WO2006085612A1 - Method for preparation of neural stem cells - Google Patents

Method for preparation of neural stem cells Download PDF

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Publication number
WO2006085612A1
WO2006085612A1 PCT/JP2006/302350 JP2006302350W WO2006085612A1 WO 2006085612 A1 WO2006085612 A1 WO 2006085612A1 JP 2006302350 W JP2006302350 W JP 2006302350W WO 2006085612 A1 WO2006085612 A1 WO 2006085612A1
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cerebral infarction
mouse
cells
neural stem
bone marrow
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PCT/JP2006/302350
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French (fr)
Japanese (ja)
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Tomohiro Matsuyama
Akihiko Taguchi
Hiroo Yoshikawa
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The New Industry Research Organization
Japan Health Sciences Foundation
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Priority to JP2007502657A priority Critical patent/JP4905719B2/en
Publication of WO2006085612A1 publication Critical patent/WO2006085612A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • C12N2501/04—Immunosuppressors, e.g. cyclosporin, tacrolimus
    • 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
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • the present invention relates to a method for preparing neural stem cells, and more particularly, to a method for inducing and preparing neural stem cells from bone marrow and application to nerve regeneration therapy.
  • Cerebrovascular disorders such as cerebral infarction and neurodegenerative diseases are one of the most important issues to be solved for Japan, which is facing a super-aging society.
  • therapeutic therapies that have focused on the suppression of neuronal cell death, the results that have led to the development of effective therapies have still been obtained sufficiently.
  • Nerve regeneration therapy has the potential to improve brain functions that have already been damaged by regenerating nerves, and is expected as a new treatment method for neurological diseases.
  • nerve regeneration treatment has been carried out by transplanting neural stem cells collected from fetal brain in neurodegenerative diseases such as Parkinson's disease.
  • neural stem cells collected from fetal brain in neurodegenerative diseases such as Parkinson's disease.
  • issues that need to be resolved with this treatment including ethical issues.
  • ES cell-derived neural stem cell transplantation for the treatment of cerebral infarction has also been performed.
  • functional analysis of ES cells is inadequate, and it is currently difficult to obtain ethically and institutionally There is a problem.
  • the method of inducing differentiation of ES cells into nerves has not been established yet, and it is uncertain whether it will function in vivo.
  • neural stem cells that are expected to be used in nerve regeneration medicine are also derived from the subventricular zone (SVZ) derived from the mature brain. Can be collected.
  • SVZ subventricular zone
  • Neural stem cells derived from fetal brain differentiate into neurons.
  • neural stem cells derived from mature brain SVZ are highly differentiated into glial cells and do not differentiate into neurons under normal culture conditions.
  • nerve cells could be created from bone marrow cells by introducing genes into bone marrow stromal cells or by culturing bone marrow stromal cells with several types of growth factors. .
  • neurospheres which are neural stem cells, were obtained from bone marrow cells of human mice (see Non-Patent Documents 1 and 2 below).
  • (1)-the culture period until preparation of neurostem cells capable of eurosphering and separation takes about 2 to 6 months, and it takes a long period of time.
  • nutrient factors such as BDNF and PDGF.
  • Non-Patent Document 1 Hermann A, Gastl R, Liebau S, Oana Popa M, Fiedler J, Boehm BO, Maisel M, Lerche H, Schwarz J, Brenner R, Storch A (2004) J Cell Sci 117: 4411 —44 22.
  • Non-Patent Document 2 Kabos P, Ehtesham M, Kabosova A, Black KL, Yu JS (2002) Generati on of neural progenitor cells from whole adult bone marrow. Exp Neurol 178: 288—29 3.
  • Non-Patent Document 3 Taguchi, A., Soma, T., Tanaka, H., Kanda, T., Nisnimura, H., Yoshik awa, H., Tsukamoto, Y., Iso, H., Fujimori, Y. , Stern, DM, Naritomi, H., Matsuyam a, T. (2004) Administration of CD34 + cells post-stroke enhances angiogenesis and neurogenesis in a murine model. J. Clin. Invest., 114: 330—338.
  • neural stem cells that are expected to be applied to nerve regeneration treatment have been developed from embryonic brain-derived neural stem cells, adult brain-derived neural stem cells, ES cell-derived neural stem cells, and bone marrow adult pluripotent stem cells (multipotent Adult stem cells derived from adult progenitor cells (MAPC) have been reported.
  • MPC multipotent Adult stem cells derived from adult progenitor cells
  • problems such as the supply source, efficiency of differentiation into neurons, engraftment into the transplanted brain, and function performance.
  • methods for artificially creating nerve cells such as introducing genes into bone marrow cells, have been devised. There are problems with such as carcinogenicity, engraftment, and functionality.
  • the neural stem cells used for nerve regeneration therapy are ethically and for the avoidance of graft-versus-host disease (G VHD). It is desirable that the cell be an autograft. In addition, it is a stem cell that is highly differentiated into neurons (Neurogenesis), maintains nerve function (Functional), and is easily (eaSY), easily (Amplify), and abundant (Amplify) In other words, it is desirable to have “FANTASY” that combines these characteristics!
  • a living body has various repair functions when a tissue or organ is damaged. These are mainly performed through the division and proliferation of tissue-specific cells such as the gastrointestinal epithelium, liver, and vascular endothelial cells. Recently, it has been known that various bone marrow-derived stem cells are involved in this. It was. However, in reality, the degree to which bone marrow is involved in tissue repair in most pathological conditions has not been studied. When developing a nerve regeneration therapy by neural stem cell transplantation, we will study the mechanism of the repair mechanism as part of the homeostasis maintenance mechanism that normally occurs in vivo, and then devise a therapy according to that mechanism. It is considered effective.
  • the present invention has been made in view of the above-mentioned problems, and its purpose is to develop and establish a new nerve regeneration treatment method for cerebrovascular disorders such as cerebral infarction and neurodegenerative diseases. , Used in nerve regeneration treatment or the development of treatment methods thereof, It is intended to provide a method for preparing neural stem cells that can be prepared in a short period of time and can be expected to successfully engraft and function nerve cells in the affected brain area by transplantation.
  • the present inventors first treated an immunodeficient SCID mouse to create a cerebral infarction model mouse, and collected it from the mouse one week after the cerebral infarction. It has been clarified that the bone marrow cells can be induced to differentiate into neural stem cells (neurospheres) by culturing these bone marrow cells. In addition, as described later, important knowledge about regeneration and immunity was obtained, and a technique for inducing and preparing neural stem cells from bone marrow in a short period (about 2 weeks) was established. By this method, neural stem cells were actually derived from human normal bone marrow.
  • the present invention includes the following inventions (1) to (48) as industrially and medically useful medical inventions.
  • a method for preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressive agent and serum collected from humans or animals after cerebral infarction or other brain injury means that a part of the brain is in an ischemic state due to stenosis, occlusion, ligation or other causes of blood vessels in the brain, that is, cerebral ischemic disorder.
  • TNF a tumor necrosis factor-a
  • a differentiation-inducing factor that is a chemokine (or other site force-in) and directly or indirectly induces differentiation from bone marrow cells to neural stem cells.
  • a neural stem cell differentiation inducer comprising the differentiation inducer according to (6) above and an immunosuppressant.
  • a cerebral infarction treatment method or other nerve regeneration treatment method wherein the neural stem cell according to (10) or the nerve cell according to (11) is administered by a method such as intravenous administration.
  • bone marrow cells used for preparation of neural stem cells should be collected from the patient power to be treated. How to use.
  • FK506 tacrolimus
  • cyclosporine cyclosporine
  • anti-CD28 can be used as an immunosuppressant used for preparation of neural stem cells.
  • immunosuppressive agents that suppress T cell functions such as antibodies and anti-ICOS antibodies.
  • the anti-CD28 antibody and the anti-ICOS antibody mean blocking antibodies that inhibit the action of CD28 and ICOS, respectively, and suppress the function of T cells, and do not stimulate and activate T cells.
  • a therapeutic agent for nerve regeneration comprising the neural stem cell according to (10) above or the nerve cell according to (11) above.
  • the bone marrow cells of a maternal mouse of an immunodeficient mouse are cultured by adding serum (or chemokine or other site force in) collected from a cerebral infarction model mouse and an immunosuppressive agent, the method of.
  • neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared by an immunodeficient mouse.
  • Neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared after administration of an immunosuppressive agent to a maternal mouse of an immunodeficient mouse to make it immunocompromised. Method.
  • a cerebral infarction model animal obtained by ligating brain blood vessels of a SCID mouse maternal mouse.
  • Nerve (stem) cells or other cells are transplanted into the cerebral infarction model animal described in any one of (30) to (35) above, and the effectiveness of the transplantation treatment for cerebral infarction is screened. Method.
  • a nerve regeneration therapeutic agent comprising as an active ingredient a substance having an immunosuppressive action.
  • the therapeutic agent for nerve regeneration according to the above (38), wherein the substance having an immunosuppressive action is a substance that suppresses T cell function.
  • the nerve regeneration therapeutic agent according to (39) above, wherein the substance having an immunosuppressive effect is any of FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody.
  • a therapeutic agent for nerve regeneration comprising, as an active ingredient, a chemokine or other site force-in or a substance that promotes the action of inducing differentiation into a bone marrow cell force neural stem cell.
  • a screening method for a nerve regeneration therapeutic agent used for the treatment of cerebral infarction or other neurological diseases, which induces or promotes differentiation from bone marrow cells to neural stem cells A screening method for a therapeutic agent for nerve regeneration, wherein a candidate substance is searched using as an index whether or not it has an action to act.
  • test substance is administered to a bone marrow cell culture medium collected from humans or animals, and the test substance induces differentiation from bone marrow cells to neural stem cells or has an action of promoting the differentiation
  • the neural stem cell preparation method of the present invention can prepare neural stem cells from bone marrow cells in a simple and short period of time.
  • the obtained neural stem cells can be efficiently separated into the nerve cells, and it can be expected that the nerve cells will be engrafted and function well in the affected part of the brain by transplanting to the living body.
  • it can be used for the purpose of developing treatments using cerebral infarction model mice and the like.
  • bone marrow cells used as a material are relatively easy to collect from a living body, for example, patient-powered bone marrow after cerebral infarction is collected and cultured in the presence of an immunosuppressant to prepare neural stem cells.
  • the present invention can be used for autologous transplantation treatment in which this is transplanted into a patient by intravenous administration or the like.
  • FIG. 1 Neurospheres and neurons etc. derived from embryonic mouse brain (A, B, C) and-Eurosphere-like cell clusters and neurons derived from cerebral infarction SCID mouse bone marrow (D, E , F) is a photograph replacing the drawing. In the original C'F, MAP2-positive neurons are displayed in red, and GFAP-positive glial cells are displayed in green.
  • FIG.2 Cerebral infarction Regarding the formation of Spheroid cell mass derived from SCID mouse bone marrow, how the time until bone marrow collection after cerebral infarction and the culture period of bone marrow cells affect cell mass formation, etc. It is a graph which shows the result of having examined.
  • FIG. 3 is a photograph replacing a drawing which shows the result of examination by immunostaining of the differentiation of bone marrow-derived neurosphere-like cell clusters into neurons and glial cells.
  • FIG. 4 is a graph showing the results of examining the differentiation ability and differentiation efficiency of neurosphere-like cell clusters derived from bone marrow into neurons.
  • FIG. 6 is a photograph replacing a drawing which shows a comparison of brains removed on the 16th day after ligation of the middle cerebral artery in SCID mice and C. B17 mice.
  • FIG. 7 is a photograph replacing a drawing, showing a comparison of the results of TTC staining of brain slices extracted on the first day after ligation of the middle cerebral artery in SCID mice and C. B17 mice. The cerebral infarction area is shown in white.
  • FIG. 8 is a graph showing the results of examining the presence or absence of brain (cortex) regeneration after cerebral infarction in cerebral infarction SCID mice and cerebral infarction C. B17 mice based on CI values.
  • FIG. 11 is a photograph replacing a drawing which shows the results of examining apoptosis in cerebral infarction SCID mice (A) and cerebral infarction C.
  • necrotic cells are displayed in red and apoptotic cells are displayed in green.
  • FIG. 12 is a graph showing the results of studying the formation of neural stem cells with cerebral infarcted tissue strength in cerebral infarction SCID mice and cerebral infarction C. B17 mice.
  • FIG. 13 FK506 was intraperitoneally administered daily for 3 days before and 7 days after the creation of cerebral infarction in C. B 17 mice, and then the bone marrow and the subinfarcted tissue (cerebral infarction scar site) were collected and cultured. From the bone marrow ( ⁇ ⁇ ⁇ ) and cerebral infarction scar site (C)-Eurosphere-like cell clusters formed It is the photograph replaced with drawing which shows.
  • FIG. 14 is a photograph replacing a drawing which shows that neurosphere-like cell clusters ( ⁇ ⁇ ⁇ ) were formed when bone marrow of cerebral infarction C. B17 mice were cultured with FK506 added.
  • FIG. 15 is a graph showing the results of examining the effect of F F506 on the formation of a eurosphere-like cell cluster derived from cerebral infarction C. B17 mouse bone marrow.
  • FIG. 17 Addition of FK506 by the above method-Eurosphere-like cell mass was formed, and on the 7th day after culture, immunohistochemistry for Nestin and MAP2 (double-stained indirect fluorescent antibody) It is a photograph replacing a drawing showing the result of performing the method.
  • FIG. 18 is a photograph replacing a drawing showing that bone marrow-derived neural stem cells have engrafted and separated in brain tissue in SCID mice after cerebral infarction.
  • Bone marrow-derived GFP-positive cells green in the original map
  • PSA-NCAM positive red in the original map
  • neural progenitor cells Nege
  • Neurosphere-like cell mass obtained from normal human bone marrow cells cultured in the presence of sera and FK506 in cerebral infarction patients, and nerve cells (B) obtained by further culturing It is a photograph.
  • FIG. 20 Instead of a drawing showing the results of studying the effect of bone marrow-derived neural stem cells from cerebral infarction SCID mice on brain regeneration when transplanted to another cerebral infarction SCID mouse, and the effect of treatment of cerebral infarction It is a photograph.
  • ⁇ -NSCj is the result of administration of bone marrow-derived neural stem cells
  • PBS is the result of administration of PBS as a control.
  • FIG. 21 is a photograph replacing a drawing, showing the results of examining the effects of anti-CD28 antibody and anti-ICOS antibody on the formation of C.
  • B17 mouse bone marrow-derived eurosphere-like cell clusters Sham surgery without cerebral infarction C.
  • B17 mouse bone marrow was cultured with SCID mouse serum 1 week after cerebral infarction.
  • FK506 A
  • B anti-CD28 antibody
  • B anti-ICOS antibody
  • bone marrow cells cultured with FK506, anti-CD28 antibody, and anti-ICOS antibody also added-Eurosphere-like cell mass formed, and when cultured with serum alone (D)-Euro No sphere-like cell mass was formed.
  • FIG. 23 is a photograph replacing a drawing, showing the results of examining the effect of FK506 on the formation of eurosphere-like cell clusters stimulated by CINC-1.
  • CINC-1 and FK506 were added, cell clusters were already formed within 5 days of culture, and -Eurosphere-like cell clusters were formed after 7 days (A).
  • FK506 was not added, the cell mass stopped at the force of forming force or the state of the cell mass (B), and did not become neurosphere-like.
  • ⁇ and ⁇ are both images on day 9 of culture.
  • FIG. 26 is a graph showing the results of examining the effect of cytoforce-in (CINC-1 and TNF a) on bone marrow-derived eurosphere formation.
  • the vertical axis is formed on day 9 of culture per ldish.
  • FIG. 27 is a graph showing the results of examining the effects of anti-CD28 antibody and anti-ICOS antibody on the formation of C-B17 mouse bone marrow-derived -eurosphere-like cell clusters.
  • the vertical axis shows the number of nestin positive-eurospheres per culture ldish (4 animals per group). * P ⁇ 0.001, stude nt t test
  • the present inventors recently developed a cerebral infarction model mouse that exhibits good reproducibility and can survive for a long period of time (Japanese Patent Application No. 2004-108500). As described later, this cerebral infarction model mouse selectively disrupts the blood flow in the cortical branch of the middle cerebral artery by ligating the middle cerebral artery of the SCID mouse, which is an immunodeficient mouse, and has good reproducibility. A uniform cerebral infarction was created. Interestingly, in this cerebral infarction SCID mouse, the delayed infarct expansion after ischemia ended 3 days after the cerebral infarction, and then the brain morphology rather than the progression of cerebral atrophy recovered. To do.
  • Nerve stem cells are not induced by culturing the bone marrow of normal SCID mice without cerebral infarction! However, adding serum from cerebral infarction model mice to this bone marrow induces neural stem cells. The neural stem cells were also divided into neurons (FIG. 5C′D). As a result, a stimulating factor that induces differentiation of bone marrow cells into neural stem cells in serum after cerebral infarction (diff It is thought that in the living body, this stimulating factor is transmitted to the bone marrow through the bloodstream to produce neural stem cells.
  • one of the stimulating factors in other words, one of the inducers of neural stem cells, is a chemokine of the interleukin 8 (IL 8) family, CINC—1ZG RO ⁇ cytokine- induced neutrophil chemoattractant-1 / It has been revealed that it is a growth-related oncogene (Figs. 22 and 23).
  • IL 8 interleukin 8
  • the immunosuppressive agent FK506 (tacrolimus) was administered to the CB-17 / IcrCrlBR mice to make them immunodeficient and administration of the immunosuppressive agent was continued even after causing cerebral infarction. Then, when bone marrow cells collected from the mice were cultured, differentiation into neural stem cells could be induced (FIG. 13). Similar results were obtained when cyclosporin A (CsA), another immunosuppressant, was administered ( Figure 24). When C.B-17 / IcrCrlBR mice were infarcted, their bone marrow was collected and cultured with the addition of the immunosuppressant FK506 to form neural stem cells (FIGS. 14 and 15).
  • CsA cyclosporin A
  • (6) Cerebral infarction Neural stem cells which are also prepared from bone marrow in SCID mice, were intravenously administered to another cerebral infarction SCID mouse and examined for therapeutic effects on cerebral infarction. The effect of promoting nerve regeneration was confirmed (Fig. 20). [0026] Based on these findings, the present invention is based on these findings. (1) In order to differentiate normal bone marrow cells into neural stem cells, the stimulating factor present in the serum after cerebral infarction and the immune reaction in the bone marrow culture solution are determined. It is important to add an immunosuppressive agent to suppress. (2) In order to differentiate bone marrow cells collected from patients after cerebral infarction or immune normal model animals into neural stem cells, it is important to add an immunosuppressive agent. And (3) one of the above stimulating factors (min-inducing factors) was found to be IL-8 family chemokine CINC-1 / GRO, and the following first to third neural stem cells A method of preparation is provided.
  • First neural stem cell preparation method differentiation is induced by culturing bone marrow cells by adding an immunosuppressant and serum collected from human or animal after cerebral infarction or other brain injury, How to prepare.
  • Second Neural Stem Cell Preparation Method A method for preparing neural stem cells by inducing differentiation by culturing human bone or animal force collected after cerebral infarction or other brain injury by adding an immunosuppressive agent.
  • immunosuppressant and chemokine preferably IL-8 family chemokine
  • chemokine preferably IL-8 family chemokine
  • the bone marrow from which human or animal force was also collected according to the conventional method is supplemented with an immunosuppressant and serum after brain injury (or chemokine instead of the serum).
  • an immunosuppressant and serum after brain injury or chemokine instead of the serum.
  • it can be cultured by a method similar to the conventional method for culturing neural stem cells.
  • it can be cultured by the same method as the conventional neural stem cell culture method except that an immunosuppressive agent is added.
  • Examples of conventional culture methods include a neurosphere method, a low-density monolayer culture method, and a high-density monolayer culture method, and among these, a preferred culture method includes the neurosphere method.
  • this -eurosphere method was used in the presence of basic fibroblast growth factor (bFGF, 50 ⁇ g / ml) and epidermal growth factor (EGF, 20 ⁇ g / ml). Bone marrow cells were cultured and neural stem cells were prepared.
  • bFGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • the immunosuppressive agent added to the culture solution is well-known, such as basiliximab, azathioprine, muromonab CD3, mizoribine, mofuethyl mycofenolate, etc.
  • An immunosuppressant is a substance such as an antibody (for example, an anti-CD28 antibody or an anti-ICOS antibody) as long as an agent that suppresses cellular immunity, such as T cell function, has a preferable immunosuppressive function. Widely included in the “immunosuppressive agent” of the present invention.
  • the amount of the immunosuppressant added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 0.01 / ⁇ 8 ⁇ 1 to 1.0 ⁇ g Zml.
  • FK506 was added to the culture solution at a concentration of 0.1 ⁇ g / ml.
  • the amount of serum added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 5 ⁇ l Zml to 25 ⁇ l Zml.
  • a differentiation-inducing factor in the serum after cerebral infarction that induces bone marrow cells to differentiate into neural stem cells.
  • This differentiation-inducing factor is partially damaged by cerebral neurons due to ischemic invasion or other causes. Is considered to be produced as part of the repair mechanism of the living body. Therefore, not only the serum after cerebral infarction but also the serum after the brain thread and tissue have been partially damaged due to an accident etc. It is considered that the same effect can be obtained by culturing.
  • the neural stem cells of the present invention prepared using serum after cerebral infarction may be used not only for the treatment of cerebral infarction but also for the regeneration treatment of other neurological diseases.
  • mice When preparing neural stem cells for the development of therapeutic methods using serum collected from “animals”, the animals include mice, rats, as well as ushi, pig, hidge, goat, usagi, i Mammals such as nu, cat, guinea pig and hamster are exemplified.
  • a mouse other than a SCID mouse may be used.
  • neural stem cells were successfully prepared by using serum collected one week after cerebral infarction, so the first preparation of the present invention
  • the method uses serum 5 to 10 days after cerebral infarction (or after other cerebral disorders) It is preferable.
  • the third preparation method is a method of culturing bone marrow cells by adding chemokine as an inducing factor together with an immunosuppressant.
  • CINC lZ GRO cytokine-induced neutrophil chemoattractant- lZ growth-relat ed oncogene
  • IL—8 interleukin 8 family
  • CINC-1 is also produced by NK cells and is involved in the immune response of fertilized eggs via the uterine decidua (Biochem. Biophys. Res. Commun.
  • CINC-1 is a factor that induces differentiation of neural stem cells.
  • FK506 immunosuppressive agents
  • CINC-1 is one of the induction factors.
  • CINC-1 also has the action of agglutinating bone marrow cells in culture (FIG. 23B), and this aggregated cell mass is considered to be the nucleus to form -Eurosphere.
  • chemokines with such an aggregating action are also CINC Similar to 1, it is thought to act as a differentiation-inducing factor. Whether such chemokine acts directly on stem cells in the bone marrow, or indirectly through stromal cells, etc., it is a force that requires further analysis in the future. Addition of chemokines to cultured bone marrow together with immunosuppressants can induce differentiation into neural stem cells.
  • IL-8 family chemokines are preferred, IL-8 family chemokines derived from humans, rats, or mice, or chemokines selected from other mammalian counterparts can be mentioned.
  • human-derived IL 8 family chemokines include IL-8 / CXCL8, GRO-a, GRO- ⁇ and GRO- ⁇ , and rat-derived chemokines include CINC-1, CINC-2 ⁇ , CINC-2 Examples of j8, CINC-3, and mouse origin are KC and MIP-2.
  • Other CXC chemokines may be used.
  • the amount of chemokine added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 0.1 gZml to 1.0 gZml. In the examples below, at a concentration of 10- 5 M were ⁇ Ka ⁇ the CI NC- 1 to the cultures.
  • chemokine can be induced into neural stem cells by adding it to cultured bone marrow together with an immunosuppressive agent. Therefore, a chemokine and an immunosuppressive agent are combined to produce neural stem cells. It can be provided as a differentiation inducer.
  • immunosuppressive agents is to suppress T cell function in culture, immunosuppression can be achieved if T cell function can be suppressed by other methods such as removing T cells. It is considered possible to prepare cultured cell force neural stem cells with chemokine alone without adding any agent.
  • TNF a tumor necrosis factor-a force which is a cytokine has a differentiation inducing action from bone marrow cells to neural stem cells.
  • other site force-in such as TNFa or IL18 may be used as a differentiation-inducing factor instead of chemokine.
  • a cyto force-in such as TNF ⁇
  • it can be used under the same conditions as the above chemokine and can be used for preparation from bone marrow cells to neural stem cells.
  • the neural stem cell preparation method of the present invention has the following advantages.
  • Neural stem cells can be easily and easily prepared in a short time. As shown in the examples described later, bone marrow strength can be actually induced and prepared in a short period (about 2 weeks), and the resulting neural stem cells can then divide and proliferate for high efficiency in nerve cells. Differentiated.
  • the present inventors observed the appearance of numerous neural stem cells after cerebral infarction in cerebral infarction SCID mice, which engrafted in normal neural tissue and contributed to brain regeneration and brain function improvement. It was also clarified that these neural stem cells are derived from bone marrow.
  • the present invention prepares bone marrow-derived neural stem cells that are specifically produced during cerebral infarction repair and regenerate nerves, and can provide a large amount of cells required by the living body. It is thought that transplantation treatment along the line will be possible.
  • bone marrow cells are relatively easy to collect, for example, bone marrow is collected from a patient after cerebral infarction and cultured in the presence of an immunosuppressive agent to prepare neural stem cells, which are then used as veins. Autologous transplantation to patients by internal administration etc. is also feasible.
  • the present invention can provide the patient's own bone marrow-derived neural stem cells, which can be said to be the best in terms of engraftment in a living body and function, and is suitable for regenerative medicine.
  • the present invention is not limited to use for autologous transplantation, and can also be used for a method for preparing neural stem cells from bone marrow derived from another person for other transplantation.
  • chemokine or other site force-in which is a differentiation-inducing factor
  • a method for preparing neural stem cells by adding a cytodynamic force such as chemokine is extremely effective.
  • the use of chemokine or cytodynamic ins instead of serum is a highly desirable method in terms of safety.
  • the present invention can be used not only for the treatment of cerebral infarction but also for nerve transplantation treatment for other cerebrovascular disorders, cerebral ischemic diseases, and neurodegenerative diseases, and widely used for nerve regeneration treatments. Have potential.
  • the present invention can be directly used for nerve regeneration treatment, and can also be used in the development of therapeutic methods.
  • the neural stem cells obtained by the present invention can be used for cerebral infarction model mice under various conditions. By confirming the therapeutic effect after transplantation into a new patient, further development of therapeutic methods can be expected.
  • a method for transplanting and administering a nerve (stem) cell prepared according to the present invention to a patient as a nerve regeneration therapeutic agent for example, administration is performed by intravenous injection or intravenous infusion.
  • Such an injection is produced according to a conventional method, and generally a physiological saline, a cell culture solution, or the like can be used as a diluent.
  • bactericides, preservatives, stabilizers, tonicity agents, soothing agents and the like may be added.
  • the compounding amount of nerve (stem) cells in these preparations is not particularly limited, and may be determined according to the type of disease, the degree of symptoms, the age of the patient, the body weight, and the like.
  • the nerve (stem) cells of the present invention may be administered to a patient several times, and the dose per administration, the administration interval, etc. may be determined according to the diagnosis result.
  • the administration method may be different from the administration of nerve (stem) cells.
  • nerve (stem) cells can be used as therapeutic agents for nerve regeneration.
  • nerve (stem) cells can be used as therapeutic agents for nerve regeneration.
  • an immunosuppressive substance particularly a substance that suppresses T cell function
  • nerve regeneration can be used as a therapeutic agent for nerve regeneration.
  • substances that suppress the activity of T cells such as FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody can be used as therapeutic agents for nerve regeneration.
  • chemokines such as CI NC-1 and TNF ⁇ , which have differentiation-inducing action from bone marrow cells to neural stem cells, and site force-in, can be administered by administering these differentiation-inducing factors.
  • CI NC-1 and TNF ⁇ which have differentiation-inducing action from bone marrow cells to neural stem cells, and site force-in
  • substances that promote the differentiation-inducing action of these chemokines and cytoforce-ins can also be applied as therapeutic agents for nerve regeneration.
  • Such a substance can be searched using, for example, the screening system of the present invention described later.
  • the nerve regeneration therapeutic agent containing these immunosuppressive substances, chemokines, cytosites, etc. of the present invention as active ingredients may be oral agents, or parenteral agents such as injections, suppositories, and coating agents.
  • Oral preparations such as tablets, capsules, granules, fine granules, powders etc. are produced according to conventional methods using, for example, starch, lactose, sucrose, trehalose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, etc. Is done.
  • the compounding amount of the active ingredient in these preparations is not particularly limited and can be appropriately set. In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrances and the like can be appropriately used.
  • a parenteral agent for example, it is administered by intravenous injection, intravenous infusion, subcutaneous injection, intramuscular injection or the like.
  • This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent.
  • this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and a freeze-dried liquid solution can be re-prepared immediately before use. If necessary, disinfectants, preservatives, A constanting agent, an isotonic agent, a soothing agent and the like may be added.
  • the compounding amount of the active ingredient in these preparations is not particularly limited, as in the case of administering the nerve (stem) cells described above, depending on the type of disease, the degree of symptoms, the age of the patient, the body weight, etc. Just decide.
  • the therapeutic agent for nerve regeneration of the present invention may be administered to a patient several times, and the dose per administration, the administration interval, etc. may be determined according to the diagnosis result.
  • a new cerebral infarction model animal was developed in which the blood vessels of the brain of a maternal mouse of an immunodeficient mouse (SCID mouse) were ligated.
  • SCID mouse immunodeficient mouse
  • This cerebral infarction model animal can be prepared in the same manner as described in Japanese Patent Application No. 2004-108500, except that the maternal mouse is used instead of the SCID mouse.
  • Maternal mice of SCID mice may be SCID mice that are currently commercially available! (See, for example, FOX Chase Cancer Center) or maternal mice of this improved mouse.
  • the blood vessels in the ligation are not particularly limited as long as they are capable of developing cerebral infarction in the brain. Easily blood vessels on the epidermis side are preferred. Examples of preferable blood vessels include middle cerebral artery, internal carotid artery, vertebral basilar artery and the like.
  • the site to be ligated there is no particular limitation on the site to be ligated, but depending on the site of ligation, the selectivity of the ischemic region may deteriorate, so it is also necessary to set a site that can ensure the selectivity.
  • the blood flow in the cortical branch of the middle cerebral artery is selectively disrupted by selecting the ligation site immediately after the middle cerebral artery passes through the olfactory tract, that is, the distal Ml portion. Is possible.
  • the method of ligating the blood vessels of the brain is not particularly limited as long as it is a ligation method capable of developing cerebral infarction.
  • the method can be used. It is also necessary to select a ligation method depending on whether the ischemia is transient or permanent. For example, a permanent ligation method in which cutting is performed after electrocoagulation with coagulation tweezers, or a transient ligation method using an arterial ligation clip.
  • the mouse is anesthetized with halothane or the like, the left rib of the mouse is excised, the skull base is exposed, and a bone window having a diameter of about 1 to 5 mm is formed in the middle cerebral artery running site.
  • a dental drill the dura mater and the arachnoid membrane are removed, and the middle cerebral artery is separated and ligated.
  • the athymic mouse BALB / cAJcHiu
  • the left middle cerebral artery of the maternal mouse were each ligated, thereby achieving good reproducibility.
  • a cerebral infarction model mouse could be prepared (see Example 7 described later).
  • nude mice are immunodeficient mice whose T cell function is suppressed.
  • the bone marrow and subcerebral infarction tissue Cerebral infarction scar site
  • many nestin positive-eurospheres were formed (Fig. 25).
  • the maternal mouse control mouth
  • culturing the cells collected from the bone marrow and tissue force under cerebral infarction most of the nestin-positive eurospheres are formed. I helped.
  • This result also shows that T cells have a suppressive function on the formation of -eurosphere in bone marrow or subcerebral infarcted tissue.
  • a neural stem cell was prepared in the same manner as neural stem cells were prepared from bone marrow cells of SCID mouse maternal mice. It is considered that neural stem cells can be prepared. Furthermore, it is considered that (2) neural stem cells can be prepared from the bone marrow of T cell dysfunctional mice other than SCID mice and nude mice in the same manner as from SCID mice.
