres. All solutes can be weighed outside the hood since the SATO 100X stock will be sterile filtered before it is aliquoted and frozen down. In the interest of time efficiency, weigh out all solutes at once, make up individual stock solutions, then combine them and filter. It is also advisable to pre-label each weighing boat and glass culture tube with the substance it will contain to assure that mistakes are not made.
  • N NaOH can be used repeatedly if sterile technique is maintained.
  • f) Carefully pour 2.4 mg thyroxine powder into a 5 mL roundbottom tube.
  • h) Cap the tube and vortex until all solute is dissolved ( ⁇ 2 minutes).
  • BSA putrecine, apo-transferrin
  • SATO Constituent stock solutions are the growth factor additives for SATO-no-serum medium and, combined, are the second series of steps in the process of making oligospheres. All solutes can be weighed outside the hood since the constituent stocks will be sterile filtered before they are aliquoted and frozen down.
  • CNTF stock is now ready. This stock is usually made fresh each time we need it, but many aliquots can be made up at one time and stored at -20°C for several months.
  • NS weigh out all constituent nucleoside powders at one time outside the hood. a) Adenosine 40 mg b) Guanosine 42.5 mg c) Citidine 36.5 mg d) Uridine 36.5 mg e) Thymidine 12 mg
  • ESIM is now ready for use, will keep for -30 days, and can be used repeatedly if sterile technique is maintained.
  • This procedure is the final recipe for creating SATO-no-serum medium, the culturing medium used in creating oligospheres from EBs.
  • Oligosphere Recipe Procedural Notes It is very important to carry out all procedures in a sterile hood and with sterile instruments and tubes unless otherwise indicated. Specifically, it is advisable to use a fresh, sterile, serological pipette for each step unless repeated use is indicated. Centrifugation is not generally performed in a hood, but tube caps must be kept tight during this procedure to insure that cells remain in a sterile environment. Do not allow any liquids or pipettes to contact the necks of flasks at any time because this part of the flask can be contaminated. To prevent any medium from contacting the neck when handling cells in T25 flasks, keep flasks nearly level with a very slight tilt toward the rear of the flask.
  • caps should be tightened on flasks when they are removed from the incubator to ensure that a sterile environment is maintained.

Abstract

L'invention concerne une culture cellulaire différenciée in vitro enrichie en oligodendrocytes. Elle concerne également des procédés servant à préparer cette culture. Elle concerne, de plus, des procédés servant à utiliser des cellules neurales différenciées in vitro, de préférence, enrichies en oligodendrocytes, dans des transplantations afin de traiter des traumatismes ou de dégénérescences de la moelle épinière.
PCT/US2000/041367 1999-10-22 2000-10-21 Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation WO2001028342A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP00986805A EP1221853A4 (fr) 1999-10-22 2000-10-21 Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation
JP2001530946A JP2003511090A (ja) 1999-10-22 2000-10-21 オリゴデンドロサイト培養物、その製造方法及び使用
CA002388736A CA2388736A1 (fr) 1999-10-22 2000-10-21 Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation
AU22979/01A AU2297901A (en) 1999-10-22 2000-10-21 Oligodendrocyte cell cultures and methods for their preparation and use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US16112599P 1999-10-22 1999-10-22
US60/161,125 1999-10-22

Publications (1)

Publication Number Publication Date
WO2001028342A1 true WO2001028342A1 (fr) 2001-04-26

Family

ID=22579930

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/041367 WO2001028342A1 (fr) 1999-10-22 2000-10-21 Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation

Country Status (5)

Country Link
EP (1) EP1221853A4 (fr)
JP (1) JP2003511090A (fr)
AU (1) AU2297901A (fr)
CA (1) CA2388736A1 (fr)
WO (1) WO2001028342A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004007696A2 (fr) * 2002-07-11 2004-01-22 The Regents Of The University Of California Oligodendrocytes derives de cellules souches embryonnaires humaines pour remyelinisation et traitement de lesion de la moelle epiniere
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
US10221390B2 (en) 2008-01-30 2019-03-05 Asterias Biotherapeutics, Inc. Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells
US11603518B2 (en) 2019-01-23 2023-03-14 Asterias Biotherapeutics, Inc. Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells
US11920155B2 (en) * 2016-03-30 2024-03-05 Asterias Biotherapeutics, Inc. Oligodendrocyte progenitor cell compositions

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9177967B2 (en) 2013-12-24 2015-11-03 Intel Corporation Heterogeneous semiconductor material integration techniques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5750103A (en) * 1990-10-19 1998-05-12 The New York University Medical Center Method for transplanting cells into the brain and therapeutic uses therefor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5750103A (en) * 1990-10-19 1998-05-12 The New York University Medical Center Method for transplanting cells into the brain and therapeutic uses therefor

