WO2005056778A1 - Method of regulating the differentiation of hematopoietic stem cells or proliferating the same - Google Patents

Method of regulating the differentiation of hematopoietic stem cells or proliferating the same Download PDF

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Publication number
WO2005056778A1
WO2005056778A1 PCT/JP2004/018860 JP2004018860W WO2005056778A1 WO 2005056778 A1 WO2005056778 A1 WO 2005056778A1 JP 2004018860 W JP2004018860 W JP 2004018860W WO 2005056778 A1 WO2005056778 A1 WO 2005056778A1
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hematopoietic stem
stem cells
cells
hematopoietic
cell
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PCT/JP2004/018860
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French (fr)
Japanese (ja)
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Kazuo Todokoro
Hiroyuki Satofuka
Yoshikazu Kuwahara
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Riken
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Publication of WO2005056778A1 publication Critical patent/WO2005056778A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled

Definitions

  • the present invention relates to a method for inhibiting or proliferating hematopoietic stem cells, a method for producing hematopoietic stem cells, a hematopoietic stem cell obtained by the method, a culture system used in the culture method, and a method for screening for a substance that regulates the growth or differentiation of hematopoietic stem cells.
  • the present invention relates to an agent for suppressing differentiation or proliferating hematopoietic stem cells.
  • Wnt hematopoietic stem cells
  • Matthews et al. also show that Wnt5a and Wnt10b are expressed in mouse fetal liver, that Wnt10b also expresses hematopoietic stem cells, that Wnt5a or Wnt10a.
  • the present inventors have conducted extensive studies and found that adult mouse bone marrow stromal cells express Wnt2 and Wnt5a, and adult human bone marrow stromal cells express Wnt2, Wnt5a and Wnt5a. b was expressed, and Wnt 2 was found to be most frequently expressed. The present inventors have continued their research based on this new finding and found that the use of Wnt2 or Wnt5a can suppress or proliferate the differentiation of hematopoietic stem cells. The present invention has been completed.
  • the present invention provides the following methods for inhibiting and differentiating or proliferating hematopoietic stem cells, a method for producing hematopoietic stem cells, a method for producing hematopoietic stem cells, a culture system used in the culture method, and a method for expanding hematopoietic stem cells. Also, the present invention provides a method for screening a substance that regulates differentiation, an agent for suppressing differentiation or proliferating hematopoietic stem cells, and the like.
  • a method for inhibiting differentiation or expanding hematopoietic stem cells comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
  • a hematopoietic stem cell obtained by the method according to any one of (1) to (4).
  • a method for producing hematopoietic stem cells comprising a step of culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
  • a hematopoietic stem cell culture system characterized in that:
  • An agent for suppressing or proliferating hematopoietic stem cell differentiation which comprises one or more proteins selected from Wnt2 and Wnt5a.
  • the hematopoietic stem cell differentiation suppressing or proliferating method of the present invention it is possible to culture and proliferate hematopoietic stem cells in vitro and ex vivo.
  • the use of the hematopoietic stem cell differentiation-inhibiting or proliferating agent of the present invention allows hematopoietic stem cells to be expanded in vivo or outside of the body while remaining undifferentiated.
  • the screening method of the present invention it is possible to screen for a substance that regulates the proliferation or differentiation of hematopoietic stem cells, so that diseases at the level of hematopoietic stem cells can be elucidated and new therapeutic methods and therapeutic agents developed.
  • a substance that regulates the proliferation or differentiation of hematopoietic stem cells so that diseases at the level of hematopoietic stem cells can be elucidated and new therapeutic methods and therapeutic agents developed.
  • a method for inhibiting or expanding hematopoietic stem cells and a method for producing hematopoietic stem cells
  • hematopoietic stem cells include all cells including granulocyte cells (mye 1 oid), lymphocyte cells (1 ymph oid), erythroid cells (erythroid), megakaryocyte cells (me gakaryocyte), etc. Refers to cells that have the ability to differentiate into blood cells and have the ability to self-renew.
  • hematopoietic stem cells Cultivation of hematopoietic stem cells in vitro requires the co-presence of some hematopoietic factors or cell stimulating factors, and in the absence of such factors hematopoietic stem cells Cells die quickly. On the other hand, in the presence of such factors, hematopoietic stem cells easily differentiate. Hematopoietic stem cells lose their ability to self-renew when differentiated. To suppress the differentiation of hematopoietic stem cells means to increase the abundance ratio and / or the number of hematopoietic stem cells having self-renewal ability under conditions that normally allow differentiation of hematopoietic stem cells to proceed.
  • Proliferation (or expansion) of hematopoietic stem cells means that hematopoietic stem cells that retain self-renewal ability increase, that is, self-renewal.
  • An increase in cells differentiated from hematopoietic stem cells corresponds to proliferation of hematopoietic stem cells. do not do.
  • Wnt2 and Wnt5a used in the present invention are known proteins, and their amino acid sequences and the nucleotide sequences of genes encoding the proteins can be obtained from the following sequence databases.
  • the human Wnt2 gene and the human Wnt5a gene are registered in GenBank as Accession No. NM-003391 and Accession No. NM-003392, respectively.