  • the present invention provides (1) a method for screening a cerebral infarction model animal of the present invention by administering a test drug and screening the effectiveness of the test drug against cerebral infarction, and (2) the present The present invention provides a method for transplanting nerve (stem) cells or other cells into the cerebral infarction model animal of the invention and screening the effectiveness of the transplantation treatment for cerebral infarction.
  • Examples of the screening method of the present invention include, for example, subjecting a cerebral infarction model animal of the present invention to a subject. Changes in the size and volume of cerebral infarction lesions, morphological examinations by administration of test drugs or transplantation of neural stem cells, etc. by carbon black perfusion method, measurement of brain size, instrumental analysis such as MRI (Ratio of left and right cerebral cortex width, degree of apoptosis by TUNEL staining, number of regenerating nerves and regenerating vascular endothelial cells by BrdU labeling), behavioral test (open field test, startle reflex, maze learning, avoidance learning), etc. Compared with the control group, there is a method for evaluating the degree of inhibition of cerebral infarction lesion, recovery of brain function, and the like.
  • the present invention provides a method for screening a therapeutic agent for nerve regeneration used for the treatment of cerebral ischemic diseases such as cerebral infarction or other neurological diseases, or induces differentiation from bone marrow cells to neural stem cells, or
  • the present invention provides a screening method for searching for candidate substances, using as an index whether or not the substance has an effect of promoting the function.
  • a test substance is administered to a medium of bone marrow cells collected from a human or an animal, and the test substance is transferred from bone marrow cells to neural stem cells.
  • Candidate substances are searched for by examining whether they have the effect of inducing differentiation or promoting their differentiation.
  • the culture conditions are (1) with an immunosuppressant added, (2) with a differentiation-inducing factor (or serum collected after brain injury), (3 Appropriate conditions may be set for various conditions, such as those with both an immunosuppressant and differentiation-inducing factor (or serum after brain injury) added, and (4) those without both.
  • TNFa which is a site force-in
  • a candidate substance useful for nerve regeneration may be searched from a group of substances having an immunosuppressive action, or a site force-in such as chemokine, or these
  • a candidate useful for nerve regeneration may be searched from a group of substances that regulate activity and production.
  • the screening method of the present invention is a screening method using a culture system described in Examples below.
  • a culture system described in Examples below.
  • substances that regulate the activity and production of these molecules can be examined in various existing systems using substances such as CINC-1 and TNFo; identified as molecular weight inducers as target molecules.
  • substances such as CINC-1 and TNFo; identified as molecular weight inducers as target molecules.
  • differentiation from bone marrow cells to neural stem cells can be promoted, and a candidate substance effective for nerve regeneration therapy can be searched.
  • Example 1 Preparation of neural stem cells derived from cerebral infarction SCID mouse bone marrow
  • the mouse left rib was excised under 3% halothane anesthesia to expose the skull base.
  • a bone window with a diameter of 1.5 mm was created with a dental drill in the middle cerebral artery running site.
  • the dura mater and arachnoid membrane were removed, and the middle cerebral artery was separated to prepare for ligation.
  • As the middle cerebral artery ligation method a permanent ligation method in which cutting is performed after electrocoagulation with coagulation tweezers, or a transient ligation method using an arterial ligation clip is possible.
  • the ligation site is the distal Ml portion immediately after the artery passes through the olfactory tract, that is, the distal Ml portion. By ligating this site, it is possible to selectively disrupt the blood flow in the cortical branch of the middle cerebral artery.
  • the cerebral infarction region of the cerebral infarction model mouse (cerebral infarction SCID mouse) prepared by ligating the distal Ml portion of the left middle cerebral artery of the SCID mouse by such a middle cerebral artery ligation method is actually 2, This was examined by 3,5-triphenyltetrazolium (TTC) staining. TTC staining was performed using coronal brain slices prepared by brain slicer after excision of the mouse brain on middle cerebral artery ligation (MCO) 1, 3 and 7 days. The left side of Fig. 7 shows the results of staining of the brain extracted on the first day after ligation (MCO) by this staining method.
  • MCO middle cerebral artery ligation
  • Cerebral infarction SCID mice prepared by the above method were decapitated in a clean bench on the 7th day after ligation, and the bone strength of femur was also collected. Then, pipetting was performed in the basic culture medium (250 ⁇ 1) of DMEM and N-2 until it became a single cell, and 10 ml of the culture medium was added and centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on low cell binding plates in the presence of bFGF (50 ⁇ g / ml) and EGF (20 ⁇ g / ml) for 10-28 days. As a result, as shown in FIG. 1D, from the 7th day onward, a neurosphere-like cell cluster was observed under a cell microscope.
  • bFGF 50 ⁇ g / ml
  • EGF 20 ⁇ g / ml
  • striatal cells collected from C57BZ6 mice at 2 weeks of embryo were cultured on low cell binding plates for 10 days. As shown in Fig. 1A, neurospheres were formed. It was. When this was further cultured on a high binding plate, it was separated after 3 days (Fig. B), and the expressed protein was examined by immunohistochemistry (double-staining indirect fluorescent antibody method). The cells were separated into GFAP-positive glial cells (see Fig. C).
  • Cerebral infarction SCID mouse bone marrow-derived eurosphere-like cell mass formation, after cerebral infarction
  • the bone marrow of cerebral infarction SCID mice (MCOIW) was cultured, and on the 10th day, the neurosphere-like cell mass floating in the culture medium was collected and further cultured on a high binding plate. After 3 to 7 days, the cells separated were fixed with a fixative containing balaformaldehyde and immunostained to examine the expressed protein. As a result, Nestin-positive neural stem cells were observed at a high rate in the center of the cell mass (Fig. 3 left). In addition, MAP2-positive neurons (middle in the figure) and GFAP-positive glial cells (right in the figure) were observed.
  • neural stem cells were produced by culturing bone marrow of cerebral infarction SCID mice.
  • sera from other mice (30 ⁇ 1) was added.
  • cerebral infarction model mice are created by ligating the middle cerebral artery of SCID mice, which are immunodeficient mice, and bone marrow cells are collected and cultured after the onset of cerebral infarction (after ligation).
  • SCID mice which are immunodeficient mice
  • bone marrow cells are collected and cultured after the onset of cerebral infarction (after ligation).
  • Neurosphere could be 'derived' and prepared. It was also clarified that this -eurosphere is divided into nerve cells.
  • cerebral infarction from the bone marrow of sham-operated SCID mice-Eurosphere is a force that is not formed Neurospheroid formation by adding serum of cerebral infarction model mice to this and culturing Therefore, neurosphere formation is thought to require a stimulating factor produced by cerebral infarction.
  • C. B17 mice immunologically normal CB-17 / IcrCrlBR mice
  • a cerebral infarction model was created by the same method and compared with cerebral infarction SCID mice to examine whether neural stem cells were induced from the bone marrow.
  • C The creation of a cerebral infarction model from B17 mice is the same as the above-described method for producing cerebral infarction SCID mice. That is, the mouse left rib was excised under 3% halothane anesthesia and the skull base was exposed. A bone window with a diameter of 1.5 mm was created with a dental drill in the middle cerebral artery running site. The dura mater and arachnoid membrane were removed, and the middle cerebral artery was separated to prepare for ligation.
  • As the middle cerebral artery ligation method a permanent ligation method in which cutting is performed after electrocoagulation with coagulation tweezers, or a transient ligation method using a tip for arterial ligation is possible.
  • the ligation site is immediately after the artery passes through the olfactory tract, that is, the distal Ml portion. By ligating this site, the middle cerebral artery It is possible to selectively disrupt the blood flow in the cortical branch of the.
  • the cerebral infarction region of the cerebral infarction model mouse (cerebral infarction C. B17 mouse) prepared by ligating the distal Ml cation of the left middle cerebral artery of the C. B 17 mouse by such a ligation method of the middle cerebral artery
  • TTC staining was used. TTC staining was performed using coronary brain slices prepared by brain slicer after excision of mouse brains on days 1, 3, and 7 after ligation (MCO). As a result, 4 animals in each group, a total of 12 animals, all made infarcts selectively in the left middle cerebral artery cortical branch region, and the cerebral infarction sites were very uniform.
  • FIG. 7 shows the result of staining of the brain extracted on day 1 after ligation (MCO). In contrast to that of SCID mice (white part is the infarct site).
  • the cerebral cleft force of the infarcted cerebral cortex was also calculated by comparing the width (a) to the infarcted site with the normal side (b) and the ratio (aZb) as the cortical width index (CI value) (Fig. 20).
  • the CI value was constant at 0.34 until the third day and the seventh day in both the cerebral infarction SCID mouse and the cerebral infarction C. B17 mouse.
  • the calorie increased to 0.37 on the 28th day, and recovery 'regeneration was observed, whereas in the C.B17 mice, it was constant at 0.34 even on the 28th day. From these results, cerebral infarction C. B17 mice, unlike cerebral infarction SCID mice, showed no recovery in brain morphology and could not be said to be a brain regeneration model.
  • anti-musashi 1 antibody was used for neural stem cells
  • Doublecortin (DCX) was used to identify immature nerve cells.
  • Oligodendrocyte progenitor cells used the platelet-derived growth factor receptor (PDGFR a) and NG2 as markers, and 04 and Myelin-associated Glycoprotein (MAG) were used for pre- and immature oligodendrocytes.
  • Astrocytes were identified with anti-GFAP antibody.
  • PSA-NCAM was used as a marker for immature neurons and nerves in the process of axon elongation.
  • PSA-NCAM has been confirmed to be expressed in the cell membrane of cultured neural stem cells.
  • N-cadherin is also expressed in -eurosphere.
  • tissue under cerebral infarction is the infarcted tissue (cerebral infarction scar site) and the site around the cerebral infarction where the infarcted tissue and white matter (corpus callosum) contact.
  • This tissue under cerebral infarction is the site where numerous neural stem cells appeared (ie, brain regeneration) in cerebral infarcted SCID mice, as well as subventricular zone tissue (SVZ) (see Japanese Patent Application No. 2004-108500). ).
  • the neural stem cells in the tissue under cerebral infarction were derived from bone marrow.
  • mice were transcardially fixed with PLP fixative solution, the brain was removed, and brain sections were prepared using a vibratome. Histochemistry was performed.
  • the expression of various markers of SVZ was all different from the normal side. On day 7, it grew to Musashil-positive, PSA-NCAM-positive cell strength SVZ. On the 14th day, these neural stem cells appeared to invade the white matter from the enormous part of the ventricle. On day 35, the number of cells expressed by SVZ decreased and reached the control level. Many Musashil-positive and PSA-NCAM-positive cells were observed in the white matter under the ventricle.
  • DCX-positive immature neurons and small NeuN-positive cells were also found. From 7 days after infarction, SVZ also expressed NG2-positive, PDGFR o; -positive oligodendrocyte progenitor cells. These had no mature oligodendrocyte markers and were reduced to force 35, which was seen until day 14.
  • neural stem cells proliferate after the 7th day after the infarction and peak on the 14th day, and then decrease. Neural stem cells are considered to be the force that exists in the white matter under the ventricle until the 35th day. In SVZ, notchl-positive cells were observed on the 7th day after infarction. Since it was not observed thereafter, the SVZ cell division signal was considered to end in about one week after infarction. .
  • neural stem cells were formed by culturing cells collected from tissue force under cerebral infarction. Cerebral infarction 7 days after ligation C. B17 mice and cerebral infarction SCID mouse infarcted tissues are collected, and then pipetted into single cells in basic culture medium (250 1) of DMEM and N-2. 10 ml of the culture solution was added and centrifuged at 600 rpm for 5 minutes. Cells Resuspended in 3 ml of culture and cultured for 29 days on a 1 ow cell binding plate in the presence of bFGF (50 ⁇ g / ml) and EGF (20 ⁇ g / ml). The number of sphere-like cell clusters was counted.
  • Example 3 Bone marrow using an immunosuppressant 'subcerebral infarcted tissue (cerebral infarction scar site) Nervous stem cell preparation method
  • the immunosuppressant FK506 (1. OmgZkg) was pre-administered to B17 mice, that is, intraperitoneally administered daily for 3 days, and then a cerebral infarction due to left middle cerebral artery occlusion was created. After cerebral infarction (after ligation), administration of FK506 (1. OmgZkg) was continued. On the 7th day after cerebral infarction, bone marrow and cerebral infarct scar site were collected, and DMEM and N-2 in a basic culture solution (250 Pipette to 1 cell with ⁇ 1), add 10ml of culture and centrifuge at 600rpm for 5 minutes.
  • a basic culture solution 250 Pipette to 1 cell with ⁇ 1
  • Cells were resuspended in 3 ml of culture medium and cultured on low cell binding plates in the presence of bFGF (50 ⁇ g / ml) and EGF (20 ⁇ g / ml) for 10-28 days. As shown in FIG. 13, after the 7th day of culture, -eurosphere-like cell clusters from the bone marrow ( ⁇ ⁇ ⁇ ) and the cerebral infarction scar site (C) were observed under a cell microscope.
  • bFGF 50 ⁇ g / ml
  • EGF 20 ⁇ g / ml
  • mice showed vigorous formation of -Eurosphere-like cell clusters from cultured bone marrow that had been administered intraperitoneally daily for 10 days with FK506 (1. OmgZkg).
  • CD28 and ICOS inducible costimulatory molecule
  • ICOS inducible costimulatory molecule
  • their blocking antibodies, anti-CD28 and anti-ICOS antibodies are known to suppress T cell function.
  • anti-T cell antibody an antibody that suppresses such T cell function
  • FK5 Whether or not neural stem cells were formed was examined in the same manner as when 06 was added.
  • neural stem cells are formed in the same manner as when FK506 is added, and neural stem cells are formed from bone marrow. For this purpose, it became clear that it is important to suppress T cell function.
  • mice formed from bone marrow and subcerebral infarcted tissue (cerebral infarction scar site)
  • Example 4 Cerebral infarction SCID mouse bone marrow-derived neural stem cell transplantation and engraftment and differentiation into brain tissue.
  • Cerebral infarction The following experiment was conducted to confirm whether neural stem cells derived from bone marrow of SCID mice physiologically enter the cerebral infarction lesion and engraft and dissociate into brain tissue.
  • the bone marrow-derived neural stem cells are transplanted with the bone marrow of a GFP transgenic mouse that expresses green fluorescence protein (GFP), the force involved in nerve regeneration that occurs after cerebral infarction in SCID mice.
  • GFP green fluorescence protein
  • the brain section was reacted with a phosphate buffer solution (diluted 2000 times) containing rat monoclonal anti-GFP antibody and mouse monoclonal anti-PSA-NCAM antibody for 12 hours (first reaction), and then washed.
  • FITC-labeled goat anti-rabbit HgG and piotin-labeled goat anti-mouse IgG (second reaction) and then avidin-labeled Cy3 were reacted.
  • Visualization of GFP and PSA-NCAM was observed with a confocal laser fluorescence microscope.
  • GFP-positive cells were widely observed in subcerebral infarcted tissues, and some of them were PSA-NCAM negative and showed microglia morphology (see Fig. 18B).
  • a group of GFP positive cells widely recognized along the stem road) is considered to be PSA-NCAM positive and neural progenitor cells (see Fig. C).
  • Other PSA-NCAM positive sites were also observed in periventricular tissues. They were GFP
  • the bone marrow-derived neural stem cells appear after the infarction and enter the brain parenchyma, that is, the bone marrow-derived neural stem cells are engrafted in the tissue under the cerebral infarction. It was shown to differentiate into nerves.
  • Cerebral infarction SCID mouse strength The effect of bone marrow-derived neural stem cells obtained on brain regeneration when transplanted into another cerebral infarction SCID mouse and the therapeutic effect on cerebral infarction were examined.
  • the bone marrow strength of cerebral infarction SCID mice was prepared-Eurosphere-like cell clusters were separated into single cells, and 100,000 of them were separated into another cerebral infarction SCID mouse (second day after cerebral infarction). It was administered intravenously. On the 16th day after the cerebral infarction, the mouse was transcardially fixed with a PLP fixative solution, and the brain was removed. In the isolated brain, defects in the middle cerebral artery region were observed in all cases.
  • the cerebral cleft force of the infarct side cerebral cortex is also calculated by comparing the width (a) to the infarct site with the normal side (b) and calculating the ratio (aZb) as the cortical width index (CI value). It was 0.48 in mice treated with bone marrow-derived neural stem cells (BM—NSC) and 0.36 in control mice treated with PBS (FIG. 20). Thus, the brains of mice transplanted with neural stem cells had a larger residual cerebral cortex than the brains of control mice.
  • BM—NSC bone marrow-derived neural stem cells
  • PBS FIG. 20
  • CINC-1 was considered to be a factor that induces differentiation of neural stem cells
  • the following experiment examined the effect of CINC-1 on bone marrow cell differentiation.
  • CINC-1 which is also produced by cerebral ischemia stimulation and is also produced by NK cells, is one of the factors that induce differentiation of bone marrow cells into neural stem cells. This shows that the suppression of function is also important for maintaining its formation.
  • mice with FK506 tacrolimus hydrate: 1.0 mg / kg / day
  • FK506 Cyclosporin A CsA: 10 mg / kg / day
  • FK5O6 1.0 mg / kg
  • CsA 10 mg / kg / day
  • bone marrow and sub-infarcted tissue were collected and DMEM and N— Pipette up to 2 single cultures in the basic culture solution (250 ⁇ 1), add 10 ml of the culture solution, and centrifuge at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on low cell binding plates in the presence of bFGF ⁇ O / z gZ ml) and EGF (20 ⁇ g Zml) for 10-28 days.
  • an immunosuppressive agent that suppresses T cell function causes nerve regeneration in the bone marrow after cerebral infarction, avoiding cell death due to apoptosis, and migrating to the cerebral infarction site. It was suggested that it would become nervous.
  • mice After creating a cerebral infarction due to occlusion of the left middle cerebral artery in a nude mouse (BALB / cAJc ⁇ nu) and its maternal mouse (control), an athymic mouse, bone marrow cells and subinfarcted tissue (cerebral infarct scar site) ) Were collected and cultured to produce neural stem cell clusters (neurospheres). In other words, the mice were decapitated in a clean bench, and bone marrow cells were collected from the femur and cells were collected from the cerebral infarction scar.
  • neural stem cell clusters neural stem cell clusters
  • Floating-Eurosphere-like cell clumps were collected and further cultured on high binding plates. Cells separated after 7 days were fixed in a fixative containing baraformaldehyde, and the number of nestin-positive eurospheres was counted. In nude mice, the number of nestin-positive eurospheres was significantly higher than that of controls. There were many (Figure 25).
  • nerve regeneration occurs in the bone marrow after cerebral infarction, avoids cell death due to apoptosis, migrates and engrafts at the cerebral infarction site, and then separates it into the nerve. It was suggested that
  • T cells have a suppressive function on the formation of eurospheres in the bone marrow or cerebral infarct scar, in other words, to maintain and promote the formation of neurospheres. It was shown that it is important to suppress T cell function.
  • immunosuppressants such as FK506 and CsA in vivo
  • SCID mice and nude mice indicate that immunosuppression is important for the generation of neural stem cells.
  • the fact that immunosuppression is linked to the therapeutic effect of cerebral infarction in vivo is a clear indication that immunosuppressive substances are effective for nerve regeneration treatment such as cerebral infarction treatment.
  • CINC-1 found as one of the differentiation-inducing factors in Example 6 is a site force-in classified as a chemokine, but a molecule that acts as a differentiation-inducing factor also exists in other site force-in.
  • TNF o is also one of the factors that induce differentiation from bone marrow cells to neural stem cells.
  • This screening method using a neural stem cell culture system has been shown to be effective for screening a substance for inducing neural stem cells and for developing a nerve regeneration therapeutic agent using the substance.
  • the present invention provides a method for preparing neural stem cells in a short time in a short period of time for bone marrow, etc., and rapid recovery of brain function after the onset of cerebral infarction by cell transplantation, cerebral infarction It has wide applicability in the field of neuroregenerative medicine, such as neuroregenerative treatment for other cerebrovascular disorders and neurodegenerative diseases, or the development of treatments therefor, and is useful in various medical-related industries engaged in regenerative medicine.

Abstract

(1) A method for the preparation of neural stem cells comprising culturing marrow cells in the presence of an immunosuppressing agent or serum collected from a human or animal suffering from cerebral infarction (or, in place of the serum, a cytokine such as a chemokine); (2) a method for the preparation of neural stem cells comprising culturing marrow cells collected from a human or animal suffering from cerebral infarction in the presence of an immunosuppressing agent; and the like. These methods are useful for the nerve regenerative therapy or the development of such a therapy.

Description

明 細 書  Specification
神経幹細胞の調製法  Neural stem cell preparation method
技術分野  Technical field
[0001] 本発明は、神経幹細胞の調製法に関し、より詳細には、骨髄から神経幹細胞を誘 導'調製する方法と、神経再生治療への応用に関するものである。  [0001] The present invention relates to a method for preparing neural stem cells, and more particularly, to a method for inducing and preparing neural stem cells from bone marrow and application to nerve regeneration therapy.
背景技術  Background art
[0002] 脳梗塞などの脳血管障害や神経変性疾患は、超高齢社会を迎えつつあるわが国 にとつて解決すべき最重要課題のひとつである。しかし、これまでの神経細胞死抑制 などに焦点をあてた治療法開発の努力にもかかわらず、有効な治療法の開発に結 びつく成果は未だ十分に得られて 、な 、。  [0002] Cerebrovascular disorders such as cerebral infarction and neurodegenerative diseases are one of the most important issues to be solved for Japan, which is facing a super-aging society. However, despite the efforts to develop therapeutic therapies that have focused on the suppression of neuronal cell death, the results that have led to the development of effective therapies have still been obtained sufficiently.
[0003] 神経再生療法は、既に障害をうけている脳機能を再生神経により改善できる可能 性があり、新たな神経疾患治療法として期待されている。既に米国では、パーキンソ ン病などの神経変性疾患において胎児脳より採取された神経幹細胞移植による神経 再生治療が行われている。しかし、この治療法には倫理上の問題など解決すべき課 題が少なくない。  [0003] Nerve regeneration therapy has the potential to improve brain functions that have already been damaged by regenerating nerves, and is expected as a new treatment method for neurological diseases. Already in the United States, nerve regeneration treatment has been carried out by transplanting neural stem cells collected from fetal brain in neurodegenerative diseases such as Parkinson's disease. However, there are many issues that need to be resolved with this treatment, including ethical issues.
[0004] 脳梗塞治療を目的とした ES細胞由来の神経幹細胞移植も行われて 、るが、 ES細 胞の機能解析が不十分で、かつその入手が倫理的にも制度的にも現状困難という問 題がある。また、 ES細胞の神経への分化誘導法は未だ確立しておらず、生体内で生 着'機能するかどうかも不確かである。  [0004] ES cell-derived neural stem cell transplantation for the treatment of cerebral infarction has also been performed. However, functional analysis of ES cells is inadequate, and it is currently difficult to obtain ethically and institutionally There is a problem. In addition, the method of inducing differentiation of ES cells into nerves has not been established yet, and it is uncertain whether it will function in vivo.
[0005] 神経再生医療への利用が期待される神経幹細胞は、上述のように胎児脳や ES細 胞に由来するもののほか、成熟脳由来の脳室下帯組織（subventricular zone : SVZ) からも採取可能である。胎児脳由来の神経幹細胞は、神経細胞に分化する。一方、 成熟脳 SVZ由来の神経幹細胞は、グリア細胞に高率に分ィ匕し、通常の培養条件で は神経細胞に分化しな ヽ。成熟脳 SVZ由来の神経幹細胞を神経細胞に分化させる ためには栄養因子の添加や、分化誘導因子 (Notchlなど）の遺伝子導入が必要で ある。  [0005] In addition to those derived from fetal brain and ES cells as described above, neural stem cells that are expected to be used in nerve regeneration medicine are also derived from the subventricular zone (SVZ) derived from the mature brain. Can be collected. Neural stem cells derived from fetal brain differentiate into neurons. On the other hand, neural stem cells derived from mature brain SVZ are highly differentiated into glial cells and do not differentiate into neurons under normal culture conditions. To differentiate neural stem cells derived from mature brain SVZ into neurons, it is necessary to add trophic factors and introduce genes for differentiation-inducing factors (such as Notchl).
[0006] 神経再生医療において、このような培養神経幹細胞を移植する方法としては、直接 細胞を脳内に注入'移植する方法が主に施行されている。しかし、この方法は手技が 煩雑であり、移植法は未だ確立されたものとはいえない。一方、このように直接脳内 に移植する方法以外に、脳梗塞急性期に骨髄単核球細胞を静脈内に投与する治療 法が試みられている。もっとも、この治療法は神経幹細胞を投与するものではなぐ未 分ィ匕の骨髄細胞を静脈内投与するものであるので、神経再生の効果がどの細胞によ つて生じているのかなどメカニズムが不明で効果の予測 '評価が困難である。 [0006] In nerve regeneration medicine, as a method of transplanting such cultured neural stem cells, direct The method of injecting and transplanting cells into the brain is mainly performed. However, this method is cumbersome and the transplantation method is not yet established. On the other hand, in addition to the method of transplanting directly into the brain as described above, a treatment method in which bone marrow mononuclear cells are administered intravenously in the acute phase of cerebral infarction has been attempted. However, since this treatment method involves intravenous administration of untreated bone marrow cells that do not administer neural stem cells, the mechanism such as which cells cause the nerve regeneration effect is unknown. Predicting effect 'Evaluation is difficult.
[0007] 骨髄間質細胞に遺伝子導入することによって、あるいは数種類の成長因子を添カロ しながら骨髄間質細胞を培養することによって、骨髄細胞から神経細胞を作り出すこ とができたとの報告がある。また最近になって、ヒトゃマウスの骨髄細胞から神経幹細 胞である-ユーロスフェア（neurosphere)が得られたとの報告がある（下記の非特許文 献 1 · 2参照)。しかし、これらの方法は、（1) -ユーロスフエアおよび分ィ匕可能な神経 幹細胞を作成するまでの培養期間が 2ヶ月から 6ヶ月程度かかり、長期間を要する、 ( 2)最終的に神経細胞あるいはグリア細胞に分ィ匕させるのに BDNFや PDGFなどの 栄養因子による刺激が必要である、などの難点がある。 [0007] It has been reported that nerve cells could be created from bone marrow cells by introducing genes into bone marrow stromal cells or by culturing bone marrow stromal cells with several types of growth factors. . Recently, it has been reported that neurospheres, which are neural stem cells, were obtained from bone marrow cells of human mice (see Non-Patent Documents 1 and 2 below). However, in these methods, (1)-the culture period until preparation of neurostem cells capable of eurosphering and separation takes about 2 to 6 months, and it takes a long period of time. In order to disperse glial cells, it is necessary to stimulate with nutrient factors such as BDNF and PDGF.
[0008] また、本発明者らは、ヒト臍帯血由来の幹細胞 (CD34陽性細胞)を脳梗塞モデル マウスに静脈内投与することで、脳内での血管再生、さらに神経再生が起こることを 見出し、これを報告している（下記の非特許文献 3参照)。  [0008] Further, the present inventors have found that human umbilical cord blood-derived stem cells (CD34 positive cells) are intravenously administered to cerebral infarction model mice to cause vascular regeneration and further nerve regeneration in the brain. This has been reported (see Non-Patent Document 3 below).
[0009] 非特許文献 1： Hermann A, Gastl R, Liebau S, Oana Popa M, Fiedler J, Boehm BO, Maisel M, Lerche H, Schwarz J, Brenner R, Storch A (2004) J Cell Sci 117:4411—44 22.  [0009] Non-Patent Document 1: Hermann A, Gastl R, Liebau S, Oana Popa M, Fiedler J, Boehm BO, Maisel M, Lerche H, Schwarz J, Brenner R, Storch A (2004) J Cell Sci 117: 4411 —44 22.
非特許文献 2 : Kabos P, Ehtesham M, Kabosova A, Black KL, Yu JS (2002) Generati on of neural progenitor cells from whole adult bone marrow. Exp Neurol 178:288—29 3.  Non-Patent Document 2: Kabos P, Ehtesham M, Kabosova A, Black KL, Yu JS (2002) Generati on of neural progenitor cells from whole adult bone marrow. Exp Neurol 178: 288—29 3.
非特許文献 3 : Taguchi, A., Soma, T., Tanaka, H., Kanda, T., Nisnimura, H., Yoshik awa, H., Tsukamoto, Y., Iso, H., Fujimori, Y., Stern, D.M., Naritomi, H., Matsuyam a, T. (2004) Administration of CD34+ cells post-stroke enhances angiogenesis and neurogenesis in a murine model. J. Clin. Invest., 114:330—338.  Non-Patent Document 3: Taguchi, A., Soma, T., Tanaka, H., Kanda, T., Nisnimura, H., Yoshik awa, H., Tsukamoto, Y., Iso, H., Fujimori, Y. , Stern, DM, Naritomi, H., Matsuyam a, T. (2004) Administration of CD34 + cells post-stroke enhances angiogenesis and neurogenesis in a murine model. J. Clin. Invest., 114: 330—338.
発明の開示 発明が解決しょうとする課題 Disclosure of the invention Problems to be solved by the invention
[0010] 脳梗塞などの脳血管障害やその後遺症、パーキンソン病やアルツハイマー病など の神経変性疾患に対しては、神経機能を回復させる有効な治療法がない。神経機 能の再生をめざした治療法確立のためには再生する神経細胞が提供されなければ ならない。神経再生治療への応用が期待される神経幹細胞は、上述したように現在 まで、胎児脳由来の神経幹細胞、成体脳由来の神経幹細胞、 ES細胞由来の神経 幹細胞、骨髄成体多能性幹細胞（multipotent adult progenitor cell : MAPC)由来の 神経幹細胞などが報告されている。しかし、その供給源や神経細胞への分化効率、 さらに移植脳への生着性、機能発揮性など問題点が山積している。また、骨髄細胞 に遺伝子を導入するなど、神経細胞を人為的に作成する手法なども考案されている 力 発がん性や生着、機能性などに問題がある。  [0010] There is no effective treatment for restoring neurological function for cerebrovascular disorders such as cerebral infarction, sequelae, neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Regenerative neurons must be provided to establish treatments aimed at regenerating nerve functions. As described above, neural stem cells that are expected to be applied to nerve regeneration treatment have been developed from embryonic brain-derived neural stem cells, adult brain-derived neural stem cells, ES cell-derived neural stem cells, and bone marrow adult pluripotent stem cells (multipotent Adult stem cells derived from adult progenitor cells (MAPC) have been reported. However, there are many problems such as the supply source, efficiency of differentiation into neurons, engraftment into the transplanted brain, and function performance. In addition, methods for artificially creating nerve cells, such as introducing genes into bone marrow cells, have been devised. There are problems with such as carcinogenicity, engraftment, and functionality.
[0011] 理想的には、まず倫理的にも、また移植片対宿主病（graft- versus- host disease : G VHD)回避のためにも、神経再生治療に使用される神経幹細胞は、患者本人の細 胞であること、つまり自家移植 (Autograft)であることが望ましい。また、神経細胞に高 率に分化し（Neurogenesis)、神経機能を保ち（Functional)、かつ必要な時期に（Tim ely)、簡単に（eaSY)、大量に (Amplify)、作成される幹細胞であること、即ち、これら の特徴をあわせもった 、わば「FANTASY」であることが望まし!/、。  [0011] Ideally, the neural stem cells used for nerve regeneration therapy are ethically and for the avoidance of graft-versus-host disease (G VHD). It is desirable that the cell be an autograft. In addition, it is a stem cell that is highly differentiated into neurons (Neurogenesis), maintains nerve function (Functional), and is easily (eaSY), easily (Amplify), and abundant (Amplify) In other words, it is desirable to have “FANTASY” that combines these characteristics!