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BRUSTLE O.: "Embryonic stem cell-derived glial precursors: A source of myelinating transplants", SCIENCE, vol. 285, 30 July 1999 (1999-07-30), pages 754 - 756, XP002938502 *
LEE J.C. ET AL.: "Gliogenesis in the central nervous system", GLIA, vol. 30, no. 2, April 2000 (2000-04-01), pages 105 - 121, XP002938503 *
LIU S. ET AL.: "Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation", PROC. NATL. ACAD. SCI. USA, vol. 97, no. 11, 23 May 2000 (2000-05-23), pages 6126 - 6131, XP002938504 *
MCDONALD J.W. ET AL.: "Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord", NATURE MEDICINE, vol. 5, no. 12, December 1999 (1999-12-01), pages 1410 - 1412, XP002938505 *
MUJTABA T.: "Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells", DEVELOPMENTAL BIOLOGY, vol. 214, 1 October 1999 (1999-10-01), pages 113 - 127, XP002938000 *
RAO M.: "Multipotent and restricted precursors in the central nervous system", THE ANATOMICAL RECORD, vol. 257, 15 August 1999 (1999-08-15), pages 137 - 148, XP002938506 *
See also references of EP1221853A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2273268A2 (fr) 2002-07-11 2011-01-12 The Regents of The University of California Oligodendrocytes dérivés de cellules souches embryonnaires humaines pour remyélinisation et traitement de lésion de la moelle épinière
AU2003250477B2 (en) * 2002-07-11 2008-07-03 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
GB2407822A (en) * 2002-07-11 2005-05-11 Univ California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
JP2005532079A (ja) * 2002-07-11 2005-10-27 ザ・レジェンツ・オブ・ザ・ユニバーシティ・オブ・カリフォルニア 再ミエリン化および脊髄損傷の治療のためのヒト胚性幹細胞に由来するオリゴデンドロサイト
WO2004007696A2 (fr) * 2002-07-11 2004-01-22 The Regents Of The University Of California Oligodendrocytes derives de cellules souches embryonnaires humaines pour remyelinisation et traitement de lesion de la moelle epiniere
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
WO2004007696A3 (fr) * 2002-07-11 2004-04-22 Gabriel I Nistor Oligodendrocytes derives de cellules souches embryonnaires humaines pour remyelinisation et traitement de lesion de la moelle epiniere
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
GB2407822B (en) * 2002-07-11 2006-02-22 Univ California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
JP4823689B2 (ja) * 2002-07-11 2011-11-24 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 再ミエリン化および脊髄損傷の治療のためのヒト胚性幹細胞に由来するオリゴデンドロサイト
EP2273268A3 (fr) * 2002-07-11 2013-06-19 The Regents of The University of California Oligodendrocytes dérivés de cellules souches embryonnaires humaines pour remyélinisation et traitement de lésion de la moelle épinière
CN103396993A (zh) * 2002-07-11 2013-11-20 加利福尼亚大学董事会 衍生自灵长类胚胎干细胞的用于脊髓损伤的再髓鞘化和治疗的少突胶质细胞
US10611998B2 (en) 2002-07-11 2020-04-07 The Regents Of The University Of California Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury
US10221390B2 (en) 2008-01-30 2019-03-05 Asterias Biotherapeutics, Inc. Synthetic surfaces for culturing stem cell derived oligodendrocyte progenitor cells
US11920155B2 (en) * 2016-03-30 2024-03-05 Asterias Biotherapeutics, Inc. Oligodendrocyte progenitor cell compositions
US11603518B2 (en) 2019-01-23 2023-03-14 Asterias Biotherapeutics, Inc. Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells

Also Published As

Publication number Publication date
AU2297901A (en) 2001-04-30
EP1221853A4 (fr) 2004-08-25
EP1221853A1 (fr) 2002-07-17
JP2003511090A (ja) 2003-03-25
CA2388736A1 (fr) 2001-04-26

Similar Documents

Publication Publication Date Title
AU665012B2 (en) Novel growth factor-responsive progenitor cells which can be proliferated (in vitro)
Kukekov et al. A nestin‐negative precursor cell from the adult mouse brain gives rise to neurons and glia
AU755028B2 (en) Neural precursor cells, method for the production and use thereof in neural defect therapy
Shim et al. Generation of functional dopamine neurons from neural precursor cells isolated from the subventricular zone and white matter of the adult rat brain using Nurr1 overexpression
US6638763B1 (en) Isolated mammalian neural stem cells, methods of making such cells
US20080138895A1 (en) Cultures of gfap+ nestin+ cells that differentiate to neurons
WO2004007696A2 (fr) Oligodendrocytes derives de cellules souches embryonnaires humaines pour remyelinisation et traitement de lesion de la moelle epiniere
CN101065478B (zh) 用于繁殖神经祖细胞的组合物和方法
Staines et al. Retinoic acid treated P19 embryonal carcinoma cells differentiate into oligodendrocytes capable of myelination
AU770501B2 (en) Ependymal neural stem cells and method for their isolation
WO2001028342A1 (fr) Cultures de cellules oligodendrocytes et leurs procedes de preparation et d'utilisation
US8043853B2 (en) Postnatal gut neural crest stem cells
WO2000006700A1 (fr) Production et utilisation de cellules dopaminergiques pour traiter des deficiences dopaminergiques
WO2002014479A2 (fr) Cellules souches neuronales isolees de mammiferes et procedes de fabrication et d'utilisation de ces cellules
Korochkin et al. Neural stem cells and their role in recovery processes in the nervous system
MXPA00005880A (en) Neural precursor cells, method for the production and use thereof in neural defect therapy
US20050118143A1 (en) Isolated mammalian neural stem cells, methods of making such cells, and methods of using such cells
Kleppner In vivo and in vitro characteristics of human neuronal cells derived from the teratocarcinoma cell line NT2
IL165645A (en) Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

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

Ref document number: 22979/01

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2388736

Country of ref document: CA

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2001 530946

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2000986805

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000986805

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2000986805

Country of ref document: EP