  • Human Wnt2 protein and human Wnt5a protein are registered as NC cession No. NP- 0 03382 and Ac cession No. NP- 003383 with NCB I Entrez Protein DataBase, respectively. It has been.
  • the mouse Wnt2 gene and mouse Wnt5a gene are registered in GenBank as Accession No. BC026373 and Accession No.
  • Wnt 2 protein and mouse Wnt 5a protein were registered with NCB I Entrez Protein DataBase as Ac cession on No. P 21 552 and Ac cession on No. A AH 18425, respectively.
  • Wnt2 and Wnt5a used in the present invention may be derived from any species, but are preferably derived from humans.
  • a mutant protein comprising an amino acid sequence in which a deletion, substitution, insertion, and / or addition has occurred to the amino acid sequence of a human protein, as long as the protein has the same activity as human Wnt 2 and Wnt 5a. Can be used in the same way as Wnt 2 and Wnt 5 a.
  • the mutation site and number of amino acids are not particularly limited as long as the mutant protein retains the same activity as human Wnt2 and Wnt5a, but the number of mutations is usually within several tens of amino acids (for example, within 60 amino acids). ), Preferably within 10 amino acids, more preferably 1 to several (eg 1 to 6), even more preferably It is 1-3, more preferably 1-2.
  • Wnt2 and Wnt5a used in the present invention can be prepared from cells or tissues expressing the protein, and can be prepared using a peptide synthesizer (for example, peptide synthesizer-1433A, Applied Biosystems Japan).
  • the Wnt2 and Wnt5a used in the present invention can be used in their own form or in the form of a fusion protein with another kind of protein, and can further convert the protein into various forms. It is possible. For example, processing by various methods known to those skilled in the art, such as various chemical modifications to proteins, binding to polymers such as polyethylene glycol, binding to insoluble carriers, sugar chain modification, lipid modification, and the like can be considered.
  • the hematopoietic stem cells used in the present invention can be collected from, for example, fetal liver, bone marrow, peripheral blood, umbilical cord blood, and the like of mammals such as humans and mice, but the source of collection is not limited as long as hematopoietic stem cells are included.
  • the preparation and isolation can be carried out by a usual method using a marker of a target hematopoietic stem cell as an index. For example, in mice, of the cells that are negative for cell differentiation antigen (Linege) and positive for c-kit and Sea-1 and that show CD34 antigen negative to weakly positive, the characteristics of hematopoietic stem cells Have been found (Osawa, M., Science, 27-3: 242, 1996).
  • the CD34 antigen is known as a marker for human hematopoietic stem cells.
  • the more undifferentiated markers are known to be CD34 antigen positive, CD38 antigen negative, and cell differentiation antigen negative (Bhatiaeta 1. Sci. USA 94, 5320-5325, 1997).
  • a nucleated cell derived from human or mouse bone marrow or a stem cell fraction can be directly used for culture without isolating hematopoietic stem cells.
  • SP sin depopulation
  • the hematopoietic stem cells or hematopoietic stem cell fraction collected as described above are cultured in the presence of one or more proteins selected from Wnt2 and Wnt5a.
  • the cells can be cultured in vitro using known cell culture techniques (for example, see Shinsei Kagaku Kenkyusho 18 Cell Culture Techniques (edited by The Japanese Biochemical Society, published by Tokyo Chemical Dojin, 1989)).
  • Hematopoietic stem cells can be cultured using an appropriate medium in a culture dish, flask, or bioreactor capable of mechanically controlling the composition and pH of the medium.
  • the appropriate medium used for the culture is not particularly limited as long as the survival and proliferation of hematopoietic stem cells are not inhibited.
  • SF-02 medium (Sanko Junyaku)
  • ⁇ pti-MEM medium GI BCO BRL
  • MEM medium GIB CO BRL
  • DMEM medium GI BCO BRL
  • I MDM medium I MDM
  • PRM I 1640 medium GI BCO BRL
  • RD medium RPM
  • DMEM 1: 1 [V / V mixed medium].
  • Culture systems usually contain additional components such as insulin, transferrin, lactoferrin, 2-mercaptoethanol, ethanolamine, sodium selenite, and HE PES, serum (eg, fetal calf serum, human serum). , Poma serum), monothioglycerol, sodium pyruvate, polyethylene glycol, various vinamines, various amino acids, and various growth factors are added as needed.
  • hematopoiesis can be more effectively achieved by adding a hematopoietic factor or a cell stimulating factor, or both to a culture system, in addition to one or more proteins selected from Wnt2 and Wnt5a. It can suppress stem cell differentiation and Z or self-renewal.
  • hematopoietic factor or cell stimulating factor broadly means a factor that stimulates hematopoietic cells to self-renew, proliferate, differentiate, survive, migrate, and the like.
  • the hematopoietic factor or cell stimulating factor used here is not particularly limited as long as it does not inhibit the survival and proliferation of hematopoietic stem cells.
  • hematopoietic factors or cell stimulating factors include, for example, SCF (stem cell growth factor), ⁇ (thrompopoetin), IL-3 (interleukin-13), and IL-11 (interleukin).