[0012] ところで、生体には組織あるいは臓器障害時に際して様々な修復機能が備わって いる。これらは主に消化管上皮や肝臓、血管内皮細胞など組織固有の細胞の分裂 や増殖を介して行われているものである力 最近種々の骨髄由来の幹細胞がこれに 関与することが知られてきた。し力しながら、ほとんどの病態において骨髄が組織修 復にどの程度関与するかの検討はなされていないのが実情である。神経幹細胞移植 による神経再生治療法の開発に際して、このような生体内で通常起こっているホメォ スターシス維持機構の一環としての修復機構のメカニズムを研究した上で、その機構 に添った治療法を考案することが有効 '適切と考えられる。  [0012] By the way, a living body has various repair functions when a tissue or organ is damaged. These are mainly performed through the division and proliferation of tissue-specific cells such as the gastrointestinal epithelium, liver, and vascular endothelial cells. Recently, it has been known that various bone marrow-derived stem cells are involved in this. It was. However, in reality, the degree to which bone marrow is involved in tissue repair in most pathological conditions has not been studied. When developing a nerve regeneration therapy by neural stem cell transplantation, we will study the mechanism of the repair mechanism as part of the homeostasis maintenance mechanism that normally occurs in vivo, and then devise a therapy according to that mechanism. It is considered effective.
[0013] 本発明は、上記の問題点に鑑みなされたものであって、その目的は、脳梗塞などの 脳血管障害や神経変性疾患に対する新たな神経再生治療法を開発'確立すること、 そのため、神経再生治療又はその治療法開発において好適に用いられ、簡便かつ 短期間に調製可能であり、し力もその移植により良好に神経細胞を脳患部に生着、 機能させることが期待できる神経幹細胞の調製法等を提供することにある。 [0013] The present invention has been made in view of the above-mentioned problems, and its purpose is to develop and establish a new nerve regeneration treatment method for cerebrovascular disorders such as cerebral infarction and neurodegenerative diseases. , Used in nerve regeneration treatment or the development of treatment methods thereof, It is intended to provide a method for preparing neural stem cells that can be prepared in a short period of time and can be expected to successfully engraft and function nerve cells in the affected brain area by transplantation.
課題を解決するための手段  Means for solving the problem
[0014] 本発明者らは、上記の課題に鑑み鋭意研究を進めた結果、まず免疫不全の SCID マウスを処置して脳梗塞モデルマウスを作出し、脳梗塞 1週間後の当該マウスより採 取した骨髄細胞を培養することで、骨髄細胞から神経幹細胞 (ニューロスフェア）に分 化誘導できることを明らかにした。さらに、後述するように再生と免疫に関する重要な 知見が得られ、骨髄から短期間（2週間程度）に神経幹細胞を誘導'調製する技術を 確立した。この方法によって、実際にヒトの正常骨髄から神経幹細胞を誘導'調製す ることにも成功した。この方法により、患者骨髄から神経幹細胞を調製し、これを自家 移植として静脈内投与することによって、再生神経を良好に脳患部に生着、機能させ る神経再生治療法が実現可能であること等を見出し、本発明を完成させるに至った。  [0014] As a result of diligent research in view of the above problems, the present inventors first treated an immunodeficient SCID mouse to create a cerebral infarction model mouse, and collected it from the mouse one week after the cerebral infarction. It has been clarified that the bone marrow cells can be induced to differentiate into neural stem cells (neurospheres) by culturing these bone marrow cells. In addition, as described later, important knowledge about regeneration and immunity was obtained, and a technique for inducing and preparing neural stem cells from bone marrow in a short period (about 2 weeks) was established. By this method, neural stem cells were actually derived from human normal bone marrow. By preparing neural stem cells from patient bone marrow by this method and administering them intravenously as autologous transplants, it is possible to realize a nerve regeneration therapy that allows the regenerative nerves to engraft and function well in the affected area of the brain, etc. As a result, the present invention has been completed.
[0015] 即ち、本発明は、産業上および医学'医療上有用な発明として、下記（1)〜(48)の 発明を包含するものである。  That is, the present invention includes the following inventions (1) to (48) as industrially and medically useful medical inventions.
(1)免疫抑制剤、および、脳梗塞その他脳障害後のヒト又は動物から採取した血清 を添加して骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。 ここで、脳障害とは、脳の血管の狭窄、閉塞、結紮又はその他の原因で脳の一部 が虚血状態になっていること、すなわち脳虚血障害を意味する。  (1) A method for preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressive agent and serum collected from humans or animals after cerebral infarction or other brain injury. Here, the brain disorder means that a part of the brain is in an ischemic state due to stenosis, occlusion, ligation or other causes of blood vessels in the brain, that is, cerebral ischemic disorder.
(2)脳梗塞その他脳障害後のヒト又は動物から採取した骨髄細胞を、免疫抑制剤を 添加して培養することにより分化誘導し、神経幹細胞を調製する方法。  (2) A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells collected from humans or animals after cerebral infarction or other brain injury by adding an immunosuppressant.
(3)免疫抑制剤、および、ケモカイン (もしくはその他のサイト力イン)を添加して骨髄 細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。  (3) A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressant and chemokine (or other cytodynamic in).
(4)ケモカインとして、インターロイキン 8 (IL 8)ファミリーのケモカインを添カ卩する 、上記（3)記載の方法。  (4) The method according to (3) above, wherein an interleukin 8 (IL 8) family chemokine is added as a chemokine.
(5)サイト力インとして、 TNF a (tumor necrosis factor- a：腫瘍壊死因子）を添カロす る、上記（3)記載の方法。  (5) The method according to (3) above, wherein TNF a (tumor necrosis factor-a) is added as site force in.
(6)ケモカイン (もしくはその他のサイト力イン)であり、骨髄細胞から神経幹細胞への 分化を直接的又は間接的に誘導する分化誘導因子。 (7)上記 (6)記載の分ィ匕誘導因子を用いて、培養細胞から神経幹細胞を調製する方 法。 (6) A differentiation-inducing factor that is a chemokine (or other site force-in) and directly or indirectly induces differentiation from bone marrow cells to neural stem cells. (7) A method of preparing neural stem cells from cultured cells using the differentiation inducer described in (6) above.
(8)上記 (6)記載の分化誘導因子と免疫抑制剤とからなる神経幹細胞分化誘導剤。 (8) A neural stem cell differentiation inducer comprising the differentiation inducer according to (6) above and an immunosuppressant.
(9)神経幹細胞として、ニューロスフェア（neurosphere)または-ユーロスフェア様細 胞塊を調製する、上記（1)〜（5)又は（7)の 、ずれかに記載の方法。 (9) The method according to any one of (1) to (5) or (7) above, wherein a neurosphere or a neurosphere-like cell mass is prepared as a neural stem cell.
(10)上記（1)〜（5)又は（7)の 、ずれかに記載の方法により神経幹細胞を生産する 方法。  (10) A method for producing neural stem cells by the method according to any one of (1) to (5) or (7) above.
( 11 )上記（ 10)記載の神経幹細胞をさらに培養することにより分化誘導し、神経細胞 を生産する方法。  (11) A method for producing a neural cell by further inducing differentiation by further culturing the neural stem cell according to (10).
( 12)上記（10)記載の神経幹細胞もしくは上記（11)記載の神経細胞を、脳梗塞治 療または他の神経再生治療に使用する方法。  (12) A method of using the neural stem cell according to (10) or the neural cell according to (11) for cerebral infarction treatment or other nerve regeneration treatment.
(13)上記（10)記載の神経幹細胞もしくは上記（11)記載の神経細胞を、静脈内投 与などの方法により投与する、脳梗塞治療法または他の神経再生治療法。  (13) A cerebral infarction treatment method or other nerve regeneration treatment method, wherein the neural stem cell according to (10) or the nerve cell according to (11) is administered by a method such as intravenous administration.
(14)上記（1)記載の方法にお!、て、神経幹細胞の調製に用いる骨髄細胞および血 清には、治療対象の患者カゝら採取したものを使用する方法。  (14) The method described in (1) above, wherein bone marrow cells and blood used for preparation of neural stem cells are collected from the patient to be treated.
(15)上記（2)〜（5)又は（7)の 、ずれかに記載の方法にお!、て、神経幹細胞の調 製に用いる骨髄細胞には、治療対象の患者力も採取したものを使用する方法。 (15) In the method described in any one of (2) to (5) or (7) above, bone marrow cells used for preparation of neural stem cells should be collected from the patient power to be treated. How to use.
(16)上記（1)〜（5)又は（7)の 、ずれかに記載の方法にお!、て、神経幹細胞の調 製に用いる免疫抑制剤に、 FK506 (タクロリムス）、シクロスポリン、抗 CD28抗体およ び抗 ICOS抗体などの T細胞機能を抑制する免疫抑制剤を使用する方法。 (16) In the method described in any one of (1) to (5) or (7) above, FK506 (tacrolimus), cyclosporine, anti-CD28 can be used as an immunosuppressant used for preparation of neural stem cells. Use of immunosuppressive agents that suppress T cell functions such as antibodies and anti-ICOS antibodies.
ここで、抗 CD28抗体および抗 ICOS抗体とは、それぞれ CD28、 ICOSの作用を 阻害し、 T細胞機能を抑制するブロッキング抗体を意味し、 T細胞を刺激し活性ィ匕す るものではない。  Here, the anti-CD28 antibody and the anti-ICOS antibody mean blocking antibodies that inhibit the action of CD28 and ICOS, respectively, and suppress the function of T cells, and do not stimulate and activate T cells.
(17)上記（13)記載の神経再生治療法にお!、て、神経 (幹)細胞の投与と同時また は異時に、免疫抑制剤を投与する方法。  (17) A method of administering an immunosuppressive agent to the nerve regeneration treatment method according to (13) above, at the same time or at the same time as the administration of nerve (stem) cells.
(18)上記（13)記載の神経再生治療法にお!、て、神経 (幹)細胞の投与と同時また は異時に、ヒト臍帯血由来 CD34陽性細胞、血管内皮前駆細胞 (EPC)などの血管 形成能を有する細胞を投与する方法。 ( 19)上記（10)記載の神経幹細胞もしくは上記（11)記載の神経細胞を含む脳梗塞 治療剤。 (18) In the nerve regeneration treatment method according to (13) above, human umbilical cord blood-derived CD34-positive cells, vascular endothelial progenitor cells (EPC), etc. A method of administering a cell having an angiogenic ability. (19) A therapeutic agent for cerebral infarction comprising the neural stem cell according to (10) above or the neural cell according to (11) above.
(20)上記（10)記載の神経幹細胞もしくは上記（11)記載の神経細胞を含む神経再 生治療剤。  (20) A therapeutic agent for nerve regeneration comprising the neural stem cell according to (10) above or the nerve cell according to (11) above.
(21)免疫不全マウス（SCIDマウスおよびヌードマウスを含む）、その母系マウス、又 はこれらのマウスカゝら作成した脳梗塞モデルマウスの骨髄細胞を培養することにより 分化誘導し、神経幹細胞を調製する方法。  (21) Inducing differentiation by culturing bone marrow cells of immunodeficient mice (including SCID mice and nude mice), their maternal mice, or cerebral infarction model mice prepared by these mice, and preparing neural stem cells Method.
(22)免疫不全マウスの骨髄細胞を、脳梗塞モデルマウス力 採取した血清、又はケ モカイン (もしくはその他のサイト力イン)を添加して培養する、上記（21)記載の方法  (22) The method according to (21) above, wherein the bone marrow cells of immunodeficient mice are cultured with the addition of serum collected from cerebral infarction model mouse force or chemokine (or other site force in).
(23)免疫不全マウスの母系マウスの骨髄細胞を、脳梗塞モデルマウス力 採取した 血清 (又は、ケモカインもしくはその他のサイト力イン）と免疫抑制剤とを添加して培養 する、上記（21)記載の方法。 (23) The bone marrow cells of a maternal mouse of an immunodeficient mouse are cultured by adding serum (or chemokine or other site force in) collected from a cerebral infarction model mouse and an immunosuppressive agent, the method of.
(24)免疫不全マウスの母系マウス力も作成した脳梗塞モデルマウスの骨髄細胞を、 免疫抑制剤を添加して培養する、上記（21)記載の方法。  (24) The method according to (21) above, wherein bone marrow cells of a cerebral infarction model mouse, in which a maternal mouse force of an immunodeficient mouse is also prepared, is cultured by adding an immunosuppressive agent.
(25)免疫不全マウスカゝら作成した脳梗塞モデルマウスの骨髄細胞を培養することに より神経幹細胞を調製する、上記（21)記載の方法。  (25) The method according to (21) above, wherein neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared by an immunodeficient mouse.
(26)免疫不全マウスの母系マウスに免疫抑制剤を投与して免疫不全状態にした後 作成した脳梗塞モデルマウスの骨髄細胞を培養することにより神経幹細胞を調製す る、上記（21)記載の方法。  (26) Neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared after administration of an immunosuppressive agent to a maternal mouse of an immunodeficient mouse to make it immunocompromised. Method.
(27)免疫不全マウスが、 C.B- 17/IcrCrlBRマウスを母系とする SCIDマウスである、 上記（21)〜（26)の!、ずれかに記載の方法。  (27) The method according to any one of (21) to (26) above, wherein the immunodeficient mouse is a SCID mouse whose parent is a C.B-17 / IcrCrlBR mouse.
(28)免疫不全マウスが、 BALB/cAJclマウスを母系とするヌードマウスである、上記（ 21)〜（26)の!、ずれかに記載の方法。  (28) The method according to any one of (21) to (26) above, wherein the immunodeficient mouse is a nude mouse whose mother line is a BALB / cAJcl mouse.
(29)上記（21)〜（28)の 、ずれかに記載の方法により調製された神経幹細胞。 (29) A neural stem cell prepared by the method according to any one of (21) to (28) above.
(30) SCIDマウスの母系マウスの脳の血管を結紮してなる脳梗塞モデル動物。(30) A cerebral infarction model animal obtained by ligating brain blood vessels of a SCID mouse maternal mouse.
(31) SCIDマウスの母系マウスの脳の血管が、中大脳動脈である上記（30)記載の 脳梗塞モデル動物。 (32) SCIDマウスの母系マウスが、 C.B- 17/IcrCrlBRマウスである上記（30)又は（31(31) The cerebral infarction model animal according to the above (30), wherein the blood vessel of the brain of the SCID mouse maternal mouse is a middle cerebral artery. (32) The above (30) or (31), wherein the maternal mouse of the SCID mouse is a CB-17 / IcrCrlBR mouse
)記載の脳梗塞モデル動物。 ) The cerebral infarction model animal of description.
(33)ヌードマウス又はその母系マウスの脳の血管を結紮してなる脳梗塞モデル動物  (33) Cerebral infarction model animal formed by ligating the blood vessels of the brain of a nude mouse or its mother mouse
(34)ヌードマウス又はその母系マウスの脳の血管力 中大脳動脈である上記（33) 記載の脳梗塞モデル動物。 (34) The cerebral infarction model animal according to the above (33), which is a cerebral vascular force of a nude mouse or a mother mouse thereof.
(35)ヌードマウスの母系マウス力 BALB/cAJclマウスである上記（33)又は（34)記 載の脳梗塞モデル動物。  (35) Maternal mouse strength of nude mice The cerebral infarction model animal according to (33) or (34) above, which is a BALB / cAJcl mouse.
(36)上記（30)〜（35)の 、ずれかに記載の脳梗塞モデル動物に、被検薬物を投与 して、当該被検薬物の脳梗塞に対する有効性をスクリーニングする方法。  (36) A method for screening the effectiveness of a test drug against cerebral infarction by administering a test drug to the cerebral infarction model animal described in any one of (30) to (35) above.
(37)上記（30)〜（35)の 、ずれかに記載の脳梗塞モデル動物に、神経 (幹)細胞 又は他の細胞を移植して、当該移植治療の脳梗塞に対する有効性をスクリーニング する方法。  (37) Nerve (stem) cells or other cells are transplanted into the cerebral infarction model animal described in any one of (30) to (35) above, and the effectiveness of the transplantation treatment for cerebral infarction is screened. Method.
(38)免疫抑制作用を有する物質を有効成分とする神経再生治療剤。  (38) A nerve regeneration therapeutic agent comprising as an active ingredient a substance having an immunosuppressive action.
(39)免疫抑制作用を有する物質が、 T細胞機能を抑制する物質である、上記 (38) 記載の神経再生治療剤。  (39) The therapeutic agent for nerve regeneration according to the above (38), wherein the substance having an immunosuppressive action is a substance that suppresses T cell function.
(40)免疫抑制作用を有する物質が、 FK506 (タクロリムス）、シクロスポリン、抗 CD2 8抗体および抗 ICOS抗体の 、ずれかである、上記（39)記載の神経再生治療剤。 (40) The nerve regeneration therapeutic agent according to (39) above, wherein the substance having an immunosuppressive effect is any of FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody.
(41)ケモカインその他のサイト力イン、又はこれらの骨髄細胞力 神経幹細胞への 分化を誘導する作用を促進する物質を有効成分とする神経再生治療剤。 (41) A therapeutic agent for nerve regeneration comprising, as an active ingredient, a chemokine or other site force-in or a substance that promotes the action of inducing differentiation into a bone marrow cell force neural stem cell.
(42)ケモカインカ、インターロイキン 8 (IL— 8)ファミリーのケモカインである、上記 (41)記載の神経再生治療剤。  (42) The nerve regeneration therapeutic agent according to the above (41), which is a chemokine of the chemokine, interleukin 8 (IL-8) family.
(43)サイト力インカ、 TNF a (tumor necrosis factor- a：腫瘍壊死因子）である、上 記 (41)記載の神経再生治療剤。  (43) The nerve regeneration therapeutic agent according to the above (41), which is a site force inca, TNF a (tumor necrosis factor-a).
(44)脳梗塞又は他の神経疾患の治療に用いられる、上記（38)〜 (43)のいずれか に記載の神経再生治療剤。  (44) The therapeutic agent for nerve regeneration according to any one of the above (38) to (43), which is used for treatment of cerebral infarction or other neurological diseases.
(45)脳梗塞又は他の神経疾患の治療に用いられる神経再生治療剤のスクリーニン グ方法であって、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進 する作用を有するかどうかを指標に、候補物質を探索することを特徴とする神経再生 治療剤のスクリーニング方法。 (45) A screening method for a nerve regeneration therapeutic agent used for the treatment of cerebral infarction or other neurological diseases, which induces or promotes differentiation from bone marrow cells to neural stem cells A screening method for a therapeutic agent for nerve regeneration, wherein a candidate substance is searched using as an index whether or not it has an action to act.
(46)ヒト又は動物から採取した骨髄細胞の培地に被検物質を投与し、当該被検物 質が、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進する作用を 有するかどうかを調べることを特徴とする上記 (45)記載の神経再生治療剤のスクリー ユング方法。  (46) Whether a test substance is administered to a bone marrow cell culture medium collected from humans or animals, and the test substance induces differentiation from bone marrow cells to neural stem cells or has an action of promoting the differentiation The screening method for a therapeutic agent for nerve regeneration according to the above (45), characterized by examining whether or not.
(47)免疫抑制作用を有する物質群の中から、候補物質を探索することを特徴とする 上記 (45)又は (46)記載の神経再生治療剤のスクリーニング方法。  (47) The screening method for a therapeutic agent for nerve regeneration according to the above (45) or (46), wherein a candidate substance is searched from a group of substances having an immunosuppressive action.
(48)ケモカインその他のサイト力イン、又はこれらの活性もしくは産生を調節する作 用を有する物質群の中から、候補物質を探索することを特徴とする上記 (45)又は (4 6)記載の神経再生治療剤のスクリーニング方法。  (48) The above-mentioned (45) or (46), characterized in that a candidate substance is searched from a group of substances having chemokine or other cytodynamic ins, or an activity that regulates these activities or production. A screening method for therapeutic agents for nerve regeneration.
発明の効果  The invention's effect
[0016] 本発明の神経幹細胞調製法は、簡易かつ短期間に、骨髄細胞から神経幹細胞を 調製することができる。得られた神経幹細胞は、神経細胞に高効率に分ィ匕可能であ り、生体への移植により脳患部において良好に神経細胞を生着、機能発揮させること が期待できるので、神経再生治療、あるいは脳梗塞モデルマウス等を用いた治療法 開発の目的に使用できる。また、材料となる骨髄細胞は生体からの採取が比較的容 易であるので、例えば脳梗塞後の患者力 骨髄を採取し、免疫抑制剤の存在下に培 養を行って神経幹細胞を調製し、これを静脈内投与などで患者に移植する自家移植 治療に本発明を利用することができる。  [0016] The neural stem cell preparation method of the present invention can prepare neural stem cells from bone marrow cells in a simple and short period of time. The obtained neural stem cells can be efficiently separated into the nerve cells, and it can be expected that the nerve cells will be engrafted and function well in the affected part of the brain by transplanting to the living body. Alternatively, it can be used for the purpose of developing treatments using cerebral infarction model mice and the like. In addition, since bone marrow cells used as a material are relatively easy to collect from a living body, for example, patient-powered bone marrow after cerebral infarction is collected and cultured in the presence of an immunosuppressant to prepare neural stem cells. The present invention can be used for autologous transplantation treatment in which this is transplanted into a patient by intravenous administration or the like.
図面の簡単な説明  Brief Description of Drawings
[0017] [図 1]胎生期マウス脳由来のニューロスフ アおよび神経細胞等 (A, B, C)と、脳梗 塞 SCIDマウス骨髄由来の-ユーロスフェア様細胞塊および神経細胞等 (D, E, F) を比較して示す図面に代わる写真である。原図の C 'Fでは、 MAP2陽性の神経細 胞は赤で、 GFAP陽性のグリア細胞は緑で表示される。  [0017] [Fig. 1] Neurospheres and neurons etc. derived from embryonic mouse brain (A, B, C) and-Eurosphere-like cell clusters and neurons derived from cerebral infarction SCID mouse bone marrow (D, E , F) is a photograph replacing the drawing. In the original C'F, MAP2-positive neurons are displayed in red, and GFAP-positive glial cells are displayed in green.
[図 2]脳梗塞 SCIDマウス骨髄由来の-ユーロスフェア様細胞塊形成に関して、脳梗 塞後骨髄を採取するまでの期間や骨髄細胞の培養期間が、細胞塊形成にどのよう に影響するか等を検討した結果を示すグラフである。 [図 3]骨髄由来のニューロスフ ア様細胞塊から神経細胞、グリア細胞への分ィ匕を免 疫染色により検討した結果を示す図面に代わる写真である。 [Fig.2] Cerebral infarction Regarding the formation of Spheroid cell mass derived from SCID mouse bone marrow, how the time until bone marrow collection after cerebral infarction and the culture period of bone marrow cells affect cell mass formation, etc. It is a graph which shows the result of having examined. FIG. 3 is a photograph replacing a drawing which shows the result of examination by immunostaining of the differentiation of bone marrow-derived neurosphere-like cell clusters into neurons and glial cells.
[図 4]骨髄由来のニューロスフェア様細胞塊の神経細胞への分化能、分化効率を検 討した結果を示すグラフである。  FIG. 4 is a graph showing the results of examining the differentiation ability and differentiation efficiency of neurosphere-like cell clusters derived from bone marrow into neurons.
[図 5]偽手術 SCIDマウスの骨髄に脳梗塞 SCIDマウス（MCOIW)の血清を添加す ると-ユーロスフェア様細胞塊ができ ( A · B)、さらに神経細胞にも分ィ匕した (C · D)こ とを示す図面に代わる写真である。  [Figure 5] When serum from cerebral infarction SCID mice (MCOIW) is added to the bone marrow of sham-operated SCID mice-Eurosphere-like cell clusters are formed (AB), and they are also separated into neurons (C · D) This is a photo to replace this drawing.
[図 6]SCIDマウスと C. B17マウスにおける中大脳動脈結紮後 16日目に摘出した脳 を比較して示す図面に代わる写真である。  FIG. 6 is a photograph replacing a drawing which shows a comparison of brains removed on the 16th day after ligation of the middle cerebral artery in SCID mice and C. B17 mice.
[図 7]SCIDマウスと C. B17マウスにおける中大脳動脈結紮後 1日目に摘出した脳切 片の TTC染色の結果を比較して示す図面に代わる写真である。脳梗塞領域が白く 示される。  FIG. 7 is a photograph replacing a drawing, showing a comparison of the results of TTC staining of brain slices extracted on the first day after ligation of the middle cerebral artery in SCID mice and C. B17 mice. The cerebral infarction area is shown in white.
[図 8]脳梗塞 SCIDマウスおよび脳梗塞 C. B17マウスの脳梗塞後における脳（皮質） 再生の有無を、 CI値をもとに検討した結果を示すグラフである。  FIG. 8 is a graph showing the results of examining the presence or absence of brain (cortex) regeneration after cerebral infarction in cerebral infarction SCID mice and cerebral infarction C. B17 mice based on CI values.
圆 9]脳梗塞 SCIDマウスの梗塞下組織では NeuN陽性の神経前駆細胞が多数観察 されたが (A)、脳梗塞 C. B17マウスの梗塞下組織ではほとんど観察されなカゝつた (B )ことを示す図面に代わる写真である。 圆 9] Cerebral infarction Many NeuN-positive neural progenitor cells were observed in the infarcted tissue of SCID mice (A), but cerebral infarction was rarely observed in the infarcted tissue of C. B17 mice (B). It is the photograph replaced with drawing which shows.
[図 10]脳梗塞 SCIDマウスの骨髄からは-ユーロスフェア様細胞塊が形成され (Α·Β )、一方、脳梗塞 C. B17マウスの骨髄からは培養開始 7日目までにわずかに-ユー ロスフェア様細胞塊が形成された (C · D)ことを示す図面に代わる写真である。  [Fig. 10] From the bone marrow of cerebral infarction SCID mice-Eurosphere-like cell clusters were formed (Α · Β), while from the bone marrow of cerebral infarction C. B17 mice, a slight It is a photograph replacing a drawing showing that a sphere-like cell cluster is formed (C · D).
[図 11]脳梗塞 SCIDマウス (A)および脳梗塞 C. B17マウス（B)の骨髄由来-ユーロ スフエア様細胞塊におけるアポトーシスを検討した結果を示す図面に代わる写真で ある。原図では、ネクローシス細胞が赤で、アポトーシス細胞が緑で表示される。 FIG. 11 is a photograph replacing a drawing which shows the results of examining apoptosis in cerebral infarction SCID mice (A) and cerebral infarction C. B17 mice (B) in bone marrow-derived eurosphere-like cell clusters. In the original figure, necrotic cells are displayed in red and apoptotic cells are displayed in green.
[図 12]脳梗塞 SCIDマウスと脳梗塞 C. B17マウスの脳梗塞下組織力もの神経幹細 胞の形成を検討した結果を示すグラフである。 FIG. 12 is a graph showing the results of studying the formation of neural stem cells with cerebral infarcted tissue strength in cerebral infarction SCID mice and cerebral infarction C. B17 mice.
[図 13]C. B 17マウスの脳梗塞作成前 3日間と後 7日間に FK506を連日腹腔内投与 し、その後、骨髄と脳梗塞下組織 (脳梗塞瘢痕部位)を採取して培養したところ、骨髄 (Α·Β)および脳梗塞瘢痕部位 (C)から-ユーロスフェア様細胞塊が形成されたこと を示す図面に代わる写真である。 [Fig. 13] FK506 was intraperitoneally administered daily for 3 days before and 7 days after the creation of cerebral infarction in C. B 17 mice, and then the bone marrow and the subinfarcted tissue (cerebral infarction scar site) were collected and cultured. From the bone marrow (Α · Β) and cerebral infarction scar site (C)-Eurosphere-like cell clusters formed It is the photograph replaced with drawing which shows.
[図 14]脳梗塞 C. B17マウスの骨髄を、 FK506を添カ卩して培養したところ、ニューロス フェア様細胞塊 (Α·Β)が形成されたことを示す図面に代わる写真である。  FIG. 14 is a photograph replacing a drawing which shows that neurosphere-like cell clusters (Α · Β) were formed when bone marrow of cerebral infarction C. B17 mice were cultured with FK506 added.
[図 15]脳梗塞 C. B17マウス骨髄由来の-ユーロスフェア様細胞塊の形成に対する F Κ506の影響を検討した結果を示すグラフである。 FIG. 15 is a graph showing the results of examining the effect of F F506 on the formation of a eurosphere-like cell cluster derived from cerebral infarction C. B17 mouse bone marrow.
[図 16]脳梗塞を起こしていない偽手術 C. B17マウスの骨髄を、脳梗塞 SCIDマウス の血清および FK506を添カ卩して培養したしたところ、ニューロスフェア様細胞塊が形 成され (B'C)、脳梗塞 SCIDマウスの血清のみ添カ卩して培養した場合は一旦-ユー 口スフ ア様細胞塊が形成されるが (A)、すぐに排除されたことを示す図面に代わる 写真である。  [Figure 16] Sham operation without cerebral infarction C. B17 mouse bone marrow was cultured with cerebral infarction SCID mouse serum and FK506, and neurosphere-like cell clusters were formed (B 'C) When cerebral infarction SCID mouse serum is added and cultured, once the mouth-spore-like cell mass is formed (A), it replaces the drawing showing that it was immediately eliminated. It is.
[図 17]上記方法により FK506を添カ卩して-ユーロスフェア様細胞塊を形成させ、さら に培養後 7日目に、 Nestinと MAP2に対する免疫組織ィ匕学（二重染色間接蛍光抗 体法)を行った結果を示す図面に代わる写真である。  [Fig. 17] Addition of FK506 by the above method-Eurosphere-like cell mass was formed, and on the 7th day after culture, immunohistochemistry for Nestin and MAP2 (double-stained indirect fluorescent antibody) It is a photograph replacing a drawing showing the result of performing the method.
[図 18]脳梗塞後 SCIDマウスにおいて、骨髄由来の神経幹細胞が脳組織へ生着し、 分ィ匕したことを示す図面に代わる写真である。骨髄由来の GFP陽性細胞 (原図では 緑）は脳梗塞下組織に広く認められ、同時に PSA— NCAM陽性 (原図では赤)で、 神経前駆細胞であった（Merge)。  FIG. 18 is a photograph replacing a drawing showing that bone marrow-derived neural stem cells have engrafted and separated in brain tissue in SCID mice after cerebral infarction. Bone marrow-derived GFP-positive cells (green in the original map) were widely observed in subcerebral infarcted tissues, and at the same time were PSA-NCAM positive (red in the original map) and neural progenitor cells (Merge).
圆 19]脳梗塞患者の血清と FK506存在下で培養した正常ヒト骨髄細胞力も得られた ニューロスフェア様細胞塊 (A)、およびさらに培養して得られた神経細胞 (B)を示す 図面に代わる写真である。 圆 19] Neurosphere-like cell mass (A) obtained from normal human bone marrow cells cultured in the presence of sera and FK506 in cerebral infarction patients, and nerve cells (B) obtained by further culturing It is a photograph.
[図 20]脳梗塞 SCIDマウスの骨髄由来の神経幹細胞を、別の脳梗塞 SCIDマウスに 移植した場合の脳再生に与える影響、脳梗塞治療効果につ 、て検討した結果を示 す図面に代わる写真である。 ΓΒΜ-NSCjは骨髄由来の神経幹細胞を投与した結 果、「PBS」はコントロールで PBSを投与した結果、である。  [Fig. 20] Instead of a drawing showing the results of studying the effect of bone marrow-derived neural stem cells from cerebral infarction SCID mice on brain regeneration when transplanted to another cerebral infarction SCID mouse, and the effect of treatment of cerebral infarction It is a photograph. ΓΒΜ-NSCj is the result of administration of bone marrow-derived neural stem cells, and “PBS” is the result of administration of PBS as a control.
[図 21]C. B17マウス骨髄由来の-ユーロスフェア様細胞塊の形成における抗 CD28 抗体、抗 ICOS抗体の影響を検討した結果を示す図面に代わる写真である。脳梗塞 を起こしていない偽手術 C. B17マウスの骨髄を、脳梗塞後 1週間目の SCIDマウス の血清を添カ卩して培養した。これにさらに、 FK506 (A)、あるいは、抗 CD28抗体（B )又は抗 ICOS抗体 (C)を各々添カ卩した。培養 9日目には FK506、抗 CD28抗体、 抗 ICOS抗体を添加して培養した骨髄細胞力も-ユーロスフェア様細胞塊が形成さ れ、血清のみを添加して培養した場合 (D)は-ユーロスフェア様細胞塊は形成され なかった。 FIG. 21 is a photograph replacing a drawing, showing the results of examining the effects of anti-CD28 antibody and anti-ICOS antibody on the formation of C. B17 mouse bone marrow-derived eurosphere-like cell clusters. Sham surgery without cerebral infarction C. B17 mouse bone marrow was cultured with SCID mouse serum 1 week after cerebral infarction. In addition to this, FK506 (A) or anti-CD28 antibody (B ) Or anti-ICOS antibody (C). On day 9 of culture, bone marrow cells cultured with FK506, anti-CD28 antibody, and anti-ICOS antibody also added-Eurosphere-like cell mass formed, and when cultured with serum alone (D)-Euro No sphere-like cell mass was formed.
