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WO2006085612A1 - Méthode de préparation de cellules souches neurales - Google Patents

Méthode de préparation de cellules souches neurales 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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Japanese (ja)
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Tomohiro Matsuyama
Akihiko Taguchi
Hiroo Yoshikawa
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New Industry Research Organization NIRO
Japan Health Sciences Foundation
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New Industry Research Organization NIRO
Japan Health Sciences Foundation
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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.

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Abstract

La présente invention a pour objet (1) une méthode de préparation de cellules souches neurales comprenant la culture de cellules de moelle osseuse en présence d'un agent immunosuppresseur ou de sérum collecté chez un humain ou un animal ayant subi un infarctus cérébral (ou, au lieu du sérum, une cytokine telle qu'une chimiokine) ; (2) une méthode de préparation de cellules souches neurales comprenant la culture de cellules de moelle osseuse collectées chez un humain ou un animal ayant subi un infarctus cérébral en présence d'un agent immunosuppresseur ; etc. Ces méthodes peuvent être employées dans une thérapie de régénération nerveuse ou pour le développement d'une telle thérapie.
PCT/JP2006/302350 2005-02-10 2006-02-10 Méthode de préparation de cellules souches neurales Ceased WO2006085612A1 (fr)

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JP2007502657A JP4905719B2 (ja) 2005-02-10 2006-02-10 神経幹細胞の調製法

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JP2005-035032 2005-02-10
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
JP2008063290A (ja) * 2006-09-08 2008-03-21 Japan Science & Technology Agency 間葉系に関連する細胞を含んでなる、パーキンソン病またはパーキンソン症候群のための治療薬およびこれを用いた治療方法
WO2015033558A1 (fr) * 2013-09-04 2015-03-12 株式会社大塚製薬工場 Procédé de préparation de cellules souches pluripotentes
JP2015533087A (ja) * 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 虚血血清を含む幹細胞活性化促進用組成物及び幹細胞の活性化促進方法

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WO2003035855A1 (fr) * 2001-10-25 2003-05-01 Cedars-Sinai Medical Center Differentiation de moelle osseuse totale
WO2003038075A1 (fr) * 2001-10-30 2003-05-08 Renomedix Institute Inc. Procede permettant d'induire une differenciation de cellules souches embroyonnaires mesodermiques, de cellules es ou de cellules immortalisees dans des cellules du systeme nerveux
JP2005287430A (ja) * 2004-03-31 2005-10-20 Japan Science & Technology Agency 脳梗塞疾患モデルマウス

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WO2003035855A1 (fr) * 2001-10-25 2003-05-01 Cedars-Sinai Medical Center Differentiation de moelle osseuse totale
WO2003038075A1 (fr) * 2001-10-30 2003-05-08 Renomedix Institute Inc. Procede permettant d'induire une differenciation de cellules souches embroyonnaires mesodermiques, de cellules es ou de cellules immortalisees dans des cellules du systeme nerveux
JP2005287430A (ja) * 2004-03-31 2005-10-20 Japan Science & Technology Agency 脳梗塞疾患モデルマウス

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008063290A (ja) * 2006-09-08 2008-03-21 Japan Science & Technology Agency 間葉系に関連する細胞を含んでなる、パーキンソン病またはパーキンソン症候群のための治療薬およびこれを用いた治療方法
JP2015533087A (ja) * 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 虚血血清を含む幹細胞活性化促進用組成物及び幹細胞の活性化促進方法
EP2905331A4 (fr) * 2012-10-05 2016-04-06 Samsung Life Public Welfare Foundation Composition comprenant un sérum ischémique pour favoriser l'activation d'une cellule souche et procédé favorisant l'activation d'une cellule souche
WO2015033558A1 (fr) * 2013-09-04 2015-03-12 株式会社大塚製薬工場 Procédé de préparation de cellules souches pluripotentes
JPWO2015033558A1 (ja) * 2013-09-04 2017-03-02 株式会社大塚製薬工場 多能性幹細胞の調製方法
US9765296B2 (en) 2013-09-04 2017-09-19 Otsuka Pharmaceutical Factory, Inc. Method for preparing pluripotent stem cells
JP2018023401A (ja) * 2013-09-04 2018-02-15 株式会社大塚製薬工場 多能性幹細胞の調製方法
US10370639B2 (en) 2013-09-04 2019-08-06 Otsuka Pharmaceutical Factory, Inc. Method for preparing pluripotent stem cells
US11155782B2 (en) 2013-09-04 2021-10-26 Otsuka Pharmaceutical Factory, Inc. Method for preparing pluripotent stem cells

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