  • SCF stem cell growth factor
  • ⁇ thrompopoetin
  • IL-3 interleukin-13
  • IL-11 interleukin
  • the hematopoietic factor or cell stimulating factor used in the present invention includes proteins derived from mammals such as humans and mice produced by genetic recombination techniques, peptide synthesis methods, cell culture methods, and the like. Furthermore, the hematopoietic factor or the cell stimulating factor used herein is such that a part (for example, 1 to several (for example, 1 to 6), preferably 1 to 3) of the amino acid sequence is substituted, inserted, added and substituted. Even a mutant protein which has been deleted can be used as long as it retains its activity. Alternatively, a feeder cell expressing each factor may be used.
  • the amount of hematopoietic or cell stimulating factor added to the culture system is generally factor-specific, but the factor is usually in the hematopoietic stem cell culture medium at about 1 ng Zm1 to about 100 ng / m1. , Preferably at a concentration of about 5 ng / ml to about 50 ng / ml, more preferably at a concentration of about 5 ng / ml to about 30 ng / ml.
  • one or more proteins selected from Wnt2 and Wnt5a and one or more proteins selected from various hematopoietic factors or cell stimulating factors can be added to the culture system.
  • Hematopoietic stem cells may be co-cultured on a single feeder expressing proteins and factors.
  • the feeder cells preferably used here include bone marrow cells or bone marrow stromal cells whose growth ability has been lost by X-ray treatment or the like, cells derived from AGM regions, fibroblasts, cells derived from fetal liver, mesenchymal stem cells, Vascular endothelial cells, preadipocytes and the like can be used.
  • the culture is performed in a serum-free medium without adding serum to the culture system.
  • a feeder cell that is particularly preferably used in a serum-free medium includes C127 cells.
  • the C127 cells which can be used in the present invention and can survive, long-term culture, and / or proliferate in a serum-free medium can be obtained, for example, as follows. First, C127 cells were treated with DMEM containing 1% FCS + 5% KSR for 1 month. Select the cells that will survive, and then expand the cells that survive in DMEM containing 0.1% FCS + 5% KSR. C127 cells capable of surviving, long-term culture, and Z or proliferating in serum-free medium are then cloned by limiting dilution of cells that have been amplified with complete serum-free DMEM + 5% KSR.
  • Expression of one or more proteins selected from Wnt2 and Wnt5a in a feeder cell and various hematopoietic factors or cell stimulating factors include DNA encoding the amino acid sequence of those proteins or factors.
  • a recombinant vector may be prepared and the recombinant vector may be introduced into a feeder cell.
  • the cultivation is performed, for example, at a temperature of 33 to 39 (preferably 37 ° C.) under 3 to 6% CO 2 (preferably 5%) for 5 to 50 days.
  • a temperature of 33 to 39 preferably 37 ° C.
  • 3 to 6% CO 2 preferably 5%
  • the results of the differentiation inhibition and / or self-renewal of hematopoietic stem cells are described in Seaberg, LA rweir's Handbook of Experimental Immunology, 5th edition, Blackwell Science Inc. 1997, Spangrude, GJ, Proc. Natl. Acad. Sci. USA, 87 : 7433-7437, 1990; Visser, JMW, Flow cytometry in hematology], Academic Press, p9-29, 1992, etc., and can be confirmed using cell surface antigens as an index.
  • a marker for human hematopoietic stem cells at least CD34 antigen positive, preferably CD34 antigen positive, CD38 antigen negative, cell differentiation antigen negative can be used as an indicator of human hematopoietic stem cells.
  • a transplantation experiment using an irradiated mouse or an in vitro (in vitro) colony formation method can also be used.
  • a bone marrow cell or hematopoietic stem cell-containing fraction isolated from another mouse is transplanted into a mouse (recipient) irradiated with radiation and damaging the hematopoietic system.
  • the proportion of hematopoietic cells derived from recipients and donors is used as an indicator to confirm the presence of hematopoietic stem cells with long-term bone marrow remodeling ability (Osawa, M., Science, 273: 242-245). Natl. Ac ad. Sci. USA 94, 5320-5325, 1997).
  • hematopoietic progenitor cells in which the differentiation direction has been determined can be a small number or a single cell. Contains only differentiated cells Hematopoietic stem cells that form no colonies but have pluripotency are able to form colonies that include blood cells of multiple differentiation lineages. In particular, the formation of a mixed colony containing red blood cells (CFU-Emix) is regarded as an indicator of hematopoietic stem cells in humans.
  • CFU-Emix mixed colony containing red blood cells
  • the present invention provides a method for producing hematopoietic stem cells, characterized by using the above-described culture method of the present invention.
  • the production method of the present invention is characterized by comprising the step of (a) culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
  • the production method of the present invention may further include the following steps, but is not limited thereto, and may appropriately include the steps according to the culture method and culture system of the present invention; (b) -A step of culturing in the presence of at least one or more hematopoietic factors or cell stimulating factors, in the absence of serum, (c) a step of co-culturing hematopoietic stem cells with one feeder cell, and (d) a Wnt t A step of expressing one or more proteins selected from 2 and Wnt5a. 2. Hematopoietic stem cells obtained by the above culture method
  • the hematopoietic stem cells obtained by the above method of the present invention can be used for hematopoietic stem cell transplantation instead of conventional bone marrow transplantation or cord blood transplantation.
  • the stem cell expansion technology according to the present invention can be used to expand autologous or non-autologous stem cells.