[図 22]脳梗塞を起こして!/、な!/、偽手術 C. B 17マウス骨髄に CINC - 1と FK506を添 加して培養すると、培養 5日目に細胞塊が形成され (Α· Β)、培養 7日目には-ユーロ スフエア様細胞塊が形成された (C ' D)ことを示す図面に代わる写真である。 Bは Aの 、 Dは Cの拡大像である。  [Figure 22] Caused cerebral infarction! /, Na! /, Sham operation C. BNC mice cultured with CINC-1 and FK506 added to bone marrow formed a cell mass on the fifth day of culture (培養· Β) On the 7th day of culturing-a photograph replacing the drawing showing that a eurosphere-like cell mass was formed (C'D). B is an enlarged image of A and D is an enlarged image of C.
[図 23]CINC— 1により刺激誘導された-ユーロスフェア様細胞塊形成における FK5 06の影響を検討した結果を示す図面に代わる写真である。脳梗塞を起こして 、な!/ヽ 偽手術 C. B17マウス骨髄に CINC— 1を添加、あるいは CINC— 1と FK506を添カロ して培養した。 CINC— 1と FK506を添加すると培養 5日目以内にすでに細胞塊が 形成され、 7日目以降には-ユーロスフェア様細胞塊が形成された (A)。一方、 FK5 06を添加しない場合は、細胞塊は形成される力 排除される力、あるいは細胞塊の 状態で止まっており（B)、ニューロスフェア様にはならなかった。 Α· Βはいずれも培養 9日目の像である。  FIG. 23 is a photograph replacing a drawing, showing the results of examining the effect of FK506 on the formation of eurosphere-like cell clusters stimulated by CINC-1. After cerebral infarction, sham operation C. B17 Bone marrow bone marrow was added with CINC-1 or cultured with CINC-1 and FK506. When CINC-1 and FK506 were added, cell clusters were already formed within 5 days of culture, and -Eurosphere-like cell clusters were formed after 7 days (A). On the other hand, when FK506 was not added, the cell mass stopped at the force of forming force or the state of the cell mass (B), and did not become neurosphere-like. Α and Β are both images on day 9 of culture.
圆 24]C. B17マウスの骨髄 (a)および脳梗塞下組織 (脳梗塞瘢痕部位）（b)由来- ユーロスフエア形成における免疫抑制剤 (FK506とシクロスポリン A)の影響を検討し た結果を示すグラフである。縦軸は、 ldishあたりの培養 9日目の nestin陽性-ユーロ スフエア数を示す（各群 4匹ずつ）。 * P< 0.001, student t test 圆 24] C. B17 mice derived from bone marrow (a) and subcerebral infarcted tissue (cerebral infarct scar site) (b)-graph showing the results of examining the effects of immunosuppressive agents (FK506 and cyclosporin A) on eurosphere formation It is. The vertical axis shows the number of nestin positive-Eurosphere on day 9 of culture per ldish (4 animals per group). * P <0.001, student t test
圆 25]ヌードマウスの骨髄由来および脳梗塞下組織 (脳梗塞瘢痕部位）由来-ユーロ スフエア形成能を検討した結果を示すグラフである。縦軸は、培養 9日目の骨髄 (a)と 脳梗塞下組織 (b)力も形成された nestin陽性-ユーロスフェア数を示す (各群 4匹ず つ）。培養 ldishあたりの-ユーロスフェア数はヌードマウスではそれぞれ 13 ±4個、 42 ±5個であったのに対し、コントロールでは 1.5 ± 1.5個、 2.0± 1.5で、有意にヌードマ ウスで多かった。 * P< 0.001, student t test [25] This is a graph showing the results of examining the ability to form eurosphere derived from bone marrow and tissue derived from cerebral infarction (cerebral infarction scar site) in nude mice. The vertical axis shows the number of nestin positive-Eurospheres (4 animals in each group) in which the bone marrow (a) and subcerebral infarcted tissue (b) force on day 9 of culture were also formed. The number of -Eurospheres per culture ldish was 13 ± 4 and 42 ± 5 for nude mice, respectively, 1.5 ± 1.5 and 2.0 ± 1.5 for controls, which were significantly higher in nude mice. * P <0.001, student t test
[図 26]サイト力イン（CINC— 1と TNF a )の骨髄由来-ユーロスフエア形成に及ぼす 影響を検討した結果を示すグラフである。縦軸は、 ldishあたりの培養 9日目に形成さ れた nestin陽性-ユーロスフェア数を示す（各群 4匹ずつ）。 * P< 0.001, student t te st FIG. 26 is a graph showing the results of examining the effect of cytoforce-in (CINC-1 and TNF a) on bone marrow-derived eurosphere formation. The vertical axis is formed on day 9 of culture per ldish. Nestin positive-eurosphere counts (4 per group). * P <0.001, student t te st
[図 27]C. B17マウス骨髄由来の-ユーロスフェア様細胞塊の形成における抗 CD28 抗体、抗 ICOS抗体の影響を検討した結果を示すグラフである。縦軸は、培養 ldish あたりの nestin陽性-ユーロスフェアの数を示す（各群 4匹ずつ）。 * P< 0.001, stude nt t test  FIG. 27 is a graph showing the results of examining the effects of anti-CD28 antibody and anti-ICOS antibody on the formation of C-B17 mouse bone marrow-derived -eurosphere-like cell clusters. The vertical axis shows the number of nestin positive-eurospheres per culture ldish (4 animals per group). * P <0.001, stude nt t test
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 以下、本発明の具体的態様、技術的範囲等について説明する。  [0018] Specific embodiments and technical scope of the present invention will be described below.
[ 1 ]骨髄からの新たな神経幹細胞調製法の開発  [1] Development of new neural stem cell preparation method from bone marrow
本発明者らは最近、良好な再現性を示し、しかも長期間生存可能な脳梗塞モデル マウスを開発した (特願 2004— 108500号)。この脳梗塞モデルマウスは、後述する ように、免疫不全マウスである SCIDマウスの中大脳動脈を結紮することにより、中大 脳動脈の皮質枝の血流を選択的に途絶させ、再現性のよい均一な脳梗塞を作成し たものである。興味深いことに、この脳梗塞 SCIDマウスでは、虚血後の脳梗塞の進 展（delayed infarct expansion)は脳梗塞後 3日で終了し、その後は脳萎縮の進展で はなぐ脳形態上はむしろ回復する。免疫組織ィ匕学による検討の結果、このマウスの 脳梗塞下組織 (脳梗塞周囲の瘢痕部位）にお ヽて脳梗塞後多数の神経幹細胞の出 現が観察され、これが正常神経組織に生着して脳再生、脳機能改善に貢献している こと、さらに、この神経幹細胞は骨髄に由来するものであることを明らかにした。  The present inventors recently developed a cerebral infarction model mouse that exhibits good reproducibility and can survive for a long period of time (Japanese Patent Application No. 2004-108500). As described later, this cerebral infarction model mouse selectively disrupts the blood flow in the cortical branch of the middle cerebral artery by ligating the middle cerebral artery of the SCID mouse, which is an immunodeficient mouse, and has good reproducibility. A uniform cerebral infarction was created. Interestingly, in this cerebral infarction SCID mouse, the delayed infarct expansion after ischemia ended 3 days after the cerebral infarction, and then the brain morphology rather than the progression of cerebral atrophy recovered. To do. As a result of examination by immunohistochemistry, the appearance of numerous neural stem cells after cerebral infarction was observed in the sub-cerebral infarction tissue (the scar site around the cerebral infarction) of this mouse, and this engrafted in the normal neural tissue. It has been clarified that it contributes to brain regeneration and brain function improvement, and that these neural stem cells are derived from bone marrow.
[0019] そこで、上記脳梗塞 SCIDマウスから脳梗塞 1週間後に採取した骨髄細胞を培養し たところ、上述のように、神経幹細胞 (ニューロスフェア）に分化誘導することができた (図 1D)。またその後も培養を続けると、神経幹細胞は分裂'増殖し、神経細胞に分 化誘導された（同図 E)。さらに、神経再生と免疫に関する下記（1)〜 (6)の重要な知 見が得られた (なお、各実験の詳細にっ 、ては後述する）。  [0019] Thus, when bone marrow cells collected one week after cerebral infarction from the above-mentioned cerebral infarction SCID mouse were cultured, it was possible to induce differentiation into neural stem cells (neurospheres) as described above (Fig. 1D). When the culture was continued thereafter, the neural stem cells proliferated and proliferated and were induced to differentiate into neurons (Fig. E). Furthermore, the following important findings (1) to (6) regarding nerve regeneration and immunity were obtained (details of each experiment will be described later).
[0020] (1) 脳梗塞を起こしていない正常 SCIDマウスの骨髄を培養しても神経幹細胞は誘 導されな!ヽが、この骨髄に脳梗塞モデルマウスの血清を添加すると神経幹細胞が誘 導され（図 2、図 5Α· Β)、神経幹細胞は神経細胞にも分ィ匕した（図 5C ' D)。このこと より、脳梗塞後の血清中に、骨髄細胞を神経幹細胞に分化誘導させる刺激因子 (diff erentiation factor)が存在し、生体ではこの刺激因子が血流を介して骨髄に伝えられ 、神経幹細胞が産生されると考えられる。 [0020] (1) Nerve stem cells are not induced by culturing the bone marrow of normal SCID mice without cerebral infarction! However, adding serum from cerebral infarction model mice to this bone marrow induces neural stem cells. The neural stem cells were also divided into neurons (FIG. 5C′D). As a result, a stimulating factor that induces differentiation of bone marrow cells into neural stem cells in serum after cerebral infarction (diff It is thought that in the living body, this stimulating factor is transmitted to the bone marrow through the bloodstream to produce neural stem cells.
さらに解析の結果、この刺激因子、換言すれば神経幹細胞への分ィヒ誘導因子の ひとつは、インターロイキン 8 (IL 8)ファミリーのケモカインである、 CINC—1ZG RO ^cytokine- induced neutrophil chemoattractant- 1/ growth-related oncogeneノで あることを明らかにした（図 22、 23)。  As a result of analysis, one of the stimulating factors, in other words, one of the inducers of neural stem cells, is a chemokine of the interleukin 8 (IL 8) family, CINC—1ZG RO ^ cytokine- induced neutrophil chemoattractant-1 / It has been revealed that it is a growth-related oncogene (Figs. 22 and 23).
[0021] (2) SCIDマウスの母系である免疫機能が正常な C.B-17/IcrCrlBRマウスの中大脳 動脈を結紮して脳梗塞モデルを作成した後、その骨髄を採取して培養すると、神経 幹細胞がー且形成されるが（図 lOC'D)、これらは 2週間以内にすべて消失してしま う（図 15)。 [2] (2) After creating a cerebral infarction model by ligating the middle cerebral artery of a CB-17 / IcrCrlBR mouse with normal immune function, which is the mother system of SCID mice, neural stem cells are collected and cultured. They are formed (Figure lOC'D), but they all disappear within two weeks (Figure 15).
[0022] (3) 一方、上記 C.B- 17/IcrCrlBRマウスに対して、免疫抑制剤 FK506 (タクロリムス )を投与して免疫不全状態にし、脳梗塞を起こさせた後も免疫抑制剤の投与を続け、 その後当該マウスから採取した骨髄細胞を培養したところ、神経幹細胞に分化誘導 させることができた（図 13)。他の免疫抑制剤であるシクロスポリン A (CsA)を投与し た場合も同様の結果が得られた（図 24)。また、 C.B-17/IcrCrlBRマウスを脳梗塞後、 その骨髄を採取し、免疫抑制剤 FK506を添加して培養すると、神経幹細胞が形成さ れた（図 14、図 15)。  [0022] (3) On the other hand, the immunosuppressive agent FK506 (tacrolimus) was administered to the CB-17 / IcrCrlBR mice to make them immunodeficient and administration of the immunosuppressive agent was continued even after causing cerebral infarction. Then, when bone marrow cells collected from the mice were cultured, differentiation into neural stem cells could be induced (FIG. 13). Similar results were obtained when cyclosporin A (CsA), another immunosuppressant, was administered (Figure 24). When C.B-17 / IcrCrlBR mice were infarcted, their bone marrow was collected and cultured with the addition of the immunosuppressant FK506 to form neural stem cells (FIGS. 14 and 15).
[0023] (4) 脳梗塞を起こしていない正常 C.B-17/IcrCrlBRマウスの骨髄細胞を異なる条件 下で培養したところ、免疫抑制剤 FK506および脳梗塞モデルマウスの血清を添加し た場合にのみ、神経幹細胞が形成された（図 16)。 FK506の代わりに、 T細胞機能 を抑制する抗体 (抗 T細胞抗体)を添加しても同様に神経幹細胞が形成された（図 21 、図 27)。  [0023] (4) When bone marrow cells of normal CB-17 / IcrCrlBR mice without cerebral infarction were cultured under different conditions, only when immunosuppressant FK506 and serum of cerebral infarction model mice were added, Neural stem cells were formed (Figure 16). In place of FK506, neural stem cells were similarly formed when an antibody that suppresses T cell function (anti-T cell antibody) was added (FIGS. 21 and 27).
[0024] (5) さらに、ヒトの正常骨髄細胞を、免疫抑制剤 FK506および脳梗塞患者の血清 を添加して培養したところ、神経幹細胞に分ィ匕誘導させることができた（図 19A)。こ れをさらに培養すると、神経細胞に分化した（同図 B)。  (5) Furthermore, when human normal bone marrow cells were cultured with the addition of immunosuppressant FK506 and serum from a cerebral infarction patient, they were able to induce neural stem cells (FIG. 19A). When this was further cultured, it differentiated into neurons (Fig. B).
[0025] (6) 脳梗塞 SCIDマウスの骨髄力も調製された神経幹細胞を、別の脳梗塞 SCIDマ ウスに静脈内投与してその脳梗塞治療効果を調べたところ、脳形態上の回復が観察 され、神経再生の促進効果が認められた（図 20)。 [0026] 本発明は、これらの知見に基づき、（1)正常骨髄細胞を神経幹細胞へ分化させる ためには、脳梗塞後の血清中に存在する刺激因子と、骨髄培養液中の免疫反応を 抑制させる免疫抑制剤の添加が重要であり、（2)脳梗塞後の患者又は免疫正常なモ デル動物から採取した骨髄細胞を神経幹細胞へ分化させるためには、免疫抑制剤 の添加が重要であること、および、（3)上記の刺激因子 (分ィ匕誘導因子）のひとつは、 IL— 8ファミリーのケモカイン CINC— 1/GROであることを見出し、以下の第 1〜第 3の神経幹細胞調製法を提供するものである。 [0025] (6) Cerebral infarction Neural stem cells, which are also prepared from bone marrow in SCID mice, were intravenously administered to another cerebral infarction SCID mouse and examined for therapeutic effects on cerebral infarction. The effect of promoting nerve regeneration was confirmed (Fig. 20). [0026] Based on these findings, the present invention is based on these findings. (1) In order to differentiate normal bone marrow cells into neural stem cells, the stimulating factor present in the serum after cerebral infarction and the immune reaction in the bone marrow culture solution are determined. It is important to add an immunosuppressive agent to suppress. (2) In order to differentiate bone marrow cells collected from patients after cerebral infarction or immune normal model animals into neural stem cells, it is important to add an immunosuppressive agent. And (3) one of the above stimulating factors (min-inducing factors) was found to be IL-8 family chemokine CINC-1 / GRO, and the following first to third neural stem cells A method of preparation is provided.
[0027] 第 1の神経幹細胞調製法:免疫抑制剤、および、脳梗塞その他脳障害後のヒト又は 動物から採取した血清を添加して骨髄細胞を培養することにより分化誘導し、神経幹 細胞を調製する方法。  [0027] First neural stem cell preparation method: differentiation is induced by culturing bone marrow cells by adding an immunosuppressant and serum collected from human or animal after cerebral infarction or other brain injury, How to prepare.
第 2の神経幹細胞調製法:脳梗塞その他脳障害後のヒト又は動物力 採取した骨 髄細胞を、免疫抑制剤を添加して培養することにより分化誘導し、神経幹細胞を調 製する方法。  Second Neural Stem Cell Preparation Method: A method for preparing neural stem cells by inducing differentiation by culturing human bone or animal force collected after cerebral infarction or other brain injury by adding an immunosuppressive agent.
第 3の神経幹細胞調製法:免疫抑制剤、および、ケモカイン (好ましくは、 IL 8ファ ミリ一のケモカイン)を添加して骨髄細胞を培養することにより分ィ匕誘導し、神経幹細 胞を調製する方法。  Third method of neural stem cell preparation: immunosuppressant and chemokine (preferably IL-8 family chemokine) is added to induce bone marrow cell culture to prepare neural stem cells how to.
[0028] 第 1 (および第 3)の調製法において、常法にしたがってヒト又は動物力も採取した 骨髄は、免疫抑制剤および脳障害後の血清 (又は、当該血清の代わりにケモカイン） を添加する以外、従来の神経幹細胞の培養方法と同様の方法により培養可能である 。また、第 2の調製法においても、免疫抑制剤を添加する以外、従来の神経幹細胞 の培養方法と同様の方法により培養可能である。従来の培養方法としては、ニューロ スフエア (neurosphere)法、低密度単層培養法、高密度単層培養法などが例示され、 このうち好ましい培養法としては、ニューロスフェア法が挙げられる。後述の実施例に おいては、この-ユーロスフェア法を用いて、塩基性線維芽細胞成長因子 (bFGF, 5 0 μ g/ml)および上皮成長因子 (EGF, 20 μ g/ml)存在下骨髄細胞を培養し、神 経幹細胞を調製した。  [0028] In the first (and third) preparation method, the bone marrow from which human or animal force was also collected according to the conventional method is supplemented with an immunosuppressant and serum after brain injury (or chemokine instead of the serum). Other than that, it can be cultured by a method similar to the conventional method for culturing neural stem cells. Further, in the second preparation method, it can be cultured by the same method as the conventional neural stem cell culture method except that an immunosuppressive agent is added. Examples of conventional culture methods include a neurosphere method, a low-density monolayer culture method, and a high-density monolayer culture method, and among these, a preferred culture method includes the neurosphere method. In the examples described below, this -eurosphere method was used in the presence of basic fibroblast growth factor (bFGF, 50 μg / ml) and epidermal growth factor (EGF, 20 μg / ml). Bone marrow cells were cultured and neural stem cells were prepared.
[0029] 以下、本発明の調製法において使用する免疫抑制剤、血清および分化誘導因子 であるケモカインにっ 、て説明する。 [1-A]免疫抑制剤 [0029] Hereinafter, the immunosuppressant, serum, and chemokine that is a differentiation-inducing factor used in the preparation method of the present invention will be described. [1-A] Immunosuppressant
培養液に添加する免疫抑制剤は、実施例で用いた FK506 (タクロリムス）およびシ クロスポリンを使用することができる力 そのほかに、バシリキシマブ、ァザチォプリン、 ムロモナブ CD3、ミゾリビン、ミコフエノール酸モフエチル、など公知の免疫抑制剤の 使用が考えられる。免疫抑制剤は、 T細胞機能など細胞性免疫を抑制する作用 ·性 質を有するものが好ましぐ免疫抑制機能を有する限りにおいて、抗体 (例えば抗 CD 28抗体、抗 ICOS抗体)などの物質も広く本発明の「免疫抑制剤」に含まれる。  In addition to the ability to use FK506 (tacrolimus) and cyclosporine used in the examples, the immunosuppressive agent added to the culture solution is well-known, such as basiliximab, azathioprine, muromonab CD3, mizoribine, mofuethyl mycofenolate, etc. The use of immunosuppressants is considered. An immunosuppressant is a substance such as an antibody (for example, an anti-CD28 antibody or an anti-ICOS antibody) as long as an agent that suppresses cellular immunity, such as T cell function, has a preferable immunosuppressive function. Widely included in the “immunosuppressive agent” of the present invention.
培養液への免疫抑制剤の添加量は、特に限定されないが、 0. 01 /ζ 8Ζπι1〜1. 0 μ gZml程度の濃度で添加することが好ましい。後述の実施例では、 0. 1 μ g/ml の濃度で FK506を培養液に添加した。 The amount of the immunosuppressant added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 0.01 / ζ 8 Ζπι1 to 1.0 μg Zml. In the examples described below, FK506 was added to the culture solution at a concentration of 0.1 μg / ml.
[1-B]血清 [1-B] Serum
第 1の調製法にぉ 、て、培養液へ添加する血清の量も特に限定されるものではな いが、 5 μ lZml〜25 μ lZml程度の濃度で添加することが好ましい。上述のように、 脳梗塞後の血清中には、骨髄細胞を神経幹細胞に分化誘導させる分化誘導因子が 存在し、この分化誘導因子は、虚血侵襲や他の原因により脳神経細胞が一部障害を 受けることによって、生体の有する修復機構の一環として産生されるものと考えられる 。したがって、脳梗塞後の血清に限らず、事故等により脳糸且織が一部障害を受けた 後の血清中にもこの分ィ匕誘導因子は出現し、当該血清を添加して骨髄細胞を培養 することで同様の効果が得られると考えられる。  In the first preparation method, the amount of serum added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 5 μl Zml to 25 μl Zml. As described above, there is a differentiation-inducing factor in the serum after cerebral infarction that induces bone marrow cells to differentiate into neural stem cells. This differentiation-inducing factor is partially damaged by cerebral neurons due to ischemic invasion or other causes. Is considered to be produced as part of the repair mechanism of the living body. Therefore, not only the serum after cerebral infarction but also the serum after the brain thread and tissue have been partially damaged due to an accident etc. It is considered that the same effect can be obtained by culturing.
また、例えば脳梗塞後の血清を使用して調製した本発明の神経幹細胞を、脳梗塞 の治療に限らず、他の神経疾患の再生治療に利用してもよ 、。  For example, the neural stem cells of the present invention prepared using serum after cerebral infarction may be used not only for the treatment of cerebral infarction but also for the regeneration treatment of other neurological diseases.
「動物」から採取した血清を使用して、治療法の開発用などに神経幹細胞を調製す る場合、当該動物としてはマウスやラットのほか、ゥシ、ブタ、ヒッジ、ャギ、ゥサギ、ィ ヌ、ネコ、モルモット、ハムスターなどの哺乳動物が例示される。マウスは、 SCIDマウ ス以外のものを使用してもよい。  When preparing neural stem cells for the development of therapeutic methods using serum collected from “animals”, the animals include mice, rats, as well as ushi, pig, hidge, goat, usagi, i Mammals such as nu, cat, guinea pig and hamster are exemplified. A mouse other than a SCID mouse may be used.
なお、後述の実施例に示すように、ヒトおよびマウスの場合、脳梗塞後 1週間目に採 取した血清を使用することで良好に神経幹細胞を調製できたので、本発明の第 1の 調製法においては、脳梗塞後（または他の脳障害後） 5日〜10日の血清を使用する ことが好ましい。 As shown in the Examples below, in the case of humans and mice, neural stem cells were successfully prepared by using serum collected one week after cerebral infarction, so the first preparation of the present invention The method uses serum 5 to 10 days after cerebral infarction (or after other cerebral disorders) It is preferable.
[0031] [1-C]分ィ匕誘導因子  [0031] [1-C] min-inducing factor
第 3の調製法は、免疫抑制剤とともに、分ィ匕誘導因子としてケモカインを添加して骨 髄細胞を培養する方法である。  The third preparation method is a method of culturing bone marrow cells by adding chemokine as an inducing factor together with an immunosuppressant.
上述のように、脳梗塞後の血清中には、骨髄細胞を神経幹細胞へ分化させる何ら かの分化誘導因子が存在し、この分化誘導因子が血流を介して骨髄に伝えられ、神 経幹細胞が産生されると考えられる。本発明者は、この分ィ匕誘導因子の探索に際し て、 SCIDマウスの免疫反応の特徴に注目した。すなわち、 SCIDマウスは T細胞が 欠落しているが NK細胞は十分存在しているため、 SCIDマウスが C. B17マウスより 骨髄での神経幹細胞産生に適した条件を備えて 、るとすると、 NK細胞にその原因 を求めるのが妥当と考えた。さらに、骨髄細胞の分ィ匕に関与し、かつ虚血などの生体 への侵襲刺激に際して血中に放出される因子を探索した。  As described above, in the serum after cerebral infarction, there is some differentiation inducing factor for differentiating bone marrow cells into neural stem cells, and this differentiation inducing factor is transmitted to the bone marrow through the bloodstream, and the neural stem cell Is thought to be produced. The present inventor paid attention to the characteristics of the immune response of SCID mice in searching for the differentiation-inducing factor. In other words, SCID mice lack T cells but sufficient NK cells, so SCID mice have better conditions for neural stem cell production in bone marrow than C.B17 mice. We thought it appropriate to ask the cells for the cause. Furthermore, we searched for factors involved in bone marrow cell sorting and released into the blood upon invasive stimulation of living bodies such as ischemia.
[0032] CINC lZ GRO (cytokine- induced neutrophil chemoattractant- lZ growth- relat ed oncogene)は、インターロイキン 8 (IL— 8)ファミリーのケモカインであり、コノレヒチ ン刺激などの noxious stimulationにより脳の視床下部神経で産生亢進され、下垂体 後葉を経て血中に放出される（Neurosci. Res. (1997) 27:181-184.)。 CINC— 1は、 NK細胞からも産生されて子宮の脱落膜を介した受精卵の免疫反応に関与し (Bioch em. Biophys. Res. Commun. (1994) 200:378- 383.)、また骨髄間質（stroma)細胞から も産生される（Exp. Cell Res. (2004) 299:383-392.) 0さらに、本発明者は独自に、脳 虚血負荷に際しても視床下部や下垂体後葉において CINC— 1産生が亢進するとい う調査結果を得ていた。 [0032] CINC lZ GRO (cytokine-induced neutrophil chemoattractant- lZ growth-relat ed oncogene) is a chemokine of the interleukin 8 (IL—8) family, which is induced in the hypothalamic nerve of the brain by noxious stimulation such as conoleicin stimulation. Production is increased and released into the blood via the posterior lobe of the pituitary gland (Neurosci. Res. (1997) 27: 181-184.). CINC-1 is also produced by NK cells and is involved in the immune response of fertilized eggs via the uterine decidua (Biochem. Biophys. Res. Commun. (1994) 200: 378-383.) It is also produced from stroma cells (Exp. Cell Res. (2004) 299: 383-392.) 0 In addition, the inventor independently developed in the hypothalamus and the posterior pituitary gland during cerebral ischemic load. The survey results showed that CINC-1 production was increased.
そこで、 CINC— 1が神経幹細胞への分ィ匕誘導因子である可能性を検討した。その 結果、脳梗塞を起こしていない正常 C.B-17/IcrCrlBRマウス力も採取した骨髄に、免 疫抑制剤 FK506および CINC— 1を添加して培養すると、骨髄から神経幹細胞へ分 化誘導させることができた（図 22、 23)。この結果から、 CINC— 1が分ィ匕誘導因子の ひとつであることが示された。また、 CINC— 1は、培養中の骨髄細胞を凝集させる作 用を有しており（図 23B)、この凝集した細胞塊が核となって-ユーロスフェアが形成 されると考えられる。したがって、このような凝集作用を有する他のケモカインも CINC 1と同様に分化誘導因子として作用すると考えられる。このようなケモカインの作用 が骨髄中の幹細胞に直接作用しているの力、あるいは間質細胞などを介して間接的 に作用しているのかは今後更なる解析を必要とする力 いずれにせよ、ケモカインを 免疫抑制剤とともに培養骨髄中に添加することで、神経幹細胞への分ィ匕誘導が可能 である。 Therefore, we examined the possibility that CINC-1 is a factor that induces differentiation of neural stem cells. As a result, when bone marrow from which normal CB-17 / IcrCrlBR mice without cerebral infarction were collected is cultured with the addition of the immunosuppressive agents FK506 and CINC-1, it can induce differentiation from bone marrow into neural stem cells. (Figures 22 and 23). From this result, it was shown that CINC-1 is one of the induction factors. CINC-1 also has the action of agglutinating bone marrow cells in culture (FIG. 23B), and this aggregated cell mass is considered to be the nucleus to form -Eurosphere. Therefore, other chemokines with such an aggregating action are also CINC Similar to 1, it is thought to act as a differentiation-inducing factor. Whether such chemokine acts directly on stem cells in the bone marrow, or indirectly through stromal cells, etc., it is a force that requires further analysis in the future. Addition of chemokines to cultured bone marrow together with immunosuppressants can induce differentiation into neural stem cells.
[0033] ケモカインは IL— 8ファミリーのケモカインの使用が好ましぐヒト、ラット又はマウス 由来の IL— 8ファミリーのケモカイン、あるいは他の哺乳動物のカウンターパートから 選ばれるケモカインを添加する方法が挙げられる。ヒト由来 IL 8ファミリーのケモカ インとしては、 IL— 8/CXCL8、 GRO - a、 GRO— β及び GRO— γなどが例示さ れ、ラット由来は、 CINC— 1、 CINC— 2 α、 CINC— 2 j8、 CINC— 3、マウス由来は 、 KC、 MIP— 2などが例示される。その他の CXCケモカインの使用も考えられる。  [0033] For chemokines, IL-8 family chemokines are preferred, IL-8 family chemokines derived from humans, rats, or mice, or chemokines selected from other mammalian counterparts can be mentioned. . Examples of human-derived IL 8 family chemokines include IL-8 / CXCL8, GRO-a, GRO-β and GRO-γ, and rat-derived chemokines include CINC-1, CINC-2 α, CINC-2 Examples of j8, CINC-3, and mouse origin are KC and MIP-2. Other CXC chemokines may be used.
[0034] 培養液へのケモカインの添加量は、特に限定されないが、 0. 1 gZml〜l. 0 g Zml程度の濃度で添加することが好ましい。後述の実施例では、 10— 5Mの濃度で CI NC— 1を培養液に添カ卩した。 [0034] The amount of chemokine added to the culture solution is not particularly limited, but it is preferably added at a concentration of about 0.1 gZml to 1.0 gZml. In the examples below, at a concentration of 10- 5 M were添Ka卩the CI NC- 1 to the cultures.
上述のように、ケモカインは、免疫抑制剤とともに培養骨髄中に添加することによつ て、神経幹細胞への分ィヒ誘導が可能であるので、ケモカインと免疫抑制剤とを組み 合わせ、神経幹細胞分化誘導剤として提供できる。また、免疫抑制剤の添加は培養 下の T細胞機能を抑制するためであるから、あら力じめ T細胞を除去するなどして他 の方法で T細胞機能を抑制することができれば、免疫抑制剤を添加せずにケモカイ ンのみで培養細胞力 神経幹細胞を調製することも可能と考えられる。  As described above, chemokine can be induced into neural stem cells by adding it to cultured bone marrow together with an immunosuppressive agent. Therefore, a chemokine and an immunosuppressive agent are combined to produce neural stem cells. It can be provided as a differentiation inducer. In addition, because the addition of immunosuppressive agents is to suppress T cell function in culture, immunosuppression can be achieved if T cell function can be suppressed by other methods such as removing T cells. It is considered possible to prepare cultured cell force neural stem cells with chemokine alone without adding any agent.
[0035] さらに後述するように、本発明者らによる探索の結果、上記ケモカイン以外に、サイ トカインである TNF a (tumor necrosis factor- a )力 骨髄細胞から神経幹細胞への 分化誘導作用を有することを明らかにした（図 26)。従って、本発明の上記第 3の調 製法において、ケモカインの代わりに、 TNF a又は IL18などその他のサイト力インを 分ィ匕誘導因子として使用してもよい。 TNF αなどのサイト力インを分ィ匕誘導因子とし て使用する場合も、上記ケモカインと同様の方法'条件で使用可能であり、骨髄細胞 から神経幹細胞への調製に利用することができる。  [0035] As will be described later, as a result of the search by the present inventors, in addition to the chemokine, TNF a (tumor necrosis factor-a) force which is a cytokine has a differentiation inducing action from bone marrow cells to neural stem cells. (Figure 26). Therefore, in the third preparation method of the present invention, other site force-in such as TNFa or IL18 may be used as a differentiation-inducing factor instead of chemokine. When a cyto force-in such as TNFα is used as a differentiation-inducing factor, it can be used under the same conditions as the above chemokine and can be used for preparation from bone marrow cells to neural stem cells.