  • the hematopoietic stem cells obtained by the method of the present invention can be used in combination with systemic X-ray therapy or advanced chemotherapy for various leukemias.
  • hematopoietic stem cells are obtained by collecting bone marrow prior to surgery and performing hematopoietic stem cell expansion in vitro, for example, when performing chemotherapy, radiation therapy, or other treatments that cause bone marrow suppression as a side effect in patients with solid cancer. By returning to the patient after the procedure, hematopoietic damage due to side effects can be recovered early, and stronger chemotherapy can be performed.
  • the present invention is used to treat a patient with insufficient formation of various blood cells by dividing a patient or another person's hematopoietic stem cells into various blood cells and transferring them into the patient's body. Can be.
  • the culture obtained by the culture method of the present invention. Blood stem cells can ameliorate hematopoietic failure caused by bone marrow hypoplasia presenting anemia such as aplastic anemia.
  • hematopoietic stem cell transplantation obtained by the culture method of the present invention includes chronic granulomatous disease, double immunodeficiency syndrome, cancer-free magropurinemia, Wisko tt—Aldrich syndrome, acquired Immunodeficiency syndrome (AIDS) and other immune deficiency syndromes, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, Gaucher disease, lysosomal storage disease such as mucopolysaccharidosis, adrenal white matter Degeneration, various cancers or tumors, and the like.
  • the transplantation of hematopoietic stem cells can be performed in the same manner as conventional bone marrow transplantation or cord blood transplantation, except for the cells used.
  • the graft of the present invention can be used as a composition containing a buffer solution and the like in addition to the cell components containing hematopoietic stem cells grown by the method of the present invention.
  • a feeder cell (1 :) one or more proteins selected from 11 cells and 11c5a, and (c) one or more hematopoietic factors or cells.
  • a hematopoietic stem cell culture system containing a stimulating factor and containing no serum.
  • a preferred feeder cell is a C 127 cell that has been prepared to survive in serum free.
  • Such a serum-free culture system can be used for a method of screening for a substance that regulates differentiation or proliferation of hematopoietic stem cells.
  • the method of screening for a substance that regulates the proliferation or differentiation of hematopoietic stem cells of the present invention is characterized by using a feeder cell that can survive in serum-free, and (a) comprises a feeder cell and a hematopoietic stem cell.
  • one or more proteins selected from Wnt2 and Wnt5a are included in the culture system (addition or feeder).
  • the proliferation promoting activity of the test substance can be more effectively tested in a state where the differentiation of the stem cells is suppressed.
  • one or more hematopoietic factors or cell stimulating factors may be included in the culture system.
  • the regulation of proliferation or differentiation means any of growth suppression, growth promotion, differentiation suppression and differentiation promotion, and growth promotion is particularly preferred.
  • Test substances used in this screening include cell culture supernatants, purified proteins or peptides, synthetic compounds, natural products derived from microorganisms and plants, and factors that promote the growth or differentiation of hematopoietic stem cells. Examples include proteins and biological materials that are expected to be produced by supporting cells.
  • the activity of the test substance can be measured by calculating the ratio and Z or number of hematopoietic stem cells contained in the cells before and after the treatment in the presence and absence of the test substance in the culture system.
  • the proportion and / or number of hematopoietic stem cells contained in the cells can be determined by FACS.
  • the differentiation of hematopoietic stem cells can be measured by FACS or a fluorescent antibody method using an antibody against an antigen specific to the differentiated cells.
  • B220 can be used as a specific antigen for B cells, Gr-1 as a specific antigen for granulocytes, and TER119 as a specific antigen for erythroid cells.
  • it can be measured by colony assay such as CFU-GM, CFU-Mix, and CFU-S.
  • colony assay such as CFU-GM, CFU-Mix, and CFU-S.
  • a hematopoietic stem cell differentiation inhibitory or proliferating agent containing one or more proteins selected from Wnt2 and Wnt5a (hereinafter, also simply referred to as "hematopoietic stem cell proliferating agent”) ) Is provided.
  • the growth agent of the present invention preferably comprises, in addition to one or more proteins selected from Wnt2 and Wnt5a, Alternatively, it contains one or more cell stimulating factors, for example, SCF, TPO or both.
  • the hematopoietic stem cell proliferating agent of the present invention can proliferate hematopoietic stem cells in vivo or outside of the body while undifferentiated, it can be used in the above-mentioned method for culturing and expanding hematopoietic stem cells in vitro, and in addition to radiation therapy.
  • Chemotherapeutic drugs such as anticancer drugs and anticancer drugs to prevent cytopenia caused by lymphopenia, treatment of bone marrow disorders such as bone marrow aplasia and bone marrow suppression, leukemia, severe renal disorders, bone marrow suppression, etc. It can be used for the treatment of bone marrow diseases, the treatment of hypocytosis caused by genetic diseases, the in vitro culture of recombinant stem cells during gene therapy, and the like.
  • the hematopoietic stem cell proliferating agent of the present invention can be administered orally, parenterally, locally, such as bone marrow, or any other suitable route.
  • the desired dose of the hematopoietic stem cell proliferating agent is 1 to 10 per day as its active ingredient (the Wnt protein and, if necessary, each hematopoietic factor or cell stimulating factor is referred to as “active ingredient”). It is about 0 i GZ kg body weight, preferably about 5 to 500 ⁇ 71 ⁇ 8 body weight, but naturally varies depending on the weight and symptoms of the patient and individual administration route. In some cases, a dose lower than the lower limit of the above range may be appropriate, and even if the dose is higher than the above range, if it is divided into several times a day and administered in small amounts, adverse side effects may occur. Sometimes it does not happen.
  • the active ingredient used in the present invention can be administered alone or together with a pharmaceutically or pharmaceutically acceptable carrier or diluent by any of the above three administration routes. It can be performed once or in several times. More specifically, the proliferating agent of the present invention can be administered in various types of administration forms, for example, tablets, capsules, medicinal drops in combination with various pharmaceutically acceptable inert carriers. Troches, hard candy, powders, sprays, creams, salves, suppositories, jellies, jewels, pastes, lotions, ointments, aqueous suspensions, injections, elixirs, syrups and the like. These carriers include solid diluents or excipients, sterile aqueous media, various non-toxic organic solvents, and the like.
  • Example 1 Example 1
  • Mouse bone marrow cells were injected into C57BL / 6 mice at 8 weeks of age by rapidly pouring phosphate-buffered saline (PBS) containing 2% inactivated fetal calf serum (FCS) into the marrow cavity with a 26-gauge needle. Prepared from mice (Charles River). 1 Adult human bone marrow cells were obtained from the posterior iliac crest (pos terior il iac crest) of normal donors based on informed consent and institutional ethics committee approval. The cell suspension was placed on Ly hop rep (Nycomed) and centrifuged at 800 g for 20 minutes. Mononuclear cells were collected and washed with PBS containing 2% FCS.
  • PBS phosphate-buffered saline
  • FCS inactivated fetal calf serum
  • the adherent cells were cultured for 10 days in an IMD culture solution (Gibco-BRL) containing 10% FCS. After isolating total RNA by the AGPC method, the type of Wnt expressed was identified by degenerative RT-PCR.
  • RT-PCR Reverse transcription polymerase chain reaction
  • Nucleated cells were prepared from bone marrow cells of 7-8 week old lacZ transgenic mice (B6; S129-Gt (R0SA26) Sor) by centrifugation using Lymphoprep and suspended in PBS containing 2% FCS. Cells (1 ⁇ 10 6 cells / ml) were transferred to Hoechs t33342 (l.
  • SP Side population cells
  • FACS Vantage SE cell sorter (trademark: Beckton Dickinson Bioscience). Approximately 4000 SP cells were isolated from one mouse. From lacZ transgenic mice, Lin-negative c-Kit-positive Seal-positive CD34-negative or low cell fractions, mouse bone marrow hematopoietic stem cells, were isolated using FACS. Approximately 10,000 cells were isolated from one mouse.
  • the full-length mouse or human Wnt2 and Wnt5a cDNAs were isolated by RT-PCR using K0D + DNA polymerase, and the complete nucleic acid sequence of the Wnt cDNA was identical to the sequence registered in the database overnight. confirmed.
  • Recombinant mouse or human Wnt cDNAs in PCDNA3.1 expression vector transfect C127 cells using Lipofect Amine 2000 (invitrogen), resistant to G418 (l mg / ml)
  • the stably expressing cells were maintained in a serum-free medium (DMEM + 5% Knockout serum replacement (KSR) (Gibco)).
  • DMEM + 5% Knockout serum replacement (KSR) Knockout serum replacement
  • the isolated lacZ transgenic mouse-derived bone marrow hematopoietic stem cells were implanted at 500 / well, and Co-cultured.
  • the medium used was a serum-free medium or a serum-free medium supplemented with TP0 and SCF. The medium was changed every two days, and the cells were collected by trypsin digestion on the sixth day.
  • Cells derived from lacZ transgenic mice are fractionated by fluorescence using fluoroscein di-jQ-galactopyranoside as a substrate, and the percentage of Lin-negative c-Kit-positive Seal-positive cells (hematopoietic stem cells) is calculated. did.
  • Hematopoietic stem cells are differentiated or proliferated in vivo or in vitro without undifferentiation by using the method for suppressing or proliferating hematopoietic stem cells of the present invention, the method for producing hematopoietic stem cells, and the agent for suppressing or proliferating hematopoietic stem cells of the present invention Can be because of It can be used to improve cytopenia, prevent infectious diseases caused by lymphopenia, treat bone marrow disease, and treat bone marrow diseases such as leukemia, severe renal impairment, and bone marrow suppression.
  • a substance that regulates the proliferation or differentiation of hematopoietic stem cells can be screened, which is useful for elucidating diseases at the level of hematopoietic stem cells and for developing new therapeutic methods and therapeutic agents. is there.

Abstract

It is intended to provide a method of regulating hematopoietic stem cells in the undifferentiated state or proliferating the same, an agent for regulating the differentiation of hematopoietic stem cells or proliferating the same, and so on. More specifically speaking, it is intended to provide a method of regulating the differentiation of hematopoietic stem cells or proliferating the same characterized by comprising culturing the hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a; a hematopoietic stem cell culture system characterized by containing (a) feeder cells, (b) one or more proteins selected from Wnt2 and Wnt5a and (c) or more hematopoietic factors or cell stimulating factors but being free from serum; an agent for regulating the differentiation of hematopoietic stem cells or proliferating the same characterized by containing one or more proteins selected from Wnt2 and Wnt5a; and so on.