[0036] 本発明の神経幹細胞調製法は、次のような利点'特徴を有する。 (1) 簡便かつ短期間に、神経幹細胞の誘導'調製が可能である。後述の実施例に 示すように、実際に骨髄力も短期間（2週間程度)で神経幹細胞を誘導'調製すること ができ、得られた神経幹細胞はその後分裂 ·増殖して、神経細胞に高効率に分化し た。 [0036] The neural stem cell preparation method of the present invention has the following advantages. (1) Neural stem cells can be easily and easily prepared in a short time. As shown in the examples described later, bone marrow strength can be actually induced and prepared in a short period (about 2 weeks), and the resulting neural stem cells can then divide and proliferate for high efficiency in nerve cells. Differentiated.
神経再生治療に応用する場合、治療開始は 2週間以内（即ち、幹細胞の産生およ び生着に最も適した時期である急性期）が好ましぐ従来この期間内に大量の幹細 胞を準備'調製することは困難であった。短期間に幹細胞の調製可能な本発明の方 法は、神経再生治療の臨床応用に最も適した方法と!/、える。  When applied to nerve regeneration therapy, it is preferable to start treatment within 2 weeks (ie, the acute phase, which is the most suitable time for stem cell production and engraftment). Preparation 'It was difficult to prepare. The method of the present invention that can prepare stem cells in a short period of time is the most suitable method for clinical application of nerve regeneration treatment!
[0037] (2) 脳患部において良好に神経細胞を生着、機能発揮させることが期待できる。実 際に、本方法により調製された神経幹細胞を移植 (静脈内投与)したところ、脳形態 上の回復が観察され、幹細胞移植により神経細胞が脳組織に生着したものと考えら れる。 [0037] (2) It can be expected that neuronal cells are successfully engrafted and functioned in the affected brain region. In fact, when the neural stem cells prepared by this method were transplanted (intravenous administration), recovery in the brain morphology was observed, and it is considered that the neurons were engrafted in the brain tissue by the stem cell transplantation.
上述のように、本発明者らは、脳梗塞 SCIDマウスにおいて、脳梗塞後多数の神経 幹細胞の出現が観察され、これが正常神経組織に生着して脳再生、脳機能改善に 貢献していること、および、この神経幹細胞は骨髄に由来するものであることを明らか にした。本発明は、このように脳梗塞修復時に特異的に産生され、神経を再生する骨 髄由来の神経幹細胞を調製するものであり、生体が要求する細胞を大量に提供する ことができ、生体機構に沿った移植治療が可能になると考えられる。  As described above, the present inventors observed the appearance of numerous neural stem cells after cerebral infarction in cerebral infarction SCID mice, which engrafted in normal neural tissue and contributed to brain regeneration and brain function improvement. It was also clarified that these neural stem cells are derived from bone marrow. The present invention prepares bone marrow-derived neural stem cells that are specifically produced during cerebral infarction repair and regenerate nerves, and can provide a large amount of cells required by the living body. It is thought that transplantation treatment along the line will be possible.
[0038] (3) 骨髄細胞は比較的採取が容易であるので、例えば脳梗塞後の患者から骨髄を 採取し、免疫抑制剤の存在下に培養を行って神経幹細胞を調製し、これを静脈内投 与などで患者に移植する自家移植も十分実現可能である。  [0038] (3) Since bone marrow cells are relatively easy to collect, for example, bone marrow is collected from a patient after cerebral infarction and cultured in the presence of an immunosuppressive agent to prepare neural stem cells, which are then used as veins. Autologous transplantation to patients by internal administration etc. is also feasible.
細胞移植は、基本的に自家移植が倫理的にも医学的にも最良の方法と考えられる 。本発明は、生体への生着性と機能発揮性の点で最良ともいえる患者自身の骨髄由 来神経幹細胞を提供することができ、再生医療に適している。ただし、本発明は、自 家移植への利用のみに限定されるものではなぐ他家移植用に他者由来の骨髄から 神経幹細胞を調製する方法にも利用可能である。  Cell transplantation is basically considered to be the best ethical and medical method for autotransplantation. The present invention can provide the patient's own bone marrow-derived neural stem cells, which can be said to be the best in terms of engraftment in a living body and function, and is suitable for regenerative medicine. However, the present invention is not limited to use for autologous transplantation, and can also be used for a method for preparing neural stem cells from bone marrow derived from another person for other transplantation.
[0039] (4) 分ィ匕誘導因子であるケモカイン (もしくはその他のサイト力イン)の使用によって 、より簡便かつ安全性を向上できる。また、慢性期などの患者で骨髄又は血清中に 分ィ匕誘導因子としてのケモカインやサイト力インがまったく或いは殆ど存在しな 、場 合に、ケモカインなどのサイト力インを添加して神経幹細胞を調製する方法は極めて 効果的である。そのほか、他家移植用に他者由来の骨髄から神経幹細胞を調製する 方法においても、血清の代わりにケモカインやサイト力インを使用することは、安全性 の面力も非常に望ましい方法である。 [0039] (4) Use of a chemokine (or other site force-in) which is a differentiation-inducing factor can improve convenience and safety. Also, in patients such as chronic stage in bone marrow or serum When there is no or almost no chemokine or cytodynamic force-in as a differentiation factor, a method for preparing neural stem cells by adding a cytodynamic force such as chemokine is extremely effective. In addition, in the method of preparing neural stem cells from bone marrow derived from another person for transplantation in another family, the use of chemokine or cytodynamic ins instead of serum is a highly desirable method in terms of safety.
[0040] (5) 本発明は、脳梗塞の治療に限らず、その他の脳血管障害、脳虚血性疾患、神 経変性疾患に対する神経移植治療にも利用可能であり、神経再生治療に広く利用 可能性を有する。  [0040] (5) The present invention can be used not only for the treatment of cerebral infarction but also for nerve transplantation treatment for other cerebrovascular disorders, cerebral ischemic diseases, and neurodegenerative diseases, and widely used for nerve regeneration treatments. Have potential.
[0041] (6) 本発明は、神経再生治療へ直接利用可能であるほか、治療法開発においても 利用可能であり、例えば本発明により得られた神経幹細胞を様々な条件下、脳梗塞 モデルマウスに移植してその治療効果を確認することで、更なる治療法開発'研究進 展が期待できる。  [0041] (6) The present invention can be directly used for nerve regeneration treatment, and can also be used in the development of therapeutic methods. For example, the neural stem cells obtained by the present invention can be used for cerebral infarction model mice under various conditions. By confirming the therapeutic effect after transplantation into a new patient, further development of therapeutic methods can be expected.
[0042] また、本発明により調製した神経 (幹)細胞を神経再生治療剤として患者に移植 ·投 与する方法としては、例えば、静注、点滴静注などによって投与する。このような注射 剤は常法に従って製造され、希釈剤として一般に生理食塩水、細胞培養液などを用 いることができる。さらに必要に応じて、殺菌剤、防腐剤、安定剤、等張化剤、無痛化 剤などを加えてもよい。これら製剤中の神経 (幹)細胞の配合量は特に限定されるも のではなぐ疾患の種類、症状の程度、患者の年齢、体重などに応じて決定すれば よい。また、本発明の神経 (幹)細胞を、数回にわたり患者に投与してもよいし、 1回当 たりの投与量、投与間隔などを診断結果に応じて決定してもよい。  [0042] In addition, as a method for transplanting and administering a nerve (stem) cell prepared according to the present invention to a patient as a nerve regeneration therapeutic agent, for example, administration is performed by intravenous injection or intravenous infusion. Such an injection is produced according to a conventional method, and generally a physiological saline, a cell culture solution, or the like can be used as a diluent. Further, if necessary, bactericides, preservatives, stabilizers, tonicity agents, soothing agents and the like may be added. The compounding amount of nerve (stem) cells in these preparations is not particularly limited, and may be determined according to the type of disease, the degree of symptoms, the age of the patient, the body weight, and the like. In addition, the nerve (stem) cells of the present invention may be administered to a patient several times, and the dose per administration, the administration interval, etc. may be determined according to the diagnosis result.
[0043] 神経 (幹)細胞に対する患者の免疫応答を抑制するため、神経 (幹)細胞の投与と 同時またはその投与前 (もしくは投与後）に、免疫抑制剤を投与することが望ましい。 投与方法は、神経 (幹)細胞の投与と異なる方法であってもよ 、。  [0043] In order to suppress a patient's immune response to nerve (stem) cells, it is desirable to administer an immunosuppressant simultaneously with or before (or after) administration of nerve (stem) cells. The administration method may be different from the administration of nerve (stem) cells.
[0044] また、脳内での血管再生、神経再生を向上させるため、神経 (幹)細胞の投与と同 時またはその投与前 (もしくは投与後）に、ヒト臍帯血由来 CD34陽性細胞 (J.Clin.Inv est. 114:330-338 (2004)参照）、血管内皮前駆細胞 (EPC)などの血管形成能を有 する細胞を投与する方法は、好ましい方法である。  [0044] In order to improve blood vessel regeneration and nerve regeneration in the brain, human umbilical cord blood-derived CD34-positive cells (J. Clin. Invest. 114: 330-338 (2004)), and a method of administering cells having angiogenic ability such as vascular endothelial progenitor cells (EPC) is a preferred method.
[0045] 以上のように、神経 (幹)細胞を神経再生治療剤として用いることができるが、これ以 外に、後述の実施例の結果から、 in vitroにおいて、また in vivoにおいても、骨髄由 来神経幹細胞の形成を維持'促進し、神経再生治療を実現するためには、免疫機能 を抑制すること、特に T細胞機能を抑制することが重要であることが示されたので、免 疫抑制物質 (特に T細胞機能を抑制する物質)を神経再生治療剤として用いることが 可能である。 [0045] As described above, nerve (stem) cells can be used as therapeutic agents for nerve regeneration. In addition, from the results of the examples described later, in order to maintain and promote the formation of bone marrow-derived neural stem cells both in vitro and in vivo, and to realize nerve regeneration treatment, suppress immune function. In particular, since it was shown that it is important to suppress T cell function, an immunosuppressive substance (particularly a substance that suppresses T cell function) can be used as a therapeutic agent for nerve regeneration.
[0046] 例えば、 FK506 (タクロリムス）、シクロスポリン、抗 CD28抗体および抗 ICOS抗体 など、 T細胞の活性を抑制する物質を神経再生治療剤として用いることができる。  [0046] For example, substances that suppress the activity of T cells such as FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody can be used as therapeutic agents for nerve regeneration.
[0047] そのほか、前述のように、骨髄細胞から神経幹細胞への分化誘導作用を有する CI NC—1、 TNF αといったケモカイン、サイト力インについても、これらの分化誘導因 子を投与することによって、 in vitroと同様に、 in vivoでの骨髄由来神経幹細胞の形 成が促進されると考えられるので、神経再生治療剤としての応用が可能である。  [0047] In addition, as described above, chemokines such as CI NC-1 and TNFα, which have differentiation-inducing action from bone marrow cells to neural stem cells, and site force-in, can be administered by administering these differentiation-inducing factors. As in vitro, it is thought that the formation of bone marrow-derived neural stem cells in vivo is promoted, so that it can be applied as a therapeutic agent for nerve regeneration.
[0048] さらに、これらケモカイン、サイト力インの分化誘導作用を促進する物質 (これらケモ 力イン、サイト力インの産生を促進する物質を含む)も神経再生治療剤としての応用 が可能である。このような物質は、例えば後述する本発明のスクリーニング系を利用 して探索することができる。  [0048] Furthermore, substances that promote the differentiation-inducing action of these chemokines and cytoforce-ins (including substances that promote the production of these chemokines and cytoforce-ins) can also be applied as therapeutic agents for nerve regeneration. Such a substance can be searched using, for example, the screening system of the present invention described later.
[0049] 本発明のこれら免疫抑制物質、ケモカイン、サイト力イン等を有効成分とする神経 再生治療剤は、経口剤でもよいし、注射剤、坐剤、塗布剤等の非経口剤でもよい。錠 剤、カプセル剤、顆粒剤、細粒剤、散剤等の経口剤は、例えば、デンプン、乳糖、白 糖、トレハロース、マンニット、カルボキシメチルセルロース、コーンスターチ、無機塩 類等を用いて常法に従って製造される。これらの製剤中の有効成分の配合量は特に 制限されるものではなく適宜設定できる。この種の製剤には、結合剤、崩壊剤、界面 活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、香料等を適宜に使用することが できる。  [0049] The nerve regeneration therapeutic agent containing these immunosuppressive substances, chemokines, cytosites, etc. of the present invention as active ingredients may be oral agents, or parenteral agents such as injections, suppositories, and coating agents. Oral preparations such as tablets, capsules, granules, fine granules, powders etc. are produced according to conventional methods using, for example, starch, lactose, sucrose, trehalose, mannitol, carboxymethylcellulose, corn starch, inorganic salts, etc. Is done. The compounding amount of the active ingredient in these preparations is not particularly limited and can be appropriately set. In this type of preparation, binders, disintegrants, surfactants, lubricants, fluidity promoters, corrigents, colorants, fragrances and the like can be appropriately used.
[0050] 非経口剤の場合は、例えば、静注、点滴静注、皮下注射、筋肉注射などによって 投与する。この非経口剤は常法に従って製造され、希釈剤として一般に注射用蒸留 水、生理食塩水等を用いることができる。また、この非経口剤は安定性の点から、バ ィアル等に充填後冷凍し、通常の凍結乾燥処理により水分を除き、使用直前に凍結 乾燥物力 液剤を再調製することもできる。さらに必要に応じて、殺菌剤、防腐剤、安 定剤、等張化剤、無痛化剤などを加えてもよい。これら製剤中の有効成分の配合量 は特に制限されるものではなぐ前述の神経 (幹)細胞を投与する場合と同様に、疾 患の種類、症状の程度、患者の年齢、体重などに応じて決定すればよい。また、本 発明の神経再生治療剤を、数回にわたり患者に投与してもよいし、 1回当たりの投与 量、投与間隔などを診断結果に応じて決定してもよ 、。 [0050] In the case of a parenteral agent, for example, it is administered by intravenous injection, intravenous infusion, subcutaneous injection, intramuscular injection or the like. This parenteral preparation is produced according to a conventional method, and distilled water for injection, physiological saline and the like can be generally used as a diluent. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water can be removed by ordinary freeze-drying treatment, and a freeze-dried liquid solution can be re-prepared immediately before use. If necessary, disinfectants, preservatives, A constanting agent, an isotonic agent, a soothing agent and the like may be added. The compounding amount of the active ingredient in these preparations is not particularly limited, as in the case of administering the nerve (stem) cells described above, depending on the type of disease, the degree of symptoms, the age of the patient, the body weight, etc. Just decide. Moreover, the therapeutic agent for nerve regeneration of the present invention may be administered to a patient several times, and the dose per administration, the administration interval, etc. may be determined according to the diagnosis result.
[0051] [2]脳梗塞モデル動物の開発  [0051] [2] Development of cerebral infarction model animals
上述した本発明の神経幹細胞調製法の開発に伴!、、免疫不全マウス（SCIDマウ ス)の母系マウスの脳の血管を結紮してなる新たな脳梗塞モデル動物を開発した。こ の脳梗塞モデル動物は、 SCIDマウスの代わりに、その母系マウスを使用すること以 外、特願 2004— 108500号記載の方法と同様に作製可能である。  Along with the development of the above-described method for preparing neural stem cells of the present invention, a new cerebral infarction model animal was developed in which the blood vessels of the brain of a maternal mouse of an immunodeficient mouse (SCID mouse) were ligated. This cerebral infarction model animal can be prepared in the same manner as described in Japanese Patent Application No. 2004-108500, except that the maternal mouse is used instead of the SCID mouse.
[0052] SCIDマウスの母系マウスとしては、現在市販されて!、る（例えば、 FOX Chase Can cer Centerなど参照のこと） SCIDマウス、または、この改良型のマウスの母系マウスで あればよい。結紮する脳の血管としては、脳内の脳梗塞を発症させることができる血 管であれば、いずれの血管であっても特に制限はないが、発症例の多い血管や、ま た実験の行いやすい表皮側の血管が好ましい。好ましい血管としては、中大脳動脈 、内頸動脈、椎骨脳底動脈などが例示される。  [0052] Maternal mice of SCID mice may be SCID mice that are currently commercially available! (See, for example, FOX Chase Cancer Center) or maternal mice of this improved mouse. The blood vessels in the ligation are not particularly limited as long as they are capable of developing cerebral infarction in the brain. Easily blood vessels on the epidermis side are preferred. Examples of preferable blood vessels include middle cerebral artery, internal carotid artery, vertebral basilar artery and the like.
[0053] また、結紮する部位としても特に制限はないが、結紮部位によっては、虚血領域の 選択性が悪くなることもあることから、選択性を確保できる部位を設定することも必要 である。例えば、結紮部位として、中大脳動脈が嗅索を通過した直後、すなわち遠位 側 Ml部位（distal Ml portion)を選択することにより、中大脳動脈の皮質枝の血流を 選択的に途絶させることが可能である。  [0053] Also, there is no particular limitation on the site to be ligated, but depending on the site of ligation, the selectivity of the ischemic region may deteriorate, so it is also necessary to set a site that can ensure the selectivity. . For example, the blood flow in the cortical branch of the middle cerebral artery is selectively disrupted by selecting the ligation site immediately after the middle cerebral artery passes through the olfactory tract, that is, the distal Ml portion. Is possible.
[0054] 脳の血管を結紮する手法としては、脳梗塞が発症できる結紮方法であれば特に制 限はなぐ例えば、クリップ法、血管の凝固'切断法、血管内栓子法などの各種の手 法を使用することができる。虚血を一過性にする力、永久的にするかにより、結紮方 法を選定することも必要である。例えば、凝固用ピンセットにて電気凝固後に切断す る永久結紮法、動脈瑠結紮用クリップを用いた一過性結紮法などが挙げられる。  [0054] The method of ligating the blood vessels of the brain is not particularly limited as long as it is a ligation method capable of developing cerebral infarction. The method can be used. It is also necessary to select a ligation method depending on whether the ischemia is transient or permanent. For example, a permanent ligation method in which cutting is performed after electrocoagulation with coagulation tweezers, or a transient ligation method using an arterial ligation clip.
[0055] 具体的な結紮方法としては、例えば、ハロセンなどでマウスを麻酔し、マウスの左頰 骨を切除し、頭蓋底を露出させ、中大脳動脈走行部位に直径 l〜5mm程度の骨窓 を歯科用ドリルで作成し、硬膜、クモ膜を剥離し、中大脳動脈を分離して結紮を行うこ とがでさる。 [0055] As a specific ligation method, for example, the mouse is anesthetized with halothane or the like, the left rib of the mouse is excised, the skull base is exposed, and a bone window having a diameter of about 1 to 5 mm is formed in the middle cerebral artery running site. Can be made with a dental drill, the dura mater and the arachnoid membrane are removed, and the middle cerebral artery is separated and ligated.
[0056] 上記方法にしたがって、実際に SCIDマウスの母系マウスである C.B- 17/IcrCrlBR マウスの脳の血管を結紮することにより、良好な再現性を示し、しかも長期間生存可 能な脳梗塞モデルマウスを作製することができた (後述の実施例 2参照)。  [0056] According to the above method, a cerebral infarction model that shows good reproducibility and can survive for a long time by ligating the blood vessels of the brain of CB-17 / IcrCrlBR mouse, which is actually a maternal mouse of SCID mouse A mouse could be produced (see Example 2 below).
[0057] さらに、上記脳梗塞モデルマウスの作製方法と同様に、無胸腺マウスであるヌード マウス（BALB/cAJcHiu)およびその母系マウスの左中大脳動脈をそれぞれ結紮する ことにより、良好な再現性で脳梗塞モデルマウスを作製することができた (後述の実施 例 7参照)。  [0057] Further, in the same manner as the method for producing the above-mentioned cerebral infarction model mouse, the athymic mouse (BALB / cAJcHiu) and the left middle cerebral artery of the maternal mouse were each ligated, thereby achieving good reproducibility. A cerebral infarction model mouse could be prepared (see Example 7 described later).
[0058] SCIDマウスと同様に、ヌードマウスは、その T細胞機能が抑制された免疫不全マウ スであり、上記方法でヌードマウス力 脳梗塞モデルを作製後、その骨髄および脳梗 塞下組織 (脳梗塞瘢痕部位)カゝら採取した細胞をそれぞれ培養したところ、 nestin陽 性の-ユーロスフェアが多数形成された（図 25)。対照的に、その母系マウス (コント口 ール)から脳梗塞モデルを作製後、その骨髄および脳梗塞下組織力ゝら採取した細胞 をそれぞれ培養したところ、 nestin陽性の-ユーロスフェアは殆ど形成されな力つた。 この結果からも、 T細胞が骨髄あるいは脳梗塞下組織における-ユーロスフェア形成 に抑制的機能を果たしていることがわかる。  [0058] Similar to SCID mice, nude mice are immunodeficient mice whose T cell function is suppressed. After creating a nude mouse force cerebral infarction model by the above method, the bone marrow and subcerebral infarction tissue ( Cerebral infarction scar site) When the collected cells were cultured, many nestin positive-eurospheres were formed (Fig. 25). In contrast, after preparing a cerebral infarction model from the maternal mouse (control mouth) and then culturing the cells collected from the bone marrow and tissue force under cerebral infarction, most of the nestin-positive eurospheres are formed. I helped. This result also shows that T cells have a suppressive function on the formation of -eurosphere in bone marrow or subcerebral infarcted tissue.
[0059] 従って、（1)T細胞機能を抑制する免疫抑制剤を利用することによって、 SCIDマウ スの母系マウスの骨髄細胞から神経幹細胞を調製したのと同様の方法で、ヌードマ ウスの母系マウスの骨髄細胞力 神経幹細胞を調製することができると考えられる。さ らに、（2) SCIDマウスおよびヌードマウス以外の T細胞機能不全マウスの骨髄からも 、 SCIDマウス等からと同様の方法で、神経幹細胞を調製することができると考えられ る。  [0059] Therefore, (1) using an immunosuppressant that suppresses T cell function, a neural stem cell was prepared in the same manner as neural stem cells were prepared from bone marrow cells of SCID mouse maternal mice. It is considered that neural stem cells can be prepared. Furthermore, it is considered that (2) neural stem cells can be prepared from the bone marrow of T cell dysfunctional mice other than SCID mice and nude mice in the same manner as from SCID mice.
[0060] また、本発明は、（1)本発明の脳梗塞モデル動物に、被検薬物を投与して、当該 被検薬物の脳梗塞に対する有効性をスクリーニングする方法、および、（2)本発明の 脳梗塞モデル動物に、神経 (幹)細胞又は他の細胞を移植して、当該移植治療の脳 梗塞に対する有効性をスクリーニングする方法、を提供するものである。  [0060] In addition, the present invention provides (1) a method for screening a cerebral infarction model animal of the present invention by administering a test drug and screening the effectiveness of the test drug against cerebral infarction, and (2) the present The present invention provides a method for transplanting nerve (stem) cells or other cells into the cerebral infarction model animal of the invention and screening the effectiveness of the transplantation treatment for cerebral infarction.
[0061] 本発明のスクリーニング方法としては、例えば、本発明の脳梗塞モデル動物に、被 検薬物を投与、あるいは神経幹細胞等を移植して、カーボンブラック灌流法、脳の大 きさの測定、 MRIなどによる機器分析などにより脳梗塞の病巣部の大きさや容積の 変化、形態学的検討 (左右大脳皮質幅の比、 TUNEL染色によるアポトーシスの程 度、 BrdU標識による再生神経や再生血管内皮細胞数）、行動テスト (オープンフィ 一ルドテストや驚愕反射、迷路学習、回避学習）などを測定し、これを対照群と比較し て脳梗塞病巣部の拡大の阻止、脳機能の回復などの程度を評価する方法が挙げら れる。 [0061] Examples of the screening method of the present invention include, for example, subjecting a cerebral infarction model animal of the present invention to a subject. Changes in the size and volume of cerebral infarction lesions, morphological examinations by administration of test drugs or transplantation of neural stem cells, etc. by carbon black perfusion method, measurement of brain size, instrumental analysis such as MRI (Ratio of left and right cerebral cortex width, degree of apoptosis by TUNEL staining, number of regenerating nerves and regenerating vascular endothelial cells by BrdU labeling), behavioral test (open field test, startle reflex, maze learning, avoidance learning), etc. Compared with the control group, there is a method for evaluating the degree of inhibition of cerebral infarction lesion, recovery of brain function, and the like.
[0062] さらに、本発明は、脳梗塞などの脳虚血性疾患、又はその他の神経疾患の治療に 用いられる神経再生治療剤のスクリーニング方法として、骨髄細胞から神経幹細胞 への分化を誘導し、又はその分ィ匕を促進する作用を有するかどうかを指標に、候補 物質を探索するスクリーニング方法を提供するものである。  [0062] Further, the present invention provides a method for screening a therapeutic agent for nerve regeneration used for the treatment of cerebral ischemic diseases such as cerebral infarction or other neurological diseases, or induces differentiation from bone marrow cells to neural stem cells, or The present invention provides a screening method for searching for candidate substances, using as an index whether or not the substance has an effect of promoting the function.
[0063] 例えば、後述の実施例記載の培養系にお 、て、ヒト又は動物から採取した骨髄細 胞の培地に被検物質を投与し、当該被検物質が、骨髄細胞から神経幹細胞への分 化を誘導し、又はその分化を促進する作用を有するかどうかを調べることによって、 候補物質を探索する。培養条件は、使用する骨髄細胞の起源などに応じて、（1)免 疫抑制剤を添加したもの、 (2)分化誘導因子 (又は脳障害後に採取した血清)を添 加したもの、（3)免疫抑制剤と分化誘導因子 (又は脳障害後血清)の両者を添加した もの、（4)両者とも添加しないもの、など種々の条件の中力 適切な条件を設定すれ ばよい。  [0063] For example, in the culture system described in the Examples below, a test substance is administered to a medium of bone marrow cells collected from a human or an animal, and the test substance is transferred from bone marrow cells to neural stem cells. Candidate substances are searched for by examining whether they have the effect of inducing differentiation or promoting their differentiation. Depending on the origin of the bone marrow cells used, the culture conditions are (1) with an immunosuppressant added, (2) with a differentiation-inducing factor (or serum collected after brain injury), (3 Appropriate conditions may be set for various conditions, such as those with both an immunosuppressant and differentiation-inducing factor (or serum after brain injury) added, and (4) those without both.
[0064] 実際に、後述の実施例記載の培養系を用いて、骨髄細胞から神経幹細胞への分 化を誘導する物質を探索したところ、サイト力インである TNF aがこのような分ィ匕誘導 作用をもつことを見出すことができ、本発明のスクリーニング方法の有効性が認めら れた。  [0064] Actually, when a substance that induces differentiation from bone marrow cells to neural stem cells was searched using the culture system described in the Examples below, TNFa, which is a site force-in, was detected in this manner. It was found to have an inducing action, and the effectiveness of the screening method of the present invention was confirmed.
[0065] 本発明のスクリーニング方法において、免疫抑制作用を有する物質群の中から、神 経再生に有用な候補物質の探索を行ってもよいし、ケモカインなどのサイト力イン、あ るいはこれらの活性や産生を調節する物質群の中から、神経再生に有用な候補物 質の探索を行ってもよい。  [0065] In the screening method of the present invention, a candidate substance useful for nerve regeneration may be searched from a group of substances having an immunosuppressive action, or a site force-in such as chemokine, or these A candidate useful for nerve regeneration may be searched from a group of substances that regulate activity and production.
[0066] また、本発明のスクリーニング方法は、後述の実施例記載の培養系を用いたスクリ 一ユング系に制限されるものではなく、その他のスクリーニング系を用いても勿論よ ヽ 。例えば、分ィ匕誘導因子として同定された CINC—1や TNF o;などの物質を標的分 子として、これらの分子の活性や産生を調節する物質を既存の様々な系で調べるこ とができ、これにより、骨髄細胞から神経幹細胞への分化を促進し、神経再生治療に 有効な候補物質を探索することができる。 [0066] In addition, the screening method of the present invention is a screening method using a culture system described in Examples below. Of course, it is not limited to one Jung system, and other screening systems may be used. For example, substances that regulate the activity and production of these molecules can be examined in various existing systems using substances such as CINC-1 and TNFo; identified as molecular weight inducers as target molecules. Thus, differentiation from bone marrow cells to neural stem cells can be promoted, and a candidate substance effective for nerve regeneration therapy can be searched.
実施例  Example
[0067] 以下、図面を参照しながら本発明の実施例について説明するが、本発明はこれら 実施例によって何ら限定されるものではない。  Hereinafter, examples of the present invention will be described with reference to the drawings, but the present invention is not limited to these examples.
〔実施例 1：脳梗塞 SCIDマウス骨髄由来の神経幹細胞の調製〕  [Example 1: Preparation of neural stem cells derived from cerebral infarction SCID mouse bone marrow]
[1-1]脳梗塞 SCIDマウスの作成  [1-1] Cerebral infarction Creation of SCID mice
特願 2004— 108500号の明細書記載の方法にしたがって、免疫不全マウスである SCIDマウス（5週齢）の中大脳動脈を結紮し、脳梗塞モデルマウスを作成した。  According to the method described in the specification of Japanese Patent Application No. 2004-108500, the middle cerebral artery of an SCID mouse (5 weeks old) which is an immunodeficient mouse was ligated to prepare a cerebral infarction model mouse.
具体的には、 3%ハロセン麻酔下にマウス左頰骨を切除し、頭蓋底を露出した。中 大脳動脈走行部位に直径 1. 5mmの骨窓を歯科用ドリルで作成した。硬膜、クモ膜 を剥離し、中大脳動脈を分離して結紮準備とした。中大脳動脈結紮法としては、凝固 用ピンセットにて電気凝固後に切断する永久結紮法、動脈瑠結紮用クリップを用い た一過性結紮法などが可能である。結紮部位は、動脈が嗅索を通過した直後、すな わち distal Ml portionである。この部位を結紮することにより、中大脳動脈の皮質枝の 血流を選択的に途絶させることが可能である。  Specifically, the mouse left rib was excised under 3% halothane anesthesia to expose the skull base. A bone window with a diameter of 1.5 mm was created with a dental drill in the middle cerebral artery running site. The dura mater and arachnoid membrane were removed, and the middle cerebral artery was separated to prepare for ligation. As the middle cerebral artery ligation method, a permanent ligation method in which cutting is performed after electrocoagulation with coagulation tweezers, or a transient ligation method using an arterial ligation clip is possible. The ligation site is the distal Ml portion immediately after the artery passes through the olfactory tract, that is, the distal Ml portion. By ligating this site, it is possible to selectively disrupt the blood flow in the cortical branch of the middle cerebral artery.