Description

明細書  Specification
造血幹細胞の分化抑制又は増殖方法 技術分野  Method for inhibiting differentiation or expanding hematopoietic stem cells
本発明は、 造血幹細胞の分化抑制又は増殖方法、 造血幹細胞の製造方法、 その 方法によって得られる造血幹細胞、 その培養法で用いられる培養系、 造血幹細胞 の増殖または分化を調節する物質をスクリーニングする方法、 造血幹細胞の分化 抑制又は増殖剤などに関する。 背景技術  The present invention relates to a method for inhibiting or proliferating hematopoietic stem cells, a method for producing hematopoietic stem cells, a hematopoietic stem cell obtained by the method, a culture system used in the culture method, and a method for screening for a substance that regulates the growth or differentiation of hematopoietic stem cells. The present invention relates to an agent for suppressing differentiation or proliferating hematopoietic stem cells. Background art
最近では、 Wn tと称されるタンパク質が造血幹細胞との関係で盛んに研究さ れている。 例えば、 We i s smanらは Wn tシグナルが幹細胞を増幅できる 可能性があると報告している （N a t u r e, VOL. 423, 2003, p p 409— 414) 。 この報告では、 従来幹細胞の自己複製に必要であるとさ れていた Ho xB 4や分化抑制シグナルに必要な No t c hの発現も誘導できる ことから、 幹細胞の増幅や維持における Wntフアミリ一の重要な役割が示された。 また、 Ma t t h ewsらは、 マウス胎児肝において Wn t 5 aと Wn t 10 bが発現していること、 Wn t 10 bは造血幹細胞 も発現していること、 Wn t 5 aまたは Wn t 10 bを発現させた細胞の培養上清は 5倍程度に幹細胞を増 幅したこと報告している （BLOOD, VOL. 89, 1997, p p 36 24— 3635) 。 Ho f f ma nらはヒト胎児骨髄に Wn t 2 b (これは Wn t 13に対応する） 、 Wn t 5 a、 Wn t 10 bが発現しており、 Wn t 5 aは ヒ卜造血幹細胞にも発現していると報告している （非特許文献 1 ； BLOOD, VOL. 92, No. 9， 1998, pp 3189— 3202) 。 しかし、 これらの Wn tは体外培養系で造血幹細胞の自己複製を促進するかどうかについ ては示されておらず、 そもそも胎児の骨髄で造血は行なわれていない。 Bha t i aらは Wn t 5 aで造血幹細胞を体外では増幅できないが、 Wn t 5 aを含む 培養上清をマウスに投与すると 3倍程度幹細胞が増幅したと報告している （非特 許文献 2 ： PNAS, VOL. 100, No. 6, 2003, p p 342 2 - 3 4 2 7 ) 。 この文献では、 Wn tによる幹細胞の体外での増幅効果は殆ど 無いと報告されている。 Recently, a protein called Wnt has been actively studied in relation to hematopoietic stem cells. For example, We is sman and colleagues have reported that Wnt signals may be able to amplify stem cells (Nature, VOL. 423, 2003, pp 409-414). In this report, we show that Wnt familly is important for stem cell expansion and maintenance because it can induce the expression of HoxB4 and Notch, which is required for stem cell self-renewal. Role indicated. Matthews et al. Also show that Wnt5a and Wnt10b are expressed in mouse fetal liver, that Wnt10b also expresses hematopoietic stem cells, that Wnt5a or Wnt10a. It has been reported that the culture supernatant of cells expressing b increased stem cells about 5-fold (BLOOD, VOL. 89, 1997, pp. 3624-3635). Hoffman et al. Express Wnt2b (which corresponds to Wnt13), Wnt5a and Wnt10b in human fetal bone marrow, and Wnt5a is expressed in human hematopoietic stem cells. (Non-Patent Document 1; BLOOD, VOL. 92, No. 9, 1998, pp 3189-3202). However, it has not been shown whether these Wnts promote self-renewal of hematopoietic stem cells in an in vitro culture system, and hematopoiesis is not performed in fetal bone marrow in the first place. Bhatia et al. Reported that hematopoietic stem cells cannot be expanded in vitro with Wnt5a, but that stem cells were expanded about three-fold when a culture supernatant containing Wnt5a was administered to mice (Non-Patent Document 2: PNAS, VOL. 100, No. 6, 2003, pp 342 2-3 4 2 7). This document reports that Wnt has almost no effect of expanding stem cells in vitro.