[0068] このような中大脳動脈結紮法により、 SCIDマウス左中大脳動脈の distal Ml portion を結紮して作成した脳梗塞モデルマウス (脳梗塞 SCIDマウス)の脳梗塞領域を、実 際に 2, 3, 5—トリフエ-ルテトラゾリゥム（2,3,5- triphenyltetrazolium (TTC) )染色法 にて検討した。 TTC染色法は、中大脳動脈結紮後（MCO) l, 3, 7日目にそれぞれ マウス脳を摘出し、 brain slicerにて作成した冠状脳スライスを用いて施行した。図 7左 には、この染色法によって、結紮後（MCO) 1日目に摘出した脳の染色結果が示され る。この方法により、左中大脳動脈皮質枝領域に選択的に梗塞（白い部分)が観察さ れ、再現性のよい均一な脳梗塞を作成することができる（特願 2004— 108500号参 照)。なお脳梗塞は、上記方法による結紮後（MCO) 3日でほぼ完成する。 [0069] [1-2]ニューロスフ ア（neurosphere)様細胞塊の形成 [0068] The cerebral infarction region of the cerebral infarction model mouse (cerebral infarction SCID mouse) prepared by ligating the distal Ml portion of the left middle cerebral artery of the SCID mouse by such a middle cerebral artery ligation method is actually 2, This was examined by 3,5-triphenyltetrazolium (TTC) staining. TTC staining was performed using coronal brain slices prepared by brain slicer after excision of the mouse brain on middle cerebral artery ligation (MCO) 1, 3 and 7 days. The left side of Fig. 7 shows the results of staining of the brain extracted on the first day after ligation (MCO) by this staining method. By this method, an infarction (white part) is selectively observed in the left middle cerebral artery cortical branch region, and a uniform cerebral infarction with good reproducibility can be created (see Japanese Patent Application No. 2004-108500). Cerebral infarction is almost completed 3 days after ligation (MCO) by the above method. [0069] [1-2] Formation of neurosphere-like cell clusters
上記方法により作成した脳梗塞 SCIDマウスを結紮後 7日目にクリーンベンチ内で 断頭し、大腿骨力も骨髄を採取した。その後、 DMEMと N— 2の基本培養液内（250 μ 1)で単細胞になるまでピペッティングし、 10mlの培養液をカ卩えて 600rpmで 5分間 遠心した。細胞を 3mlの培養液にて再浮遊させ、 bFGF (50 μ g/ml)と EGF (20 μ g/ml)存在下に low cell bindingプレート上で 10— 28日間培養を行った。その結果 、図 1Dに示すように、 7日目以降には-ユーロスフェア（neurosphere)様細胞塊が細 胞顕微鏡下で観察された。  Cerebral infarction SCID mice prepared by the above method were decapitated in a clean bench on the 7th day after ligation, and the bone strength of femur was also collected. Then, pipetting was performed in the basic culture medium (250 μ1) of DMEM and N-2 until it became a single cell, and 10 ml of the culture medium was added and centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on low cell binding plates in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. As a result, as shown in FIG. 1D, from the 7th day onward, a neurosphere-like cell cluster was observed under a cell microscope.
[0070] 比較のため、胎生 2週目の C57BZ6マウスから採取した線条体細胞を low cell bind ingプレート上で 10日間培養を行ったところ、図 1 Aに示すように、ニューロスフェアが 形成された。これを high bindingプレート上でさらに培養すると 3日後には分ィ匕し（同図 B)、発現蛋白を免疫組織化学法 (二重染色間接蛍光抗体法)にて検討すると、 MA P2陽性の神経細胞と、 GFAP陽性のグリア細胞に分ィ匕して 、た（同図 C参照)。  [0070] For comparison, striatal cells collected from C57BZ6 mice at 2 weeks of embryo were cultured on low cell binding plates for 10 days. As shown in Fig. 1A, neurospheres were formed. It was. When this was further cultured on a high binding plate, it was separated after 3 days (Fig. B), and the expressed protein was examined by immunohistochemistry (double-staining indirect fluorescent antibody method). The cells were separated into GFAP-positive glial cells (see Fig. C).
[0071] 一方、脳梗塞 SCIDマウスから採取した骨髄細胞を low cell bindingプレート上で 10 日間培養すると、上述のように、胎生期マウス脳 (線条体）由来の-ユーロスフェアとよ く似た-ユーロスフェア様細胞塊が形成された（同図 D)。これを high bindingプレート 上でさらに培養すると 3日後には突起を持った細胞に分ィ匕し（同図 E)、一部は MAP 2陽性の神経細胞と、 GFAP陽性のグリア細胞に分ィ匕して 、た（同図 F参照)。  [0071] On the other hand, when bone marrow cells collected from cerebral infarction SCID mice were cultured on a low cell binding plate for 10 days, as described above, they resembled -Eurosphere derived from embryonic mouse brain (striatum) -Eurosphere-like cell clusters were formed (Fig. D). When this is further cultured on a high binding plate, it is separated into cells with protrusions after 3 days (Fig. E), and some are separated into MAP 2 positive neurons and GFAP positive glial cells. (See Fig. F).
[0072] さらに、 SCIDマウスに対して偽手術 (骨窓まで開き、結紮はしない処置）を施行した 脳梗塞を生じさせて ヽな 、偽手術 SCIDマウス（sham SCIDマウス）から骨髄を採取し 、同様の条件で培養を行ったが、ニューロスフェア様細胞塊はほとんど形成されなか つた o  [0072] Furthermore, a sham operation (a treatment that opens to the bone window and does not ligate) was performed on the SCID mouse to cause cerebral infarction, and the bone marrow was collected from the sham operated SCID mouse (sham SCID mouse), Culture was performed under the same conditions, but almost no neurosphere-like cell mass was formed.
[0073] [1-3]ニューロスフ ア様細胞塊の形成効率  [0073] [1-3] Formation efficiency of neurosphere-like cell mass
脳梗塞 SCIDマウス骨髄由来の-ユーロスフェア様細胞塊形成に関して、脳梗塞後 Cerebral infarction: SCID mouse bone marrow-derived eurosphere-like cell mass formation, after cerebral infarction
(結紮後)骨髄を採取するまでの期間や骨髄細胞の培養期間が、細胞塊形成にどの ように影響するかを検討した。 (After ligation) We examined how the time until bone marrow collection and the culture period of bone marrow cells affect cell mass formation.
[0074] 脳梗塞作成後（結紮後） 1, 2, 3週目の SCIDマウス力も採取した骨髄をそれぞれ 2[0074] After creation of cerebral infarction (post-ligation)
8日間培養し、新たに形成される-ユーロスフェア様細胞塊数を数えた。その結果、 図 2に示すように、脳梗塞後 1週目（MCOIW)に採取した骨髄が最も多くの-ユーロ スフエア様細胞塊を形成し、培養後 10— 13日目に形成能がピークとなることが明ら カゝになった。 After 8 days of culture, the number of newly formed-Eurosphere-like cell clumps was counted. as a result, As shown in Fig. 2, bone marrow collected at 1 week after cerebral infarction (MCOIW) forms the most abundant-eurosphere-like cell mass, and the ability to form is peaked 10-13 days after culture. Apparently, I was ecstatic.
[0075] 一方、偽手術 SCIDマウスの培養骨髄からは、ニューロスフェア様細胞塊は培養 1 ヶ月を経過してもほとんど形成されなかった（Sham control)。し力し、偽手術 SCIDマ ウスの骨髄に脳梗塞 SCIDマウス（MCOIW)力 採取した血清を添カ卩して培養する と、ニューロスフェア様細胞塊が形成された（Sham control + Serum)。  [0075] On the other hand, almost no neurosphere-like cell clusters were formed from cultured bone marrow of sham-operated SCID mice even after 1 month of culture (Sham control). When the sera from SCID mice (MCOIW) were collected on the bone marrow of a sham-operated SCID mouse and cultured, the neurosphere-like cell mass was formed (Sham control + Serum).
[0076] [1-4]神経幹細胞 ·神経細胞への分化能  [0076] [1-4] Neural stem cells · Ability to differentiate into neurons
脳梗塞 SCIDマウス（MCOIW)の骨髄を培養し、 10日目に培養液に浮遊している ニューロスフェア様細胞塊を採取して high bindingプレート上でさらに培養した。 3— 7 日後に分ィ匕した細胞をバラフオルムアルデヒドを含む固定液にて固定して免疫染色 を行い、発現蛋白の検討を行った。その結果、細胞塊の中心には Nestin陽性の神 経幹細胞が高率に観察された（図 3左)。また、周囲には分ィ匕した MAP2陽性の神経 細胞（同図中央)、 GFAP陽性のグリア細胞（同図右）が観察された。  The bone marrow of cerebral infarction SCID mice (MCOIW) was cultured, and on the 10th day, the neurosphere-like cell mass floating in the culture medium was collected and further cultured on a high binding plate. After 3 to 7 days, the cells separated were fixed with a fixative containing balaformaldehyde and immunostained to examine the expressed protein. As a result, Nestin-positive neural stem cells were observed at a high rate in the center of the cell mass (Fig. 3 left). In addition, MAP2-positive neurons (middle in the figure) and GFAP-positive glial cells (right in the figure) were observed.
[0077] 次に、骨髄由来のニューロスフ ア様細胞塊の神経細胞への分ィ匕能、分化効率を 検討した。その結果、図 4に示すように、脳梗塞 SCIDマウス (MCOIW)の骨髄を培 養して得られたニューロスフェア様細胞塊の約 60%が Nestin陽性の神経幹細胞で あり、そのうち 67% (全体の-ユーロスフェア様細胞塊の約 40%)が MAP2陽性、 Ne uN陽性の神経細胞に分ィ匕した。また、 MAP2陽性の神経細胞を産生した細胞塊に は、ほぼ 100%の確率で同時に GFAP陽性のグリア細胞が観察された。  [0077] Next, the ability of the bone marrow-derived neurosphere-like cell cluster to separate into nerve cells and differentiation efficiency were examined. As a result, as shown in Fig. 4, approximately 60% of the neurosphere-like cell clusters obtained by culturing the bone marrow of cerebral infarction SCID mice (MCOIW) are Nestin-positive neural stem cells, of which 67% (total About 40% of the Eurosphere-like cell mass was divided into MAP2-positive and NeuN-positive neurons. In the cell mass that produced MAP2-positive neurons, GFAP-positive glial cells were simultaneously observed with almost 100% probability.
[0078] [1-5]神経幹細胞へ分化誘導させる刺激因子の存在  [0078] [1-5] Presence of stimulating factors that induce differentiation into neural stem cells
上述のように、脳梗塞 SCIDマウスの骨髄を培養することで、神経幹細胞が産生さ れた。このように脳梗塞 SCIDマウスの骨髄力も神経幹細胞が分ィ匕誘導されるメカ- ズムを検討する目的で、脳梗塞を起こしていない偽手術 SCIDマウスの培養骨髄液 に別のマウスの血清（30 μ 1)を添カ卩した。  As described above, neural stem cells were produced by culturing bone marrow of cerebral infarction SCID mice. Thus, in order to investigate the mechanism by which neural stem cells are also induced in cerebral infarcted SCID mice, sera from other mice (30 μ 1) was added.
[0079] その結果、偽手術 SCIDマウスの骨髄に別の偽手術 SCIDマウスの血清を添カ卩して も-ユーロスフェア様細胞塊はまったく形成されな力つた力 偽手術 SCIDマウスの骨 髄に脳梗塞 SCIDマウス（MCOIW)の血清を添加すると-ユーロスフェア様細胞塊 ができ（図 5Α·Β、図 2)、これは神経細胞にも分ィ匕した（図 5C'D)。以上の結果から 、脳梗塞を起こした後の血清には、骨髄細胞を神経幹細胞へ分化させる何らかの刺 激因子（differentiation factor)が存在し、この刺激因子が血流を介して骨髄に伝えら れ、神経幹細胞が産生されると考えられる。 [0079] As a result, even if the serum of another sham-operated SCID mouse was added to the bone marrow of a sham-operated SCID mouse, the powerful force without the formation of the eurosphere-like cell cluster was found in the bone marrow of the sham-operated SCID mouse. Cerebral infarction When adding serum from SCID mice (MCOIW)-Eurosphere-like cell mass (Fig. 5Α ・ Β, Fig. 2), which was also distributed to neurons (Fig. 5C'D). From the above results, the serum after cerebral infarction has some differentiation factor that differentiates bone marrow cells into neural stem cells, and this stimulation factor is transmitted to the bone marrow via the bloodstream. Neural stem cells are thought to be produced.
[0080] [1-6]本実施例の結果と考察  [0080] [1-6] Results and discussion of this example
以上のように、免疫不全マウスである SCIDマウスの中大脳動脈を結紮して脳梗塞 モデルマウスを作成し、脳梗塞発症後 (結紮後）に骨髄細胞を採取、培養することで 、神経幹細胞塊 (ニューロスフ ア）を誘導'調製することができた。また、この-ユーロ スフエアが神経細胞に分ィ匕することを明らかにした。  As described above, cerebral infarction model mice are created by ligating the middle cerebral artery of SCID mice, which are immunodeficient mice, and bone marrow cells are collected and cultured after the onset of cerebral infarction (after ligation). (Neurosphere) could be 'derived' and prepared. It was also clarified that this -eurosphere is divided into nerve cells.
[0081] 一方、脳梗塞を起こして 、な 、偽手術 SCIDマウスの骨髄からは-ユーロスフェア は形成されな力 た力 これに脳梗塞モデルマウスの血清を添加して培養することで ニューロスフェア形成がみられたことから、ニューロスフェア形成には脳梗塞が誘因と なって産生される刺激因子が必要と考えられる。  [0081] On the other hand, cerebral infarction, from the bone marrow of sham-operated SCID mice-Eurosphere is a force that is not formed Neurospheroid formation by adding serum of cerebral infarction model mice to this and culturing Therefore, neurosphere formation is thought to require a stimulating factor produced by cerebral infarction.
[0082] 〔実施例 2 :脳梗塞後骨髄からの神経幹細胞産生と免疫不全との関連性検討〕  [Example 2: Examination of the relationship between neural stem cell production from bone marrow after cerebral infarction and immunodeficiency]
[2-1]免疫正常マウスからの脳梗塞モデルの作成  [2-1] Creation of cerebral infarction model from immune-normal mouse
上述のように、免疫不全マウスである SCIDマウスの脳梗塞モデル力も採取した骨 髄を培養することで、多くの神経幹細胞 (ニューロスフェア）が形成された。そこで、こ の現象がマウスの免疫不全と関係があるかどうかを検討する目的で、 SCIDマウスの 母系マウスである免疫正常な C.B- 17/IcrCrlBRマウス（以下、「C. B17マウス」という。 )に対して、同様の方法で脳梗塞モデルを作成して脳梗塞 SCIDマウスと比較し、そ の骨髄から神経幹細胞が分ィ匕誘導されるか等について検討した。  As described above, many neural stem cells (neurospheres) were formed by culturing the bone marrow from which cerebral infarction model power of SCID mice, which are immunodeficient mice, was also collected. Therefore, for the purpose of examining whether this phenomenon is related to immunodeficiency in mice, immunologically normal CB-17 / IcrCrlBR mice (hereinafter referred to as “C. B17 mice”), which are maternal mice of SCID mice. On the other hand, a cerebral infarction model was created by the same method and compared with cerebral infarction SCID mice to examine whether neural stem cells were induced from the bone marrow.
[0083] C. B17マウスからの脳梗塞モデルの作成は、上述の脳梗塞 SCIDマウスの作成方 法と同様である。すなわち、 3%ハロセン麻酔下にマウス左頰骨を切除し、頭蓋底を 露出した。中大脳動脈走行部位に直径 1. 5mmの骨窓を歯科用ドリルで作成した。 硬膜、クモ膜を剥離し、中大脳動脈を分離して結紮準備とした。中大脳動脈結紮法と しては、凝固用ピンセットにて電気凝固後に切断する永久結紮法、動脈瑠結紮用タリ ップを用いた一過性結紮法などが可能である。結紮部位は、動脈が嗅索を通過した 直後、すなわち distal Ml portionである。この部位を結紮することにより、中大脳動脈 の皮質枝の血流を選択的に途絶させることが可能である。 [0083] C. The creation of a cerebral infarction model from B17 mice is the same as the above-described method for producing cerebral infarction SCID mice. That is, the mouse left rib was excised under 3% halothane anesthesia and the skull base was exposed. A bone window with a diameter of 1.5 mm was created with a dental drill in the middle cerebral artery running site. The dura mater and arachnoid membrane were removed, and the middle cerebral artery was separated to prepare for ligation. As the middle cerebral artery ligation method, a permanent ligation method in which cutting is performed after electrocoagulation with coagulation tweezers, or a transient ligation method using a tip for arterial ligation is possible. The ligation site is immediately after the artery passes through the olfactory tract, that is, the distal Ml portion. By ligating this site, the middle cerebral artery It is possible to selectively disrupt the blood flow in the cortical branch of the.
[0084] このような中大脳動脈結紮法により、 C. B 17マウス左中大脳動脈の distal Ml porti onを結紮して作成した脳梗塞モデルマウス (脳梗塞 C. B17マウス）の脳梗塞領域を 、実際に TTC染色法にて検討した。 TTC染色法は、結紮後（MCO) 1, 3, 7日目に それぞれマウス脳を摘出し、 brain slicerにて作成した冠状脳スライスを用いて施行し た。その結果、各群 4匹、計 12匹はすべて左中大脳動脈皮質枝領域に選択的に梗 塞が作成され、脳梗塞部位は極めて均一であった。図 7には、結紮後（MCO) 1日目 に摘出した脳の染色結果力 脳梗塞 SCIDマウスのものと対比して示される（白い部 分が梗塞部位)。  [0084] The cerebral infarction region of the cerebral infarction model mouse (cerebral infarction C. B17 mouse) prepared by ligating the distal Ml cation of the left middle cerebral artery of the C. B 17 mouse by such a ligation method of the middle cerebral artery Actually, TTC staining was used. TTC staining was performed using coronary brain slices prepared by brain slicer after excision of mouse brains on days 1, 3, and 7 after ligation (MCO). As a result, 4 animals in each group, a total of 12 animals, all made infarcts selectively in the left middle cerebral artery cortical branch region, and the cerebral infarction sites were very uniform. FIG. 7 shows the result of staining of the brain extracted on day 1 after ligation (MCO). In contrast to that of SCID mice (white part is the infarct site).
[0085] C. B17マウス、および SCIDマウスにおける中大脳動脈閉塞後の脳梗塞の様子を 摘出脳で観察した。マウス左中大脳動脈の distal Ml portionを結紮し、 3, 7, 16, 28 日目にマウスを経心的に PLP固定液で灌流固定後、脳を摘出した。その結果、摘出 脳は全例中大脳動脈領域の欠損が観察され（図 6には、結紮後 16日目のものが示さ れる）、 SCIDマウスの母系である C. B 17マウスを用いた場合も、 SCIDマウスと同様 に再現性のよい均一な脳梗塞モデルを作成できることが明らかになった。  [0085] The state of cerebral infarction after middle cerebral artery occlusion in C. B17 mice and SCID mice was observed in the isolated brain. The distal Ml portion of the mouse left middle cerebral artery was ligated, and the mouse was transcardially fixed with PLP fixative solution on days 3, 7, 16, and 28, and the brain was removed. As a result, in the isolated brain, a defect in the middle cerebral artery region was observed in all cases (Fig. 6 shows the 16th day after ligation), and when the C. B 17 mouse, the mother of SCID mice, was used It was also found that a uniform cerebral infarction model with good reproducibility can be created as with SCID mice.
[0086] ところで、 SCIDマウスから作成した脳梗塞モデルマウスでは、虚血後の脳梗塞の 進展（delayed infarct expansion)は脳梗塞後 3日で終了し、その後は脳萎縮の進展 ではなぐ脳形態上はむしろ回復することが認められている（特願 2004— 108500 号参照）。このことは、脳梗塞 SCIDマウスが脳梗塞モデルマウスとしてのみならず、 脳再生モデルとしても適して 、ることを示して 、る。  [0086] By the way, in the cerebral infarction model mouse prepared from the SCID mouse, the delayed infarct expansion after ischemia was completed in 3 days after the cerebral infarction, and after that, the brain morphology was not improved. Is rather allowed to recover (see Japanese Patent Application No. 2004-108500). This indicates that cerebral infarction SCID mice are suitable not only as cerebral infarction model mice but also as brain regeneration models.
[0087] そこで次に、脳梗塞 C. B17マウスにおいても、このような脳形態上の回復が認めら れるか検討した。上述のように、 C. B17マウスおよび SCIDマウスの左中大脳動脈の distal Ml portionを結紮し、 3, 7, 14, 28日目にマウスを経心的に PLP固定液で灌 流固定後、脳を摘出し、残存大脳皮質の大きさを定量ィ匕することで、脳欠損の程度、 回復 ·再生の有無を観察した。具体的には、梗塞側大脳皮質の大脳裂力も梗塞部位 までの幅（a)を正常側（b)と比較し、その比（aZb)を cortical width index (CI値）とし て算出した（図 20参照)。その結果、図 8に示すように、脳梗塞 SCIDマウス、脳梗塞 C. B17マウスとも CI値は 3日目力 7日目までは 0. 34とほぼ一定であった。しかし、 SCIDマウスでは 28日目に 0. 37と増カロし、回復 '再生が認められたのに対し、 C. B 17マウスでは 28日目でも 0. 34と一定であった。この結果から、脳梗塞 C. B17マウ スでは、脳梗塞 SCIDマウスとは異なり、脳形態上の回復は認められず、脳再生モデ ルとはいえなかった。 [0087] Then, next, it was examined whether or not such a recovery in brain morphology was also observed in cerebral infarction C. B17 mice. As described above, the distal Ml portion of the left middle cerebral artery of C. B17 mice and SCID mice was ligated, and the mice were transfusionally fixed with PLP fixative solution on days 3, 7, 14, and 28. By removing the brain and quantifying the size of the remaining cerebral cortex, we observed the degree of brain deficiency and the presence or absence of recovery / regeneration. Specifically, the cerebral cleft force of the infarcted cerebral cortex was also calculated by comparing the width (a) to the infarcted site with the normal side (b) and the ratio (aZb) as the cortical width index (CI value) (Fig. 20). As a result, as shown in FIG. 8, the CI value was constant at 0.34 until the third day and the seventh day in both the cerebral infarction SCID mouse and the cerebral infarction C. B17 mouse. But, In SCID mice, the calorie increased to 0.37 on the 28th day, and recovery 'regeneration was observed, whereas in the C.B17 mice, it was constant at 0.34 even on the 28th day. From these results, cerebral infarction C. B17 mice, unlike cerebral infarction SCID mice, showed no recovery in brain morphology and could not be said to be a brain regeneration model.
[0088] [2-2]脳梗塞 C. B17マウスにおける神経幹細胞等の発現検討  [0088] [2-2] Cerebral infarction C. Neural stem cell expression in B17 mice
脳梗塞 C. B17マウスは、 CI値では脳再生が認められなかった力 梗塞後のマウス 脳に神経幹細胞等が形成されているかどうかを、神経幹細胞や神経細胞、グリア細 胞の各種マーカーを用いた免疫組織ィ匕学で検討した。  Cerebral infarction C. B17 mice were not able to regenerate the brain with CI values. After the infarction, we used various markers of neural stem cells, nerve cells, and glial cells to determine whether neural stem cells were formed in the brain. It was examined by immunohistochemistry.
[0089] 具体的には、神経幹細胞に対しては抗ムサシ 1 (Musashil)抗体を用い、未熟神経 細胞の同定には Doublecortin (DCX)を用いた。オリゴデンドロサイト前駆細胞は Plat elet— derived Growth Factor Receptor (PDGFR a )と NG2をマーカーとし、前およ び未熟オリゴデンドロサイトに対しては 04と Myelin- associated Glycoprotein (MAG) を用いた。ァストロサイトは抗 GFAP抗体で同定した。さらに、未熟神経細胞や軸索 伸長過程にある神経のマーカーとして PSA—NCAMを用いた。 PSA—NCAMは， 培養神経幹細胞の細胞膜に発現することが確認されている。また、 Nカドヘリン (N-c adherin)も-ユーロスフ アに発現することが確認されている。これらは、ニューロスフ エアの分化誘導シグナル調節に深く関与して 、る。  [0089] Specifically, anti-musashi 1 antibody was used for neural stem cells, and Doublecortin (DCX) was used to identify immature nerve cells. Oligodendrocyte progenitor cells used the platelet-derived growth factor receptor (PDGFR a) and NG2 as markers, and 04 and Myelin-associated Glycoprotein (MAG) were used for pre- and immature oligodendrocytes. Astrocytes were identified with anti-GFAP antibody. Furthermore, PSA-NCAM was used as a marker for immature neurons and nerves in the process of axon elongation. PSA-NCAM has been confirmed to be expressed in the cell membrane of cultured neural stem cells. In addition, it has been confirmed that N-cadherin is also expressed in -eurosphere. These are deeply involved in the regulation of neuronal differentiation signal.
[0090] 上述の各種マーカーを用いて、脳梗塞 C. B17マウスの脳室下帯組織（subventricu lar zone : SVZ)、および脳梗塞下の組織における神経幹細胞等の産生について検 討した。なお、脳梗塞下の組織とは、梗塞組織 (脳梗塞瘢痕部位)および梗塞組織と 白質 (脳梁）とが接する脳梗塞周囲の部位のことである。この脳梗塞下の組織は、脳 室下帯組織 (SVZ)とともに、脳梗塞 SCIDマウスにおいて多数の神経幹細胞の出現 (即ち、脳再生）が観察された部位である（特願 2004— 108500号参照)。また、この 脳梗塞下の組織における神経幹細胞は、骨髄に由来するものであった。  [0090] Using the various markers described above, the production of neural stem cells and the like in the subventricular zone tissue (SVZ) of cerebral infarction C. B17 mice and the tissue under cerebral infarction was examined. The tissue under cerebral infarction is the infarcted tissue (cerebral infarction scar site) and the site around the cerebral infarction where the infarcted tissue and white matter (corpus callosum) contact. This tissue under cerebral infarction is the site where numerous neural stem cells appeared (ie, brain regeneration) in cerebral infarcted SCID mice, as well as subventricular zone tissue (SVZ) (see Japanese Patent Application No. 2004-108500). ). The neural stem cells in the tissue under cerebral infarction were derived from bone marrow.
[0091] [2-3]脳室下帯組織 (SVZ)における神経幹細胞等の発現  [0091] [2-3] Expression of neural stem cells in subventricular zone tissue (SVZ)
脳梗塞 C. B17マウス作成後 1, 7, 14, 35日目にマウスを経心的に PLP固定液で 灌流固定後、脳を摘出し、ビブラトームを用いて脳切片を作成し、その後、免疫組織 化学を行った。 [0092] 梗塞後 1日目には、 SVZの各種マーカー発現はすべて正常側と差はな力つた。 7 日目になると、 Musashil陽性、 PSA—NCAM陽性の細胞力 SVZに増殖するように なった。 14日目になると、これらの神経幹細胞は脳室膨大部から白質内に侵入する ようにみられた。 35日目には、 SVZでの発現細胞は減少しコントロールレベルとなつ た力 脳室下の白質には多数の Musashil陽性、 PSA— NCAM陽性の細胞が認め られた。同部位には、 DCX陽性の未熟神経細胞や、小型の NeuN陽性細胞もみら れた。また、梗塞後 7日目より SVZには NG2陽性、 PDGFR o;陽性のオリゴデンドロ サイト前駆細胞も発現するようになった。これらは成熟オリゴデンドロサイトマーカーを 有しておらず、 14日目までみられた力 35日目には減少した。 Cerebral infarction C. On days 1, 7, 14, and 35 after the creation of B17 mice, the mice were transcardially fixed with PLP fixative solution, the brain was removed, and brain sections were prepared using a vibratome. Histochemistry was performed. [0092] On the first day after infarction, the expression of various markers of SVZ was all different from the normal side. On day 7, it grew to Musashil-positive, PSA-NCAM-positive cell strength SVZ. On the 14th day, these neural stem cells appeared to invade the white matter from the enormous part of the ventricle. On day 35, the number of cells expressed by SVZ decreased and reached the control level. Many Musashil-positive and PSA-NCAM-positive cells were observed in the white matter under the ventricle. At the same site, DCX-positive immature neurons and small NeuN-positive cells were also found. From 7 days after infarction, SVZ also expressed NG2-positive, PDGFR o; -positive oligodendrocyte progenitor cells. These had no mature oligodendrocyte markers and were reduced to force 35, which was seen until day 14.
[0093] このように、 SVZでは梗塞後 7日目以降、 14日目をピークに神経幹細胞の増殖が みられ、以後減少する。神経幹細胞としては脳室下の白質に 35日目までは存在する 力 以後は成熟して神経となる力 あるいは死滅してしまうと考えられる。 SVZには梗 塞後 7日目には Notchl陽性細胞がみられた力 それ以降はみられなかったことから 、 SVZ細胞の分ィ匕増殖シグナルは、梗塞後 1週間程度で終了すると考えられた。  [0093] Thus, in SVZ, neural stem cells proliferate after the 7th day after the infarction and peak on the 14th day, and then decrease. Neural stem cells are considered to be the force that exists in the white matter under the ventricle until the 35th day. In SVZ, notchl-positive cells were observed on the 7th day after infarction. Since it was not observed thereafter, the SVZ cell division signal was considered to end in about one week after infarction. .
[0094] [2-4] C. B17マウス梗塞下組織における神経幹細胞の発現抑制  [0094] [2-4] C. Suppression of neural stem cell expression in B17 mouse infarcted tissue
C. B17マウスおよび SCIDマウスに対して脳梗塞作成後 21日目のマウス脳のビブ ラトーム切片を用いて、 NeuNに対する免疫組織ィ匕学を施行した。その結果を図 9に 示す。脳梗塞 SCIDマウスの梗塞下組織（白質領域）には多数の小型 NeuN陽性細 胞 (神経前駆細胞）が観察されたが（同図 A)、脳梗塞 C. B17マウスの梗塞下組織は 、ほとんどマクロファージで占拠されており、 PSA— NCAM陽性の神経幹細胞や小 型の NeuN陽性細胞は梗塞後 35日目までほとんど認められな力つた（同図 B)。この ことは、 C. B17マウスの正常な免疫反応が、梗塞下組織 (脳梗塞瘢痕部位を含む） における神経幹細胞発現 ·神経再生に抑制的に作用して 、ることを示唆するもので ある。  C. Immunohistochemistry for NeuN was performed using a vibratome section of the mouse brain 21 days after the creation of cerebral infarction in B17 mice and SCID mice. The results are shown in Fig. 9. Cerebral infarction Although many small NeuN positive cells (neural progenitor cells) were observed in the infarcted tissue (white matter region) of SCID mice (Fig. A), the infarcted tissue of cerebral infarction C. B17 mice is almost Occupied by macrophages, PSA-NCAM-positive neural stem cells and small NeuN-positive cells were almost unrecognized until 35 days after infarction (Fig. B). This suggests that the normal immune response of C. B17 mice suppresses neural stem cell expression and nerve regeneration in subinfarcted tissues (including cerebral infarction scar sites).
[0095] 次に、脳梗塞下組織力ゝら採取した細胞を培養して神経幹細胞が形成されるかどう か検討した。結紮後 7日目の脳梗塞 C. B17マウスおよび脳梗塞 SCIDマウスの梗塞 下組織を採取し、その後、 DMEMと N— 2の基本培養液内（250 1)で単細胞にな るまでピペッティングし、 10mlの培養液をカ卩えて 600rpmで 5分間遠心した。細胞を 3mlの培養液にて再浮遊させ、 bFGF (50 μ g/ml)と EGF (20 μ g/ml)存在下に 1 ow cell bindingプレート上で 29日間培養を行い、新たに形成される-ユーロスフェア 様細胞塊数を数えた。その結果、図 12に示すように、脳梗塞 SCIDマウスの梗塞下 組織からは培養 5日目以降に-ユーロスフェア様細胞塊の形成が観察されたが、脳 梗塞 C. B17マウスの梗塞下組織からは-ユーロスフェア様細胞塊は全く形成されな かった。 [0095] Next, it was examined whether neural stem cells were formed by culturing cells collected from tissue force under cerebral infarction. Cerebral infarction 7 days after ligation C. B17 mice and cerebral infarction SCID mouse infarcted tissues are collected, and then pipetted into single cells in basic culture medium (250 1) of DMEM and N-2. 10 ml of the culture solution was added and centrifuged at 600 rpm for 5 minutes. Cells Resuspended in 3 ml of culture and cultured for 29 days on a 1 ow cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml). The number of sphere-like cell clusters was counted. As a result, as shown in FIG. 12, from the infarcted tissue of the cerebral infarction SCID mouse, the formation of the eurosphere-like cell cluster was observed after the fifth day of culture, but the infarcted tissue of the cerebral infarction C. B17 mouse From-no eurosphere-like cell mass was formed.
[0096] [2-5]C. B17マウスにおける骨髄由来-ユーロスフェア様細胞塊の形成  [0096] [2-5] C. Formation of Bone Marrow-Eurosphere-Like Cell Mass in B17 Mice
脳梗塞作成後 7日目の SCIDマウスと C. B17マウスの骨髄を 28日間培養し、ニュ 一口スフエア様細胞塊が形成されるかどうかを観察した。その結果、 SCIDマウスの骨 髄からは培養 2週間目までに-ユーロスフェア様細胞塊が形成された（図 10Α·Β)。 一方、 C. B17マウスの骨髄からは培養開始 7日目までにわずかに-ユーロスフェア 様細胞塊が形成されたが（同図 C'D)、これらは 2週間以内にすべて消失した。  Seven days after the creation of the cerebral infarction, the bone marrow of SCID mice and C. B17 mice was cultured for 28 days, and it was observed whether or not a single-mouthed sphere-like cell mass was formed. As a result, -Eurosphere-like cell clusters were formed from the bone marrow of SCID mice by the second week of culture (FIG. 10 Α · Α). On the other hand, from the bone marrow of C.B17 mice, a slight amount of -eurosphere-like cell mass was formed by the seventh day of culture (C'D in the figure), but all of these disappeared within 2 weeks.
[0097] そこで、ニューロスフェア様細胞塊における細胞死のメカニズムを探る目的で、培養 後同時期の SCIDマウス由来と C. B17マウス由来の-ユーロスフェア様細胞塊を An exinVで標識してアポトーシスを検討した。その結果、 SCIDマウス由来の-ユーロス フェア様細胞塊では、細胞中心部が少しネクローシスに陥っているのみであつたが（ 図 11A参照）、 C. B17マウス由来の-ユーロスフェア様細胞塊では、内側の細胞は ほとんどがネクローシスで、外側の細胞はすべてアポトーシスに陥っていた（同図 B参 照）。この結果から、脳梗塞 C. B17マウス由来の-ユーロスフェア様細胞塊は、アポ トーシスで消滅することが示された。  [0097] Therefore, for the purpose of investigating the mechanism of cell death in neurosphere-like cell clusters, apoptosis was observed by labeling the eurosphere-like cell clusters derived from SCID mice and C. B17 mice at the same time after culture with AnexinV. investigated. As a result, in the -Eurosphere-like cell mass derived from SCID mice, the cell center part was only slightly necrotic (see Fig. 11A), but in the -Eurosphere-like cell mass derived from C. B17 mice, The inner cells were mostly necrotic and the outer cells were all apoptotic (see Figure B). From this result, it was shown that -Eurosphere-like cell clusters derived from cerebral infarction C. B17 mice disappeared by apoptosis.
[0098] また、脳梗塞を起こしていない偽手術 C. B17マウスの骨髄からは、ニューロスフエ ァ様細胞塊は全く形成されな力つた。さらに、偽手術 C. B17マウス力も採取した骨 髄を、脳梗塞 C. B17マウスの血清を添カ卩して培養すると、ニューロスフェア様細胞塊 がー且形成されるがすぐに排除され、ニューロスフェア様細胞塊を得ることはできな かった。  [0098] From the bone marrow of sham-operated C. B17 mice without cerebral infarction, no neurosphere-like cell mass was formed. Furthermore, when bone marrow obtained from sham-operated C. B17 mice was cultured with cerebral infarction C. B17 mouse serum, a neurosphere-like cell mass formed and was immediately eliminated, but the neurosphere was eliminated. A sphere-like cell mass could not be obtained.
[0099] [2-6]本実施例の結果と考察  [0099] [2-6] Results and discussion of this example
以上のように、免疫不全マウスである SCIDマウスの脳梗塞モデル力も採取した脳 梗塞下組織 (脳梗塞瘢痕部位)や骨髄からは、大量の-ユーロスフェアが形成された 。これに対して、その母系マウスである免疫正常な C. B17マウスの脳梗塞モデルか ら採取した脳梗塞瘢痕部位や骨髄からは、ニューロスフェアはほとんど形成されなか つた。これらの結果から、正常な免疫反応が骨髄および脳梗塞瘢痕部位における神 経幹細胞 (ニューロスフ ア）の発現に抑制的に作用していると考えられる。 As described above, a large amount of -Eurosphere was formed from subcerebral infarction tissue (cerebral infarction scar site) and bone marrow from which the brain infarction model force of SCID mice, which are immunodeficient mice, was also collected. . In contrast, almost no neurospheres were formed from the cerebral infarct scar site and bone marrow collected from the cerebral infarction model of the immunologically normal C. B17 mouse, which is the mother mouse. From these results, it is considered that a normal immune response suppresses the expression of neuronal stem cells (neurospheres) in bone marrow and cerebral infarction scar sites.
[0100] 〔実施例 3：免疫抑制剤を用いた骨髄 '脳梗塞下組織 (脳梗塞瘢痕部位)力ゝらの神経 幹細胞調製法〕 [Example 3: Bone marrow using an immunosuppressant 'subcerebral infarcted tissue (cerebral infarction scar site) Nervous stem cell preparation method]
上述のように、脳梗塞後の神経再生 (神経幹細胞の発現'形成）に免疫の関与が考 えられたので、この推論を確かめるため、脳梗塞後の骨髄'脳梗塞瘢痕部位からの 神経幹細胞の形成に対する免疫抑制の効果を in vivoと in vitro双方で検討した。  As described above, immunity was considered to be involved in nerve regeneration (expression of neural stem cells) after cerebral infarction. To confirm this reasoning, neural stem cells from cerebral infarction scar sites were confirmed. The effect of immunosuppression on the formation of phenotypes was examined both in vivo and in vitro.
[0101] [3-1]免疫抑制剤を投与した C. B17マウス骨髄'脳梗塞下組織 (脳梗塞瘢痕部位） 力 の-ユーロスフェア形成 [0101] [3-1] C-B17 mouse bone marrow administered with immunosuppressive agent 'subcerebral infarct tissue (cerebral infarction scar site)-Eurosphere formation
C. B17マウスに免疫抑制剤 FK506 (1. OmgZkg)を前投与すなわち 3日間連日 腹腔内投与し、その後、左中大脳動脈閉塞による脳梗塞を作成した。脳梗塞後 (結 紮後)も FK506 (1. OmgZkg)投与を継続し、脳梗塞後 7日目に骨髄と脳梗塞瘢痕 部位とを採取して DMEMと N— 2の基本培養液内（250 μ 1)で単細胞になるまでピ ペッティングし、 10mlの培養液をカ卩えて 600rpmで 5分間遠心した。細胞を 3mlの培 養液にて再浮遊させ、 bFGF (50 μ g/ml)と EGF (20 μ g/ml)存在下に low cell bi ndingプレート上で 10— 28日間培養を行った。図 13に示すように、培養 7日目以降 には骨髄 (Α·Β)と脳梗塞瘢痕部位 (C)から-ユーロスフェア様細胞塊が細胞顕微鏡 下で観察された。  C. The immunosuppressant FK506 (1. OmgZkg) was pre-administered to B17 mice, that is, intraperitoneally administered daily for 3 days, and then a cerebral infarction due to left middle cerebral artery occlusion was created. After cerebral infarction (after ligation), administration of FK506 (1. OmgZkg) was continued. On the 7th day after cerebral infarction, bone marrow and cerebral infarct scar site were collected, and DMEM and N-2 in a basic culture solution (250 Pipette to 1 cell with μ1), add 10ml of culture and centrifuge at 600rpm for 5 minutes. Cells were resuspended in 3 ml of culture medium and cultured on low cell binding plates in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. As shown in FIG. 13, after the 7th day of culture, -eurosphere-like cell clusters from the bone marrow (Α · Β) and the cerebral infarction scar site (C) were observed under a cell microscope.
一方、偽手術 C. B17マウスに FK506 (1. OmgZkg)を 10日間連日腹腔内投与し ただけの培養骨髄からは-ユーロスフェア様細胞塊はほとんど形成されな力つた。  On the other hand, sham-operated C. B17 mice showed vigorous formation of -Eurosphere-like cell clusters from cultured bone marrow that had been administered intraperitoneally daily for 10 days with FK506 (1. OmgZkg).
[0102] [3-2]脳梗塞 C. B17マウス骨髄に免疫抑制剤を添加した場合の-ユーロスフェア形 成 [0102] [3-2] Cerebral infarction C. Eurosphere formation when immunosuppressant is added to bone marrow of B17 mice
次に、脳梗塞作成後 7日目の C. B17マウス骨髄を培養する際に免疫抑制剤 FK5 06 (0. 1 μ g/ml)を培養液に添カ卩して-ユーロスフェア様細胞塊が形成されるかど うかを観察した。すなわち、骨髄細胞を DMEMと N— 2の基本培養液内（250 1)で 単細胞になるまでピペッティングし、 10mlの培養液を加えて 600rpmで 5分間遠心し た。細胞を FK506 (0. 1 μ g/ml)を含む 3mlの培養液にて再浮遊させ、 low cell bin dingプレート上で 10— 28日間培養を行った。すると、培養 4日目以降に-ユーロスフ ア様細胞塊が細胞顕微鏡下で観察された（図 14Α· B)。 Next, when culturing C. B17 mouse bone marrow on the 7th day after the creation of cerebral infarction, an immunosuppressant FK5 06 (0.1 μg / ml) was added to the culture solution-Eurosphere-like cell mass We observed whether or not it was formed. In other words, bone marrow cells were pipetted into single cells in DMEM and N-2 basic medium (250 1), added with 10 ml of medium, and centrifuged at 600 rpm for 5 minutes. It was. The cells were resuspended in 3 ml of medium containing FK506 (0.1 μg / ml) and cultured on low cell binding plates for 10-28 days. Then, after the fourth day of culture, a -eurosphere-like cell cluster was observed under a cell microscope (FIG. 14B).
[0103] 即ち図 15に示すように、脳梗塞作成後 7日目 . B 17マウス骨髄を培養しても、 ほとんど-ユーロスフェア様細胞塊は形成されなかったが（C.B (MC01W))、上述の ように、 FK506 (0. 1 μ g/ml)を培養液に添加すると-ユーロスフェア様細胞塊が 形成された（C.B (MC01W) + FK506) oこれは培養 4日目以降に観察され、 SCIDマ ウスの脳梗塞後における形成 (SCID (MCO 1W))よりも早期に見られた。 That is, as shown in FIG. 15, 7 days after the creation of cerebral infarction. Even though B 17 mouse bone marrow was cultured, almost no eurosphere-like cell mass was formed (CB (MC01W)). As shown above, when FK506 (0.1 μg / ml) was added to the culture solution-a eurosphere-like cell mass was formed (CB (MC01W) + FK506) o This was observed after the 4th day of culture, It was found earlier than the formation of SCID mice after cerebral infarction (SCID (MCO 1W)).
[0104] 一方、脳梗塞を起こしていない偽手術 C. B17マウス骨髄に対しては、  [0104] On the other hand, for sham operation C. B17 mouse bone marrow without cerebral infarction,
( 1) bFGF (50 g/ml)と EGF (20 μ g/ml)のみ、  (1) Only bFGF (50 g / ml) and EGF (20 μg / ml)
(2) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の SCIDマウスの 血清（50 /z l)、  (2) bFGF (50 μg / ml) and EGF (20 μg / ml) + serum of SCID mice 1 week after cerebral infarction (50 / z l),
(3) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の SCIDマウスの 血清 (50 μ 1) +FK506 (0. 1 μ g/ml)、  (3) bFGF (50 μg / ml) and EGF (20 μg / ml) + SCID mouse serum 1 week after cerebral infarction (50 μ1) + FK506 (0.1 μg / ml),
の 3種類の培養液を用意した。  Three types of culture solutions were prepared.
[0105] すると、培養 4日目以降に上記（3)の培養液中にのみ-ユーロスフェア様細胞塊が 細胞顕微鏡下で観察された（図 16B ' C)。一方、上記（2)の培養液からは-ユーロス フェア様細胞塊がー且形成される力 すぐに排除され-ユーロスフェアは得られなか つた。これらのことから、骨髄における-ユーロスフェア形成に抑制的に作用している 免疫反応を排除することにより神経幹細胞が形成されること、骨髄から異所性に神経 幹細胞が形成されるためには Τ細胞機能など細胞性免疫が抑制されなければならな いことが明らかになった。  [0105] Then, after the 4th day of culture, only-Eurosphere-like cell clusters were observed under the cell microscope in the culture solution of (3) above (Fig. 16B'C). On the other hand, from the culture solution of (2) above, the force to form and form the eurosphere-like cell mass was immediately eliminated, and the eurosphere was not obtained. From these facts, it is necessary to eliminate neural reactions that suppress the eurosphere formation in the bone marrow, thereby forming neural stem cells, and for ectopic formation of neural stem cells from the bone marrow. It became clear that cellular immunity such as cell function must be suppressed.
[0106] [3- 3] C. B17マウス骨髄に抗 T細胞抗体を添カ卩した場合の-ユーロスフェア形成  [0-3] [3-3] C. -Eurosphere formation when B17 mouse bone marrow is supplemented with anti-T cell antibody
CD28および ICOS (inducible costimulatory molecule)は、多くの T細胞依存'性免 疫反応における受容体であり、そのブロッキング抗体である抗 CD28抗体および抗 I COS抗体は、 T細胞機能を抑制することが知られている（Am. J. Respir. Cell Mol. Bi ol. (1997) 16:335- 342. ; J. Immunol. (2000) 165:5035- 5040.等）。そこで、 FK506以 外に、このような T細胞機能を抑制する抗体 (抗 T細胞抗体)を添加した場合も、 FK5 06を添加した場合と同様に神経幹細胞が形成されるかどうかを検討した。 CD28 and ICOS (inducible costimulatory molecule) are receptors in many T cell-dependent immune responses, and their blocking antibodies, anti-CD28 and anti-ICOS antibodies, are known to suppress T cell function. (Am. J. Respir. Cell Mol. Biol. (1997) 16: 335-342 .; J. Immunol. (2000) 165: 5035- 5040. etc.). Therefore, in addition to FK506, when an antibody that suppresses such T cell function (anti-T cell antibody) is added, FK5 Whether or not neural stem cells were formed was examined in the same manner as when 06 was added.
[0107] 脳梗塞を起こしていない偽手術 C. B17マウス骨髄に対して、 [0107] Sham surgery without cerebral infarction
( 1) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の SCIDマウスの 血清 (50 μ 1) +FK506 (0. 1 μ g/ml)、  (1) bFGF (50 μg / ml) and EGF (20 μg / ml) + serum of SCID mice 1 week after cerebral infarction (50 μ1) + FK506 (0.1 μg / ml),
(2) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の SCIDマウスの 血清（50 μ 1) +抗 CD28抗体（2 μ g/ml, BD Biosciences)、  (2) bFGF (50 μg / ml) and EGF (20 μg / ml) + SCID mouse serum 1 week after cerebral infarction (50 μ1) + anti-CD28 antibody (2 μg / ml, BD Biosciences ),
(3) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の SCIDマウスの 血清（50 μ 1) +抗 ICOS抗体（4 μ g/ml, BD Biosciences)、  (3) bFGF (50 μg / ml) and EGF (20 μg / ml) + SCID mouse serum 1 week after cerebral infarction (50 μ1) + anti-ICOS antibody (4 μg / ml, BD Biosciences ),
および、コントロールとして、  And as a control,
(4) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の SCIDマウスの 血清（50 1) +生理食塩水（saline)、  (4) bFGF (50 μg / ml) and EGF (20 μg / ml) + SCID mouse serum (50 1) + saline (saline) 1 week after cerebral infarction,
の 4種類の培養液を用意した。  Four types of culture solutions were prepared.
[0108] すると、培養 4日目以降に上記 (4)以外の培養液中に-ユーロスフ ア様細胞塊が 細胞顕微鏡下で観察された（図 21)。さらに、浮遊している-ユーロスフェア様細胞塊 を採取して high bindingプレート上で更に培養した。 7日後に分ィ匕した細胞をパラフォ ルムアルデヒドを含む固定液にて固定し、 nestin陽性の-ユーロスフェア数を計測し たところ、 ldishあたりの nestin陽性-ユーロスフェア数は、上記（4)の saline群に比し F K506添加群（1)ゃ抗 CD28抗体添加群（2)、抗 ICOS抗体添加群（3)で有意に多 かった（図 27)。 [0108] Then, after the 4th day of culture, -Eurosphere-like cell clusters were observed under the cell microscope in the culture solutions other than the above (4) (Fig. 21). In addition, floating-eurosphere-like cell clusters were collected and further cultured on high binding plates. Cells separated after 7 days were fixed with a fixative containing paraformaldehyde, and the number of nestin positive-eurospheres was measured. The number of nestin positive-eurospheres per ldish was as described in (4) above. Compared to the saline group, the FK506 addition group (1) was significantly more in the anti-CD28 antibody addition group (2) and the anti-ICOS antibody addition group (3) (FIG. 27).
以上の結果から、 T細胞機能を抑制する抗 T細胞抗体を添加することにより、 FK50 6を添加した場合と同様に神経幹細胞が形成されること、および、骨髄から神経幹細 胞が形成されるためには T細胞機能を抑制することが重要であることが明らかになつ た。  From the above results, by adding anti-T cell antibody that suppresses T cell function, neural stem cells are formed in the same manner as when FK506 is added, and neural stem cells are formed from bone marrow. For this purpose, it became clear that it is important to suppress T cell function.
[0109] [3-4] C. B17マウス力 調製された-ユーロスフェア様細胞塊の神経細胞への分化 能  [0109] [3-4] C. B17 mouse force -Eurosphere-like cell clusters prepared into neurons
脳梗塞を起こしていない偽手術 C. B17マウス骨髄に FK506と血清を添カ卩して培 養し、 10日目に浮遊している-ユーロスフェア様細胞塊を採取して high bindingプレ ート上でさらに培養した。 3— 7日後に分ィ匕した細胞をバラフオルムアルデヒドを含む 固定液にて固定して、 Nestinと MAP2に対する免疫組織化学（二重染色間接蛍光 抗体法)を行った。その結果、これらの中には Nestin陽性の神経幹細胞が高率に観 察された（図 17)。同図に示すように、細胞塊の中心は Nestin陽性（明灰色)で神経 幹細胞であり、周囲にはより分ィ匕した MAP2陽性の神経細胞（B音灰色）が観察された 。また、上記-ユーロスフェア様細胞塊について神経への分ィ匕能を検討した結果、骨 髄培養力 得られた-ユーロスフェア様細胞塊の約 60%が Nestin陽性の神経幹細 胞で、そのうち 67%が MAP2陽性の神経細胞に分ィ匕した。 Sham surgery without cerebral infarction C. B17 mouse bone marrow supplemented with FK506 and serum, floating on day 10-Eurosphere-like cell mass collected and high binding plate Further culture on above. 3— Cells separated after 7 days contain roseformaldehyde After fixing with a fixative, immunohistochemistry (double staining indirect fluorescent antibody method) against Nestin and MAP2 was performed. As a result, Nestin-positive neural stem cells were observed at a high rate (Fig. 17). As shown in the figure, the center of the cell cluster was Nestin positive (light gray) and neural stem cells, and more separated MAP2 positive neurons (B tone gray) were observed. In addition, as a result of examining the ability of the above-mentioned eurosphere-like cell mass to be distributed to the nerve, bone marrow culture ability was obtained.- About 60% of the eurosphere-like cell mass was Nestin-positive neural stem cells, of which 67% were divided into MAP2-positive neurons.
[0110] [3- 5]C. B17マウスの骨髄と脳梗塞下組織 (脳梗塞瘢痕部位)から形成される-ュ 一口スフエアの相違 [0110] [3-5] C. B17 mice formed from bone marrow and subcerebral infarcted tissue (cerebral infarction scar site)
上述のように、 C. B17マウスの脳梗塞後の骨髄からは-ユーロスフェア様の細胞塊 がー且は形成されるが、脳梗塞瘢痕部位からは-ユーロスフェア様細胞塊の痕跡す ら認められな力つた。このことは、骨髄では脳梗塞後に血清中の刺激因子を介して神 経幹細胞産生への誘導が生じるものの、正常な T細胞機能があるとその影響により 幹細胞が排除され、脳に到達できないものと考えられる（図 12)。  As mentioned above, the bone marrow after cerebral infarction in C. B17 mice-and the formation of eurosphere-like cell clusters-and from the cerebral infarction scar site-even the traces of the eurosphere-like cell clusters are observed. I was helped. This means that in the bone marrow, neural stem cell production is induced via a stimulating factor in serum after cerebral infarction, but if there is normal T cell function, stem cells are excluded due to the effect and cannot reach the brain. Possible (Figure 12).
[0111] [3-6]本実施例の結果と考察 [0111] [3-6] Results and discussion of this example
以上のように、 C. B17マウスに免疫抑制剤である FK506を前投与し、 T細胞機能 を十分抑制してから脳梗塞を作成すると、骨髄および脳梗塞瘢痕部位からニューロ スフエアが形成された。また、脳梗塞を起こしていない偽手術 C. B17マウスの骨髄に 脳梗塞後 1週間目の SCIDマウスの血清と FK506を添カ卩して培養すると-ユーロスフ エアが形成された。さらに、 T細胞機能抑制の目的で FK506の代わりに抗 CD28抗 体あるいは抗 ICOS抗体を添カ卩して培養すると、同様に骨髄力 -ユーロスフェアが 形成された。  As described above, when C.B17 mice were pre-administered with FK506, an immunosuppressant, and T cells function was sufficiently suppressed before creating a cerebral infarction, neurospheres were formed from bone marrow and cerebral infarction scar sites. In addition, when sham-operated C. B17 mice without cerebral infarction were cultured with SCID mouse serum and FK506 1 week after cerebral infarction, -Spherus was formed. In addition, when anti-CD28 antibody or anti-ICOS antibody was added in place of FK506 for the purpose of suppressing T cell function and cultured, bone marrow strength-eurosphere was also formed.
[0112] このことより、骨髄力ゝら異所性に神経幹細胞が形成されるためには T細胞機能など 細胞性免疫が抑制されなければならな 、こと、正常マウスでも免疫を調節 (抑制）す れば骨髄で神経再生が生じ、アポトーシスによる細胞死を回避して、脳梗塞瘢痕部 位に移行生着し、そこで分ィ匕して神経になることが示唆された。  [0112] From this, in order for neural stem cells to be formed ectopically, such as bone marrow, cellular immunity such as T cell function must be suppressed, and even normal mice regulate (suppress) immunity. This suggests that nerve regeneration occurs in the bone marrow, avoids cell death due to apoptosis, migrates to the scar site of cerebral infarction, and then breaks down into nerves.
[0113] 〔実施例 4：脳梗塞 SCIDマウス骨髄由来の神経幹細胞の移植と脳組織への生着 ·分 化〕 脳梗塞 SCIDマウスの骨髄由来の神経幹細胞が生理的に脳梗塞巣に侵入して脳 組織へ生着、分ィ匕するかどうかを確認するため、以下の実験を行った。 [0113] [Example 4: Cerebral infarction SCID mouse bone marrow-derived neural stem cell transplantation and engraftment and differentiation into brain tissue] Cerebral infarction The following experiment was conducted to confirm whether neural stem cells derived from bone marrow of SCID mice physiologically enter the cerebral infarction lesion and engraft and dissociate into brain tissue.
[0114] 即ち、骨髄由来の神経幹細胞が SCIDマウスの脳梗塞後に生じる神経再生に関与 する力どう力を、緑色蛍光タンパク質（green fluorescence protein： GFP)を発現する GFPトランスジエニックマウスの骨髄を移植した SCIDマウス脳梗塞モデルで検討し た。 [0114] That is, the bone marrow-derived neural stem cells are transplanted with the bone marrow of a GFP transgenic mouse that expresses green fluorescence protein (GFP), the force involved in nerve regeneration that occurs after cerebral infarction in SCID mice. The SCID mouse cerebral infarction model was examined.
[0115] まず、 SCIDマウスに 200radの放射線照射による骨髄抑制を施した後に GFPトラ ンスジエニックマウスの骨髄細胞（10万個）を移植した。移植後 2週間目に脳梗塞を 作成し、さらに脳梗塞後 16日目にマウスを経心的に PLP固定液で灌流固定した。脳 を摘出し、ビブラトームを用いて脳切片を作成した。その後脳切片を GFPと PSA— N CAMに対する二重染色免疫組織化学に供した。  [0115] First, bone marrow suppression by irradiation of 200 rads was performed on SCID mice, and then bone marrow cells (100,000) of GFP transgenic mice were transplanted. Cerebral infarction was created 2 weeks after transplantation, and the mouse was transcardially fixed with PLP fixative solution 16 days after cerebral infarction. The brain was removed and brain sections were prepared using a vibratome. The brain sections were then subjected to double staining immunohistochemistry for GFP and PSA-NCAM.
[0116] 具体的には、脳切片をラットモノクローナル抗 GFP抗体とマウスモノクローナル抗 P SA—NCAM抗体を含むリン酸緩衝液（2000倍希釈）にて 12時間反応させ (第一 反応)、洗浄後 FITC標識ャギ抗ラッ HgGとピオチン標識ャギ抗マウス IgG (第二反 応）、次いでアビジン標識 Cy3と反応させた。 GFPと PSA— NCAMの可視化は共焦 点レーザー蛍光顕微鏡にて観察した。 GFP陽性細胞は脳梗塞下組織に広く認めら れ、一部は PSA— NCAM陰性でミクログリアの形態を示していたが（図 18B参照）、 梗塞下の白質外側および大脳皮質断面 (本発明者らがステムロード (Stem Road)と 称する部位）に沿って広く認められた GFP陽性細胞群は、同時に PSA—NCAM陽 性で神経前駆細胞と考えられる（同図 C参照)。 PSA— NCAM陽性部位は他に脳 室周囲組織にもみられた力 ここは GFP陰性であった。  [0116] Specifically, the brain section was reacted with a phosphate buffer solution (diluted 2000 times) containing rat monoclonal anti-GFP antibody and mouse monoclonal anti-PSA-NCAM antibody for 12 hours (first reaction), and then washed. FITC-labeled goat anti-rabbit HgG and piotin-labeled goat anti-mouse IgG (second reaction) and then avidin-labeled Cy3 were reacted. Visualization of GFP and PSA-NCAM was observed with a confocal laser fluorescence microscope. GFP-positive cells were widely observed in subcerebral infarcted tissues, and some of them were PSA-NCAM negative and showed microglia morphology (see Fig. 18B). A group of GFP positive cells widely recognized along the stem road) is considered to be PSA-NCAM positive and neural progenitor cells (see Fig. C). Other PSA-NCAM positive sites were also observed in periventricular tissues. They were GFP negative.
[0117] 以上の結果から、上記ステムロードには骨髄細胞由来の神経幹細胞が梗塞後に発 現して脳実質内に侵入すること、すなわち、骨髄由来の神経幹細胞が脳梗塞下の組 織へ生着し、神経に分化することが示された。  [0117] From the above results, the bone marrow-derived neural stem cells appear after the infarction and enter the brain parenchyma, that is, the bone marrow-derived neural stem cells are engrafted in the tissue under the cerebral infarction. It was shown to differentiate into nerves.
[0118] 〔実施例 5 :ヒトへの応用と神経幹細胞移植による脳梗塞治療効果〕  [0118] [Example 5: Human application and therapeutic effect of cerebral infarction by neural stem cell transplantation]
[5-1]ヒトの骨髄由来神経幹細胞調製法  [5-1] Preparation of human bone marrow-derived neural stem cells
以上の知見をヒトに応用し、ヒト骨髄由来の神経幹細胞の調製が可能かどうかを検 Applying the above knowledge to humans, we examined whether it is possible to prepare human bone marrow-derived neural stem cells
B、Jした。 脳梗塞を起こしていない正常ヒト骨髄を採取し、その後、 DMEMと N— 2の基本培 養液内（250 μ 1)で単細胞になるまでピペッティングし、 10mlの培養液を加えて 600 rpmで 5分間遠心した。細胞を 3mlの培養液にて再浮遊させ、 low cell bindingプレー ト上で 10— 28日間培養を行った。培養液は、 B, J. Collect normal human bone marrow that has not developed cerebral infarction, and then pipet it into single cells in the basic culture solution of DMEM and N-2 (250 μ1). Centrifuge for 5 minutes. Cells were resuspended in 3 ml of culture and cultured on a low cell binding plate for 10-28 days. The culture solution is
(1) bFGF (50 g/ml)と EGF (20 μ g/ml)のみ、  (1) Only bFGF (50 g / ml) and EGF (20 μg / ml)
(2) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の患者の血清（5 0 1)、  (2) bFGF (50 μg / ml) and EGF (20 μg / ml) + serum of patient 1 week after cerebral infarction (5 0 1),
(3) bFGF (50 μ g/ml)と EGF (20 μ g/ml) +脳梗塞後 1週間目の患者の血清（5
Figure imgf000038_0001
(3) bFGF (50 μg / ml) and EGF (20 μg / ml) + serum from patient 1 week after cerebral infarction (5
Figure imgf000038_0001
の 3種類を用意した。  Three types were prepared.
[0119] すると、培養 7日目以降に上記（3)の培養液中にのみ-ユーロスフェア様細胞塊が 細胞顕微鏡下で観察された。培養 10日目に形成された-ユーロスフェア様細胞塊（ 図 19A)を high bindingプレート上でさらに 5日間培養すると、神経細胞に分化した（ 同図 B)。  [0119] Then, after the seventh day of culture, only in the culture medium of (3) above, -Eurosphere-like cell clusters were observed under a cell microscope. When the eurosphere-like cell cluster (Fig. 19A) formed on the 10th day of culture was cultured on the high binding plate for another 5 days, it differentiated into neurons (Fig. B).
[0120] [5-2]脳梗塞 SCIDマウス骨髄由来の神経幹細胞の移植  [0120] [5-2] Cerebral infarction Transplantation of neural stem cells derived from bone marrow of SCID mice
脳梗塞 SCIDマウス力 得られた骨髄由来の神経幹細胞を、別の脳梗塞 SCIDマ ウスに移植した場合の脳再生に与える影響、脳梗塞治療効果につ 、て検討した。  Cerebral infarction SCID mouse strength The effect of bone marrow-derived neural stem cells obtained on brain regeneration when transplanted into another cerebral infarction SCID mouse and the therapeutic effect on cerebral infarction were examined.
[0121] 即ち、脳梗塞 SCIDマウスの骨髄力 調製された-ユーロスフェア様細胞塊を単細 胞に分離して、その 10万個を別の脳梗塞 SCIDマウス (脳梗塞後 2日目）に静脈内投 与した。そして、その脳梗塞後 16日目に当該マウスを経心的に PLP固定液で灌流 固定後、脳を摘出した。摘出脳は、全例中大脳動脈領域の欠損が観察された。  [0121] That is, the bone marrow strength of cerebral infarction SCID mice was prepared-Eurosphere-like cell clusters were separated into single cells, and 100,000 of them were separated into another cerebral infarction SCID mouse (second day after cerebral infarction). It was administered intravenously. On the 16th day after the cerebral infarction, the mouse was transcardially fixed with a PLP fixative solution, and the brain was removed. In the isolated brain, defects in the middle cerebral artery region were observed in all cases.
[0122] 梗塞側大脳皮質の大脳裂力も梗塞部位までの幅 (a)を正常側 (b)と比較し、その 比（aZb)を cortical width index(CI値）として算出すると、 CI値は、骨髄由来神経幹 細胞（BM— NSC)投与マウスでは 0. 48で、 PBSを投与したコントロールマウスでは 0. 36であった（図 20)。このように、神経幹細胞を移植されたマウスの脳は、コント口 ールマウスの脳より残存大脳皮質が拡大して 、た。  [0122] The cerebral cleft force of the infarct side cerebral cortex is also calculated by comparing the width (a) to the infarct site with the normal side (b) and calculating the ratio (aZb) as the cortical width index (CI value). It was 0.48 in mice treated with bone marrow-derived neural stem cells (BM—NSC) and 0.36 in control mice treated with PBS (FIG. 20). Thus, the brains of mice transplanted with neural stem cells had a larger residual cerebral cortex than the brains of control mice.
[0123] 上述のように、 SCIDマウスから作成した脳梗塞モデルマウスでは、虚血後の脳梗 塞の進展（delayed infarct expansion)は脳梗塞後 3日で終了し、その後は脳萎縮の 進展ではなぐ脳形態上はむしろ回復するが、神経幹細胞を移植するとさらに回復が 進むことを示している。このことは、本神経幹細胞の移植が脳梗塞後の神経再生を促 進することを示している。 [0123] As described above, in the cerebral infarction model mouse prepared from the SCID mouse, the delayed infarct expansion after ischemia ended 3 days after the cerebral infarction, and thereafter the brain atrophy It is shown that the brain morphology rather than the progress is recovered, but the recovery is further improved by transplanting neural stem cells. This indicates that transplantation of this neural stem cell promotes nerve regeneration after cerebral infarction.