また、 N u s s eらは Wn t 3 aを可溶性タンパク質として純化に成功し、 W n t 3 a単独で、 造血幹細胞を約 6倍に増幅できたと報告している （N a t u r e , V O L . 4 2 3 , 2 0 0 3 , p p 4 4 8— 4 5 2 ) 。 しかしな力 Sら、 こ の実験では、 幹細胞が分化してしまっており、 純粋に幹細胞だけを増幅している 訳でもない。 また、 増幅の程度は、 約 1 . 7倍であり、 実用化レベルではない。 以上の報告からは、 Wn tが実際に単独で体性幹細胞の自己複製因子としての 機能を持っているのか、 W n tが全ての体性幹細胞を自己複製させる能力がある のか否か、 オートクラインで自己複製するとする矛盾をどう説明できるのか、 ま た Wn tファミリ一のどのメンパーがどの体性幹細胞を制御しているのか （特異 性） 、 などの疑問点は未だ解明されていない。  Also, Nusse et al. Reported that Wnt 3a was successfully purified as a soluble protein, and that Wnt 3a alone was able to expand hematopoietic stem cells by about 6-fold (Nature, VOL. 2 0 3, pp 4 4 8 — 4 5 2). However, in this experiment, the stem cells have differentiated, and not only pure stem cells are amplified. The degree of amplification is about 1.7 times, which is not practical. These reports indicate whether Wnt actually functions alone as a self-renewal factor for somatic stem cells, whether Wnt has the ability to self-renew all somatic stem cells, and whether autocrine Questions such as how to explain the contradiction of self-renewal in, and which member of the Wnt family controls which somatic stem cells (specificity) have not yet been elucidated.
発明の開示 Disclosure of the invention
上記したように、 造血幹細胞の増殖との関係で Wn tタンパク質は種々検討さ れてきたが、 どの Wn tタンパク質が実際に実用的レベルの造血幹細胞の分化抑 制能又は増殖能を有するかについては未だ究明されておらず、 そのようなタンパ ク質を用いて造血幹細胞を未分化に抑制する方法あるいは増殖する方法は未だ知 られていない。 従って、 造血幹細胞の分化抑制能又は増殖能を有するタンパク質 を特定することができ、 そのタンパク質を用いて造血幹細胞の分化を抑制し、 又 は増殖させることができれば、 血液関連の種々の疾患の治療あるいは研究に有効 である。  As described above, various studies have been conducted on Wnt proteins in relation to the proliferation of hematopoietic stem cells.However, which Wnt proteins actually have a practical level of inhibiting differentiation or proliferating hematopoietic stem cells is considered. The method of suppressing undifferentiated or proliferating hematopoietic stem cells using such a protein has not been known yet. Therefore, it is possible to specify a protein having the ability to inhibit differentiation or proliferation of hematopoietic stem cells, and to suppress the differentiation or proliferate hematopoietic stem cells using the protein, to treat various blood-related diseases. Or it is effective for research.
本発明者等は鋭意研究の結果、 成体マウス骨髄ストローマ細胞には Wn t 2、 Wn t 5 aが発現しており、 成体ヒト骨髄ストローマ細胞には Wn t 2、 Wn t 5 a及び Wn t 5 bが発現し、 Wn t 2が最も多く発現していることを見出し た。 本発明者等は、 この新たな知見に基づいて研究を続けた結果、 Wn t 2また は Wn t 5 aを用いることにより、 造血幹細胞の分化を抑制し、 あるいは増殖さ せることができることを見出し、 本発明を完成した。 すなわち、 本発明は、 次のような、 造血幹細胞の分化抑制及び Z又は増殖方法、 その方法によって得られる造血幹細胞、 造血幹細胞の製造方法、 その培養法で用 いられる培養系、 造血幹細胞の増殖または分化を調節する物質をスクリーニング する方法、 造血幹細胞の分化抑制又は増殖剤などを提供する。 The present inventors have conducted extensive studies and found that adult mouse bone marrow stromal cells express Wnt2 and Wnt5a, and adult human bone marrow stromal cells express Wnt2, Wnt5a and Wnt5a. b was expressed, and Wnt 2 was found to be most frequently expressed. The present inventors have continued their research based on this new finding and found that the use of Wnt2 or Wnt5a can suppress or proliferate the differentiation of hematopoietic stem cells. The present invention has been completed. That is, the present invention provides the following methods for inhibiting and differentiating or proliferating hematopoietic stem cells, a method for producing hematopoietic stem cells, a method for producing hematopoietic stem cells, a culture system used in the culture method, and a method for expanding hematopoietic stem cells. Also, the present invention provides a method for screening a substance that regulates differentiation, an agent for suppressing differentiation or proliferating hematopoietic stem cells, and the like.
(1) 造血幹細胞を、 Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク 質の存在下で培養することを特徴とする造血幹細胞の分化抑制又は増殖方法。  (1) A method for inhibiting differentiation or expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
(2) 一種以上の造血因子又は細胞刺激因子の存在下、 かっ血清の非存在下で培 養を行なう前記 （1) に記載の方法。  (2) The method according to (1) above, wherein the culture is performed in the presence of one or more hematopoietic factors or cell stimulating factors, and in the absence of serum.
(3) S CF及び TP 0から選ばれる一種以上の造血因子の存在下で培養を行な う前記 （2) に記載の方法。  (3) The method according to (2), wherein the culture is performed in the presence of one or more hematopoietic factors selected from SCF and TPO.
(4) Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質を培養系に添 加し、 あるいはそのタンパク質を発現させたフィーダ一細胞上で培養する前記 (4) adding one or more proteins selected from Wnt2 and Wnt5a to a culture system, or culturing on a feeder cell expressing the protein;
(3) に記載の方法。 The method according to (3).
(5) 前記 （1) 〜 （4) のいずれかの方法によって得られる造血幹細胞。  (5) A hematopoietic stem cell obtained by the method according to any one of (1) to (4).