[0124] 〔実施例 6 :分ィ匕誘導因子の発見とこれを用いた骨髄由来神経幹細胞の調製〕  [0124] [Example 6: Discovery of mitochondrial inducer and preparation of bone marrow-derived neural stem cells using the same]
CINC— 1が神経幹細胞への分ィ匕誘導因子である可能性が考えられたので、以下 の実験により CINC—1が骨髄細胞分ィ匕に及ぼす影響を検討した。  Since CINC-1 was considered to be a factor that induces differentiation of neural stem cells, the following experiment examined the effect of CINC-1 on bone marrow cell differentiation.
[0125] 脳梗塞を起こしていない偽手術 C. B17マウス骨髄に CINC— 1 (10— 5M :ペプチド 研究所）と FK506 (0. 1 μ g/ml)を添加して培養した。すると培養 5日目以内にす でに多くの細胞塊が形成された（図 22Α· B)。これらはまだ細胞密度が低 、細胞凝 集塊であり、ニューロスフェアとは異なっていた力 培養 7日目以降になると、ニューロ スフエア様細胞塊が形成された（図 22C'D)。 [0125] Sham operated C. B17 mouse bone marrow does not cause cerebral infarction CINC- 1 (10- 5 M: Peptide Institute) and FK506 was cultured by adding (0. 1 μ g / ml) . Many cell clusters had already formed within 5 days of culture (Fig. 22B). These are still cell agglomerates with low cell density, and after 7 days of force culture, which was different from neurospheres, neurosphere-like cell clusters were formed (Fig. 22C'D).
[0126] また、 FK506の影響を検討した結果、 CINC— 1 (10— 5M)と FK506 (0. 1 μ g/ml )を添加して培養した場合には、上述のように、培養 7日目以降に-ユーロスフェア様 細胞塊が形成された（図 23A)のに対して、 CINC— 1 (10— 5M)のみを添カ卩し、 FK5 06を添加しない場合は、細胞塊は形成されるが、殆どが排除されてしまうか、あるい は細胞塊の状態で止まっており（図 23B)、ニューロスフェア形成は認められなかった [0126] Further, the result of examining the effects of FK506, when cultured by adding CINC- 1 a (10- 5 M) and FK506 (0. 1 μ g / ml ) , as described above, cultured 7 after day th - against the euro spheres like cell cluster is formed of (Fig. 23A), CINC- 1 (10- 5 M) only by添Ka卩, if not added FK5 06, cell mass Formed, but most of it was eliminated or stopped in the form of a cell mass (Fig. 23B), and no neurosphere formation was observed.
[0127] 以上の結果は、脳虚血刺激で産生亢進し、 NK細胞でも作られる CINC— 1が骨髄 細胞の神経幹細胞への分ィ匕誘導因子のひとつであることを示すと同時に、 T細胞機 能の抑制もその形成維持には重要であることを示している。 [0127] The above results indicate that CINC-1, which is also produced by cerebral ischemia stimulation and is also produced by NK cells, is one of the factors that induce differentiation of bone marrow cells into neural stem cells. This shows that the suppression of function is also important for maintaining its formation.
[0128] 〔実施例 7 :神経幹細胞の形成における免疫抑制の重要性と免疫抑制物質の神経再 生治療への応用〕  [Example 7: Importance of immunosuppression in the formation of neural stem cells and application of immunosuppressive substances to nerve regeneration treatment]
これまでの実験結果から、 in vitroにおいて、また in vivoにおいても、骨髄由来神経 幹細胞の形成を維持'促進し、神経再生治療を実現するためには、免疫機能を抑制 すること、特に T細胞機能を抑制することが重要であると考えられた。このことをさらに 確かめるため、以下の実験を行った。  Based on the results of previous experiments, in order to maintain and promote the formation of bone marrow-derived neural stem cells both in vitro and in vivo, and to realize nerve regeneration treatment, suppress immune function, especially T cell function. It was thought that it was important to suppress this. In order to confirm this further, the following experiment was conducted.
[0129] [7-l]FK506以外の免疫抑制剤を用いたニューロスフェアの形成 [0129] Formation of neurospheres using immunosuppressive agents other than [7-l] FK506
C. B 17マウスに FK506 (tacrolimus hydrate: 1.0 mg/kg/day)あるいは FK506と 同じく T細胞機能を抑制する免疫抑制剤であるシクロスポリン A (CsA: 10mg/kg/day )を 3日間連日腹腔内投与し、左中大脳動脈閉塞による脳梗塞を作成した (3匹ずつ )。その後も FK5O6 (1.0 mg/kg)あるいは CsA(10mg/kg/day)投与を継続し、脳梗塞 後 7日目に骨髄と脳梗塞下組織 (脳梗塞瘢痕部位)を採取して DMEMと N— 2の基 本培養液内（250 μ 1)で単細胞になるまでピペッティングし、 10mlの培養液を加えて 600rpmで 5分間遠心した。細胞を 3mlの培養液にて再浮遊させ、 bFGF ^O /z gZ ml)と EGF (20 μ gZml)存在下に low cell bindingプレート上で 10— 28日間培養を 行った。 7日目以降には FK506と CsA投与群の骨髄 (BM)と脳梗塞下組織力も-ュ 一口スフ ア様細胞塊が細胞顕微鏡下で観察された。一方、生理食塩水（saline)を 1 0日間連日腹腔内投与しただけの培養骨髄からは-ユーロスフェア様細胞塊はほと んど形成されな力つた。浮遊している-ユーロスフェア様細胞塊を採取して high bindi ngプレート上でさらに培養した。 7日後に分ィ匕した細胞をバラフオルムアルデヒドを含 む固定液にて固定し、 nestin陽性の-ユーロスフェア数を計測したところ、 FK506お よび CsA投与群ではコントロールに比し、有意に nestin陽性-ユーロスフェアが多か つた（図 24)。 C. B 17 mice with FK506 (tacrolimus hydrate: 1.0 mg / kg / day) or FK506 Cyclosporin A (CsA: 10 mg / kg / day), an immunosuppressant that also suppresses T cell function, was intraperitoneally administered daily for 3 days to create cerebral infarctions due to left middle cerebral artery occlusion (3 mice each). Thereafter, administration of FK5O6 (1.0 mg / kg) or CsA (10 mg / kg / day) was continued. On the 7th day after cerebral infarction, bone marrow and sub-infarcted tissue (cerebral infarction scar site) were collected and DMEM and N— Pipette up to 2 single cultures in the basic culture solution (250 μ1), add 10 ml of the culture solution, and centrifuge at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on low cell binding plates in the presence of bFGF ^ O / z gZ ml) and EGF (20 μg Zml) for 10-28 days. From day 7 onwards, bone marrow (BM) and sub-infarct tissue strength in the FK506 and CsA-treated groups were also observed under a single-mouthed swallow-like cell mass under a cell microscope. On the other hand, almost no eurosphere-like cell clusters were formed from cultured bone marrow that had been administered intraperitoneally daily with saline for 10 days. Floating-Eurosphere-like cell clumps were harvested and further cultured on high binding plates. Cells separated after 7 days were fixed with a fixative containing baraformaldehyde, and the number of nestin-positive eurospheres was measured. -There were many eurospheres (Figure 24).
以上のことより、 T細胞機能を抑制する免疫抑制剤を投与することで脳梗塞後に骨 髄で神経再生が生じ、アポトーシスによる細胞死を回避して、脳梗塞部位に移行生 着し、そこで分ィ匕して神経になることが示唆された。  Based on the above, administration of an immunosuppressive agent that suppresses T cell function causes nerve regeneration in the bone marrow after cerebral infarction, avoiding cell death due to apoptosis, and migrating to the cerebral infarction site. It was suggested that it would become nervous.
[7- 2]T細胞機能欠如マウス (ヌードマウス）の-ユーロスフェア形成 [7-2] -Eurosphere formation in mice lacking T cell function (nude mice)
無胸腺マウスであるヌードマウス（BALB/cAJc卜 nu)とその母系マウス（コントロール） の左中大脳動脈閉塞による脳梗塞作成後、 7日目に骨髄細胞と脳梗塞下組織 (脳梗 塞瘢痕部位)を採取、培養して神経幹細胞塊 (ニューロスフェア）を作成した。すなわ ち、マウスをクリーンベンチ内で断頭し、大腿骨から骨髄細胞を、脳梗塞瘢痕部より 細胞を採取した。その後、 DMEMと N— 2の基本培養液内（250 1)で単細胞にな るまでピペッティングし、 10mlの培養液をカ卩えて 600rpmで 5分間遠心した。細胞を 3mlの培養液にて再浮遊させ、 bFGF (50 μ g/ml)と EGF (20 μ g/ml)存在下に 1 ow cell bindingプレート上で 10— 28日間培養を行った。 7日目以降には-ユーロスフ エア様細胞塊が細胞顕微鏡下で観察されたが、 T細胞が正常なコントロールマウスの 培養骨髄と脳梗塞瘢痕部からは-ユーロスフェア様細胞塊はほとんど形成されなか つた。浮遊している-ユーロスフェア様細胞塊を採取して high bindingプレート上でさ らに培養した。 7日後に分ィ匕した細胞をバラフオルムアルデヒドを含む固定液にて固 定し、 nestin陽性の-ユーロスフェア数を計測したところ、ヌードマウスではコントロー ルに比し、有意に nestin陽性-ユーロスフェアが多かった（図 25)。 After creating a cerebral infarction due to occlusion of the left middle cerebral artery in a nude mouse (BALB / cAJc 卜 nu) and its maternal mouse (control), an athymic mouse, bone marrow cells and subinfarcted tissue (cerebral infarct scar site) ) Were collected and cultured to produce neural stem cell clusters (neurospheres). In other words, the mice were decapitated in a clean bench, and bone marrow cells were collected from the femur and cells were collected from the cerebral infarction scar. Then, pipetting was performed in the basic culture medium of DMEM and N-2 (250 1) until it became a single cell, and 10 ml of the culture medium was added and centrifuged at 600 rpm for 5 minutes. The cells were resuspended in 3 ml of culture medium and cultured on a 1 ow cell binding plate in the presence of bFGF (50 μg / ml) and EGF (20 μg / ml) for 10-28 days. After 7 days-Eurosphere-like cell clusters were observed under a cell microscope, but T cells were not detected in normal control mice. From the cultured bone marrow and cerebral infarction scar,-Eurosphere-like cell cluster was hardly formed. Floating-Eurosphere-like cell clumps were collected and further cultured on high binding plates. Cells separated after 7 days were fixed in a fixative containing baraformaldehyde, and the number of nestin-positive eurospheres was counted. In nude mice, the number of nestin-positive eurospheres was significantly higher than that of controls. There were many (Figure 25).
以上のことより、 T細胞の存在がなければ、脳梗塞後に骨髄で神経再生が生じ、ァ ポトーシスによる細胞死を回避して、脳梗塞部位に移行生着し、そこで分ィ匕して神経 になることが示唆された。  Based on the above, in the absence of T cells, nerve regeneration occurs in the bone marrow after cerebral infarction, avoids cell death due to apoptosis, migrates and engrafts at the cerebral infarction site, and then separates it into the nerve. It was suggested that
[0131] 本実施例の結果から、 T細胞が骨髄あるいは脳梗塞瘢痕部における-ユーロスフエ ァ形成に抑制的機能を果たしていること、換言すれば、ニューロスフェアの形成を維 持'促進するためには T細胞機能を抑制することが重要であることが示された。すな わち、 in vivoでの FK506や CsAなどの免疫抑制剤の効果、 SCIDマウス、ヌードマウ スの効果などは、免疫抑制が神経幹細胞作成に重要であることを示すものであると 同時に、 in vivoで免疫抑制が脳梗塞治療効果に結びつくことを明ら力としている点で 、免疫抑制物質が脳梗塞治療など神経再生治療に有効であることを示すものと 、え る。 [0131] From the results of the present example, it was found that T cells have a suppressive function on the formation of eurospheres in the bone marrow or cerebral infarct scar, in other words, to maintain and promote the formation of neurospheres. It was shown that it is important to suppress T cell function. In other words, the effects of immunosuppressants such as FK506 and CsA in vivo, the effects of SCID mice and nude mice indicate that immunosuppression is important for the generation of neural stem cells. The fact that immunosuppression is linked to the therapeutic effect of cerebral infarction in vivo is a clear indication that immunosuppressive substances are effective for nerve regeneration treatment such as cerebral infarction treatment.
[0132] 〔実施例 8 :新たな分ィ匕誘導因子の発見と本スクリーニングの有効性〕  [0132] [Example 8: Discovery of a new differentiation factor inducer and effectiveness of this screening]
実施例 6で分ィ匕誘導因子のひとつとして見出された CINC—1は、ケモカインに分 類されるサイト力インであるが、分化誘導因子として作用する分子が他のサイト力イン にも存在する可能性が考えられたので、前記と同様の培養系を用いたスクリーニング によって他の分ィ匕誘導因子を探索した結果、以下のように、サイト力インの TNF o;力 S 、骨髄細胞から神経幹細胞への分ィ匕誘導作用を有することを見出した。  CINC-1 found as one of the differentiation-inducing factors in Example 6 is a site force-in classified as a chemokine, but a molecule that acts as a differentiation-inducing factor also exists in other site force-in. As a result of searching for other differentiation-inducing factors by screening using the same culture system as described above, the following results were obtained. It has been found that it has an effect of inducing differentiation into neural stem cells.
[0133] 脳梗塞を起こしていない偽手術 SCIDマウス骨髄に CINC— 1 (10"5M)を添加、あ るいは TNF a (0.1 ng/ml)を添カロして培養した。 CINC— 1、 TNF aを添カ卩すると培 養 5日目以内にすでに細胞塊が形成され 7日目以降には-ユーロスフェア様細胞塊 が形成された力 これらを添カ卩していないもの（saline添加）はほとんど細胞塊形成が みられなかった。浮遊している-ユーロスフェア様細胞塊を採取して high bindingプレ ート上でさらに培養した。 7日後に分ィ匕した細胞をバラフオルムアルデヒドを含む固定 液にて固定し、 nestin陽性の-ユーロスフェア数を計測したところ、 CINC— 1のみな らず TNF a添カ卩群でもコントロールに比し、有意に nestin陽性-ユーロスフェアが多 かった（図 26)。 [0133] Sham mice without cerebral infarction SCID mouse bone marrow was cultured with CINC-1 (10 " 5 M) or TNF a (0.1 ng / ml). When TNF a is added, the cell mass has already formed within 5 days of cultivation, and after 7 days-Eurosphere-like cell mass has been formed. Cell mass formation was not observed, floating-Eurosphere-like cell mass was harvested and further cultured on a high binding plate 7 days later, the sorted cells contained balaformaldehyde. Fixed When the number of nestin-positive-eurospheres was measured with the solution, the number of nestin-positive-eurospheres was significantly higher not only in CINC-1 but also in the TNF a supplemented group as compared to the control (Fig. 26).
[0134] 以上のことから、 TNF aで骨髄が刺激されると、骨髄で神経再生が生じることが示 され、 TNF o;も骨髄細胞から神経幹細胞への分ィヒ誘導因子のひとつであること、お よび、神経幹細胞培養系を用いた本スクリーニング法力 神経幹細胞の誘導物質の スクリーニング、さらには当該物質を用いた神経再生治療剤の開発に有効であること が示された。  [0134] These results indicate that when bone marrow is stimulated with TNF a, nerve regeneration occurs in the bone marrow, and TNF o is also one of the factors that induce differentiation from bone marrow cells to neural stem cells. This screening method using a neural stem cell culture system has been shown to be effective for screening a substance for inducing neural stem cells and for developing a nerve regeneration therapeutic agent using the substance.
産業上の利用可能性  Industrial applicability
[0135] 以上のように、本発明は、骨髄力 簡易かつ短期間に神経幹細胞を調製する方法 等を提供するものであり、細胞移植による脳梗塞発症後の速やかな脳機能の回復、 脳梗塞以外の脳血管障害、神経変性疾患に対する神経再生治療、またはそのため の治療法開発など神経再生医療の分野において広く利用可能性を有し、再生医療 などに取り組む各種の医療関連産業において有用である。 [0135] As described above, the present invention provides a method for preparing neural stem cells in a short time in a short period of time for bone marrow, etc., and rapid recovery of brain function after the onset of cerebral infarction by cell transplantation, cerebral infarction It has wide applicability in the field of neuroregenerative medicine, such as neuroregenerative treatment for other cerebrovascular disorders and neurodegenerative diseases, or the development of treatments therefor, and is useful in various medical-related industries engaged in regenerative medicine.

Claims

請求の範囲 The scope of the claims
[I] 免疫抑制剤、および、脳梗塞その他脳障害後のヒト又は動物から採取した血清を 添加して骨髄細胞を培養することにより分化誘導し、神経幹細胞を調製する方法。  [I] A method of preparing neural stem cells by inducing differentiation by adding an immunosuppressant and serum collected from humans or animals after cerebral infarction or other cerebral injury and culturing bone marrow cells.
[2] 脳梗塞その他脳障害後のヒト又は動物から採取した骨髄細胞を、免疫抑制剤を添 加して培養することにより分化誘導し、神経幹細胞を調製する方法。  [2] A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells collected from humans or animals after cerebral infarction or other cerebral disorders by adding an immunosuppressant.
[3] 免疫抑制剤、および、ケモカイン (もしくはその他のサイト力イン)を添加して骨髄細 胞を培養することにより分化誘導し、神経幹細胞を調製する方法。  [3] A method of preparing neural stem cells by inducing differentiation by culturing bone marrow cells by adding an immunosuppressant and a chemokine (or other cytodynamic in).
[4] ケモカインとして、インターロイキン一 8 (IL— 8)ファミリーのケモカインを添カ卩する、 請求項 3記載の方法。  [4] The method according to claim 3, wherein an interleukin-1 (IL-8) family chemokine is added as a chemokine.
[5] サイト力インとして、 TNF a (tumor necrosis factor- a：腫瘍壊死因子）を添カ卩する、 請求項 3記載の方法。  [5] The method according to claim 3, wherein TNF a (tumor necrosis factor-a) is added as site force-in.
[6] ケモカイン (もしくはその他のサイト力イン)であり、骨髄細胞から神経幹細胞への分 化を直接的又は間接的に誘導する分ィヒ誘導因子。  [6] A chemokine (or other site force-in) that induces differentiation of bone marrow cells into neural stem cells directly or indirectly.
[7] 請求項 6記載の分ィ匕誘導因子を用いて、培養細胞から神経幹細胞を調製する方 法。 [7] A method for preparing neural stem cells from cultured cells using the differentiation-inducing factor according to claim 6.
[8] 請求項 6記載の分ィ匕誘導因子と免疫抑制剤とからなる神経幹細胞分ィ匕誘導剤。  [8] A neural stem cell differentiation inducer comprising the differentiation inducer according to claim 6 and an immunosuppressant.
[9] 神経幹細胞として、ニューロスフェア（neurosphere)または-ユーロスフェア様細胞 塊を調製する、請求項 1〜5又は 7のいずれか 1項に記載の方法。 [9] The method according to any one of [1] to [5] or [7], wherein a neurosphere or a -eurosphere-like cell mass is prepared as a neural stem cell.
[10] 請求項 1〜5又は 7のいずれか 1項に記載の方法により神経幹細胞を生産する方法 [10] A method for producing neural stem cells by the method according to any one of claims 1 to 5 or 7.
[I I] 請求項 10記載の神経幹細胞をさらに培養することにより分化誘導し、神経細胞を 生産する方法。 [I I] A method of inducing differentiation by further culturing the neural stem cell according to claim 10 to produce a neural cell.
[12] 請求項 10記載の神経幹細胞もしくは請求項 11記載の神経細胞を、脳梗塞治療ま たは他の神経再生治療に使用する方法。  [12] A method of using the neural stem cell according to claim 10 or the neural cell according to claim 11 for cerebral infarction treatment or other nerve regeneration treatment.
[13] 請求項 10記載の神経幹細胞もしくは請求項 11記載の神経細胞を、静脈内投与な どの方法により投与する、脳梗塞治療法または他の神経再生治療法。 [13] A cerebral infarction treatment method or other nerve regeneration treatment method, wherein the neural stem cell according to claim 10 or the nerve cell according to claim 11 is administered by a method such as intravenous administration.
[14] 請求項 1記載の方法にお!、て、神経幹細胞の調製に用いる骨髄細胞および血清 には、治療対象の患者カゝら採取したものを使用する方法。 [14] The method according to claim 1, wherein the bone marrow cells and serum used for the preparation of neural stem cells are those collected from a patient to be treated.
[15] 請求項 2〜5又は 7の 、ずれ力 1項に記載の方法にお!、て、神経幹細胞の調製に 用いる骨髄細胞には、治療対象の患者力 採取したものを使用する方法。 [15] The method according to claim 2 to 5 or 7, wherein the bone marrow cells used for the preparation of neural stem cells are obtained by collecting patient force to be treated.
[16] 請求項 1〜5又は 7のいずれ力 1項に記載の方法において、神経幹細胞の調製に 用いる免疫抑制剤に、 FK506 (タクロリムス）、シクロスポリン、抗 CD28抗体および抗[16] The method according to any one of claims 1 to 5 or 7, wherein FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-CD28 antibody are used as immunosuppressive agents for preparing neural stem cells.
ICOS抗体などの T細胞機能を抑制する免疫抑制剤を使用する方法。 A method of using an immunosuppressive agent that suppresses T cell function such as an ICOS antibody.
[17] 請求項 13記載の神経再生治療法にぉ 、て、神経 (幹)細胞の投与と同時または異 時に、免疫抑制剤を投与する方法。 [17] A method for administering an immunosuppressive agent to the nerve regeneration treatment method according to claim 13, wherein the immunosuppressive agent is administered simultaneously or simultaneously with administration of nerve (stem) cells.
[18] 請求項 13記載の神経再生治療法にお 、て、神経 (幹)細胞の投与と同時または異 時に、ヒト臍帯血由来 CD34陽性細胞、血管内皮前駆細胞 (EPC)などの血管形成 能を有する細胞を投与する方法。 [18] In the nerve regeneration treatment method according to claim 13, an angiogenic ability of human umbilical cord blood-derived CD34-positive cells, vascular endothelial progenitor cells (EPCs), etc. at the same time or simultaneously with administration of nerve (stem) cells. A method of administering cells having
[19] 請求項 10記載の神経幹細胞もしくは請求項 11記載の神経細胞を含む脳梗塞治 療剤。 [19] A therapeutic agent for cerebral infarction comprising the neural stem cell according to claim 10 or the neural cell according to claim 11.
[20] 請求項 10記載の神経幹細胞もしくは請求項 11記載の神経細胞を含む神経再生 治療剤。  [20] A therapeutic agent for nerve regeneration comprising the neural stem cell according to claim 10 or the neural cell according to claim 11.
[21] 免疫不全マウス（SCIDマウスおよびヌードマウスを含む）、その母系マウス、又はこ れらのマウス力も作成した脳梗塞モデルマウスの骨髄細胞を培養することにより分ィ匕 誘導し、神経幹細胞を調製する方法。  [21] Culturing bone marrow cells from immunodeficient mice (including SCID mice and nude mice), their maternal mice, or cerebral infarction model mice that also produced these mouse forces, and induced neural stem cells. How to prepare.
[22] 免疫不全マウスの骨髄細胞を、脳梗塞モデルマウス力 採取した血清、又はケモカ イン (もしくはその他のサイト力イン)を添加して培養する、請求項 21記載の方法。 [22] The method according to claim 21, wherein the bone marrow cells of the immunodeficient mouse are cultured by adding serum collected from the force of a cerebral infarction model mouse or chemokine (or other site force in).
[23] 免疫不全マウスの母系マウスの骨髄細胞を、脳梗塞モデルマウス力も採取した血 清 (又は、ケモカインもしくはその他のサイト力イン)と免疫抑制剤とを添加して培養す る、請求項 21記載の方法。 [23] The bone marrow cells of the maternal mouse of the immunodeficient mouse are cultured with the addition of serum (or chemokine or other cytodynamic force in) collected from the cerebral infarction model mouse and an immunosuppressant. The method described.
[24] 免疫不全マウスの母系マウス力も作成した脳梗塞モデルマウスの骨髄細胞を、免 疫抑制剤を添加して培養する、請求項 21記載の方法。 [24] The method according to claim 21, wherein the bone marrow cells of a cerebral infarction model mouse, in which a maternal mouse force of an immunodeficient mouse is also prepared, is cultured with an immunosuppressive agent added thereto.
[25] 免疫不全マウスカゝら作成した脳梗塞モデルマウスの骨髄細胞を培養することにより 神経幹細胞を調製する、請求項 21記載の方法。 25. The method according to claim 21, wherein neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared by an immunodeficient mouse.
[26] 免疫不全マウスの母系マウスに免疫抑制剤を投与して免疫不全状態にした後作成 した脳梗塞モデルマウスの骨髄細胞を培養することにより神経幹細胞を調製する、請 求項 21記載の方法。 [26] Neural stem cells are prepared by culturing bone marrow cells of a cerebral infarction model mouse prepared after an immunosuppressant is administered to a maternal mouse of an immunodeficient mouse to make it immunocompromised. The method according to claim 21.
[27] 免疫不全マウスが、 C.B- 17/IcrCrlBRマウスを母系とする SCIDマウスである、請求 項 21〜26のいずれ力 1項に記載の方法。  [27] The method according to any one of [21] to [26], wherein the immunodeficient mouse is an SCID mouse having a C.B-17 / IcrCrlBR mouse as a maternal line.
[28] 免疫不全マウスが、 BALB/cAJclマウスを母系とするヌードマウスである、請求項 21[28] The immunodeficient mouse is a nude mouse whose mother line is a BALB / cAJcl mouse.
〜26の!、ずれ力 1項に記載の方法。 -26 !, the method according to item 1.
[29] 請求項 21〜28のいずれか 1項に記載の方法により調製された神経幹細胞。 [29] A neural stem cell prepared by the method according to any one of claims 21 to 28.
[30] SCIDマウスの母系マウスの脳の血管を結紮してなる脳梗塞モデル動物。 [30] A cerebral infarction model animal obtained by ligating blood vessels in the brain of a SCID mouse maternal mouse.
[31] SCIDマウスの母系マウスの脳の血管力 中大脳動脈である請求項 30記載の脳梗 塞モデル動物。 31. The cerebral infarction model animal according to claim 30, wherein the cerebral vascular force is a middle cerebral artery of a SCID mouse maternal mouse.
[32] SCIDマウスの母系マウスが、 C.B- 17/IcrCrlBRマウスである請求項 30又は 31記載 の脳梗塞モデル動物。  [32] The cerebral infarction model animal according to [30] or [31], wherein the maternal mouse of the SCID mouse is a C.B-17 / IcrCrlBR mouse.
[33] ヌードマウス又はその母系マウスの脳の血管を結紮してなる脳梗塞モデル動物。  [33] A cerebral infarction model animal obtained by ligating a brain blood vessel of a nude mouse or a mother mouse thereof.
[34] ヌードマウス又はその母系マウスの脳の血管力 中大脳動脈である請求項 33記載 の脳梗塞モデル動物。 [34] The cerebral infarction model animal according to claim 33, which is a cerebral vascular force of the brain of a nude mouse or a mother mouse thereof.
[35] ヌードマウスの母系マウス力 BALB/cAJclマウスである請求項 33又は 34記載の脳 梗塞モデル動物。  [35] The cerebral infarction model animal according to claim 33 or 34, which is a maternal mouse force of a nude mouse, a BALB / cAJcl mouse.
[36] 請求項 30〜35のいずれか 1項に記載の脳梗塞モデル動物に、被検薬物を投与し て、当該被検薬物の脳梗塞に対する有効性をスクリーニングする方法。  [36] A method for screening the effectiveness of a test drug against cerebral infarction by administering the test drug to the cerebral infarction model animal according to any one of claims 30 to 35.
[37] 請求項 30〜35のいずれか 1項に記載の脳梗塞モデル動物に、神経 (幹)細胞又 は他の細胞を移植して、当該移植治療の脳梗塞に対する有効性をスクリーニングす る方法。  [37] Nerve (stem) cells or other cells are transplanted into the cerebral infarction model animal according to any one of claims 30 to 35, and the effectiveness of the transplantation treatment for cerebral infarction is screened. Method.
[38] 免疫抑制作用を有する物質を有効成分とする神経再生治療剤。  [38] A therapeutic agent for nerve regeneration comprising a substance having an immunosuppressive action as an active ingredient.
[39] 免疫抑制作用を有する物質が、 T細胞機能を抑制する物質である、請求項 38記載 の神経再生治療剤。 [39] The therapeutic agent for nerve regeneration according to claim 38, wherein the substance having an immunosuppressive action is a substance that suppresses T cell function.
[40] 免疫抑制作用を有する物質が、 FK506 (タクロリムス）、シクロスポリン、抗 CD28抗 体および抗 ICOS抗体の 、ずれかである、請求項 39記載の神経再生治療剤。  [40] The therapeutic agent for nerve regeneration according to claim 39, wherein the substance having an immunosuppressive action is any of FK506 (tacrolimus), cyclosporine, anti-CD28 antibody and anti-ICOS antibody.
[41] ケモカインその他のサイト力イン、又はこれらの骨髄細胞から神経幹細胞への分ィ匕 を誘導する作用を促進する物質を有効成分とする神経再生治療剤。 [41] A therapeutic agent for nerve regeneration comprising, as an active ingredient, a chemokine or other cytodynamic force-in or a substance that promotes the action of inducing differentiation from bone marrow cells to neural stem cells.
[42] ケモカインが、インターロイキン一 8 (IL— 8)ファミリーのケモカインである、請求項 4[42] The chemokine is an interleukin-8 (IL-8) family chemokine.
1記載の神経再生治療剤。 1. The nerve regeneration therapeutic agent according to 1.
[43] サイト力インが、 TNF a (tumor necrosis factor- a：腫瘍壊死因子）である、請求項[43] The site force-in is TNF a (tumor necrosis factor-a).
41記載の神経再生治療剤。 41. The therapeutic agent for nerve regeneration according to 41.
[44] 脳梗塞又は他の神経疾患の治療に用いられる、請求項 38〜43のいずれか 1項に 記載の神経再生治療剤。 [44] The nerve regeneration therapeutic agent according to any one of claims 38 to 43, which is used for treatment of cerebral infarction or other neurological diseases.
[45] 脳梗塞又は他の神経疾患の治療に用いられる神経再生治療剤のスクリーニング方 法であって、骨髄細胞から神経幹細胞への分化を誘導し、又はその分ィヒを促進する 作用を有するかどうかを指標に、候補物質を探索することを特徴とする神経再生治療 剤のスクリーニング方法。 [45] A screening method for a neuroregenerative therapeutic agent used for the treatment of cerebral infarction or other neurological diseases, which has the effect of inducing differentiation from bone marrow cells to neural stem cells or promoting their differentiation. A screening method for therapeutic agents for nerve regeneration, characterized by searching for candidate substances using whether or not as an index.
[46] ヒト又は動物力 採取した骨髄細胞の培地に被検物質を投与し、当該被検物質が[46] Human or animal force A test substance is administered to a medium of collected bone marrow cells, and the test substance is
、骨髄細胞から神経幹細胞への分化を誘導し、又はその分化を促進する作用を有す るかどうかを調べることを特徴とする請求項 45記載の神経再生治療剤のスクリーニン グ方法。 46. The screening method for a therapeutic agent for nerve regeneration according to claim 45, wherein it is examined whether it has an action of inducing differentiation from bone marrow cells to neural stem cells or promoting the differentiation.
[47] 免疫抑制作用を有する物質群の中から、候補物質を探索することを特徴とする請 求項 45又は 46記載の神経再生治療剤のスクリーニング方法。  [47] The screening method for a therapeutic agent for nerve regeneration according to claim 45 or 46, wherein a candidate substance is searched from a group of substances having an immunosuppressive action.
[48] ケモカインその他のサイト力イン、又はこれらの活性もしくは産生を調節する作用を 有する物質群の中から、候補物質を探索することを特徴とする請求項 45又は 46記 載の神経再生治療剤のスクリーニング方法。  [48] The therapeutic agent for nerve regeneration according to claim 45 or 46, wherein a candidate substance is searched from a group of substances having a function of regulating chemokine or other cytodynamic ins, or their activity or production. Screening method.
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JP2015533087A (en) * 2012-10-05 2015-11-19 サムスン ライフ パブリック ウェルフェア ファンデーション Stem cell activation promoting composition containing ischemic serum and stem cell activation promoting method
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JPWO2015033558A1 (en) * 2013-09-04 2017-03-02 株式会社大塚製薬工場 Method for preparing pluripotent stem cells
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