(6) 造血幹細胞を、 Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク 質の存在下で培養する工程を含んでなる、 造血幹細胞の製造方法。  (6) A method for producing hematopoietic stem cells, comprising a step of culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
(7) (a) フィーダ一細胞、 （b) Wn t 2及び Wn t 5 aから選ばれる一種 以上のタンパク質及び （c) 一種以上の造血因子又は細胞刺激因子を含み、 かつ 血清を含まないことを特徴とする造血幹細胞培養系。  (7) (a) a feeder cell, (b) one or more proteins selected from Wnt2 and Wnt5a, and (c) one or more hematopoietic or cell stimulating factors and no serum. A hematopoietic stem cell culture system, characterized in that:
(8) フィーダ一細胞として、 無血清下で生存可能な C 127細胞を含む前記 (7) に記載の培養系。  (8) The culture system according to (7), wherein the feeder cells include C 127 cells that can survive without serum.
(9) 無血清下で生存可能なフィーダ一細胞を用いることを特徴とし、 （a) フ ィーダ一細胞および造血幹細胞を含み、 かっ血清を含まないことを特徴とする培 養系に試験物質を共存させる工程、 （b) 試験物質が共存した培養系で造血幹細 胞を培養する工程、 及び （c) 試験物質非共存下の場合と比較して、 試験物質共 存下の場合における造血幹細胞の割合および または数の変化を測定する工程を 含む、 造血幹細胞の増殖または分化を調節する物質をスクリーニングする方法。  (9) Use of feeder cells that can survive in the absence of serum; (a) Add a test substance to a culture system that contains feeder cells and hematopoietic stem cells and is free of serum. (B) culturing hematopoietic stem cells in a culture system in which the test substance is present; and (c) hematopoietic stem cells in the presence of the test substance as compared to the absence of the test substance. A method for screening a substance that regulates the proliferation or differentiation of hematopoietic stem cells, comprising a step of measuring a change in the ratio and / or number of cells.
(10) Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質を含有する 造血幹細胞分化抑制又は増殖剤。 (11) さらに、 造血因子又は細胞刺激因子を含む前記 （10) に記載の造血幹 細胞分化抑制又は増殖剤。 (10) An agent for suppressing or proliferating hematopoietic stem cell differentiation, which comprises one or more proteins selected from Wnt2 and Wnt5a. (11) The agent for suppressing or proliferating hematopoietic stem cell differentiation according to (10), further comprising a hematopoietic factor or a cell stimulating factor.
(12) 前記造血因子として、 S CF及び TP 0から選ばれる一種以上の造血因 子を含む前記 （11) に記載の造血幹細胞分化抑制又は増殖剤。  (12) The agent for suppressing or proliferating hematopoietic stem cell differentiation according to (11), wherein the agent includes one or more hematopoietic factors selected from SCF and TP0 as the hematopoietic factors.
本発明の造血幹細胞の分化抑制又は増殖方法を用いることにより、 in vitroZ ex vivoで造血幹細胞の培養、 増殖等を行なうことができる。 また、 本発明の 造血幹細胞の分化抑制又は増殖剤を用いることにより、 造血幹細胞を未分化なま ま、 体内または体外で増殖させることができるので、 放射線治療や制ガン剤等の 化学療法剤による血球減少症の改善、 リンパ球減少に起因する感染症の予防、 骨 髄形成不全症や骨髄抑制等の骨髄疾患の治療、 白血病 ·高度腎障害 ·骨髄抑制等 の骨髄疾患の治療、 遺伝的疾患に由来する低血球症の治療、 遺伝子治療時におけ る組換え幹細胞の体外培養等に用いることができる。  By using the hematopoietic stem cell differentiation suppressing or proliferating method of the present invention, it is possible to culture and proliferate hematopoietic stem cells in vitro and ex vivo. In addition, the use of the hematopoietic stem cell differentiation-inhibiting or proliferating agent of the present invention allows hematopoietic stem cells to be expanded in vivo or outside of the body while remaining undifferentiated. Improvement of illness, prevention of infectious diseases caused by lymphopenia, treatment of bone marrow diseases such as bone marrow dysplasia and myelosuppression, treatment of bone marrow diseases such as leukemia, severe renal impairment, and bone marrow suppression, derived from genetic diseases It can be used for in vitro culture of recombinant stem cells at the time of treatment of hypocytosis and gene therapy.
また、 本発明のスクリーニング法によれば、 造血幹細胞の増殖または分化を調 節する物質をスクリ一二ングすることができるので、 造血幹細胞レベルの疾患の 解明や新たな治療法 ·治療剤の開発に有用である。 発明を実施するための最良の形態  Further, according to the screening method of the present invention, it is possible to screen for a substance that regulates the proliferation or differentiation of hematopoietic stem cells, so that diseases at the level of hematopoietic stem cells can be elucidated and new therapeutic methods and therapeutic agents developed. Useful for BEST MODE FOR CARRYING OUT THE INVENTION
以下、 本発明を詳細に説明する。 1. 造血幹細胞の分化抑制又は増殖方法、 および造血幹細胞の製造方法  Hereinafter, the present invention will be described in detail. 1. A method for inhibiting or expanding hematopoietic stem cells, and a method for producing hematopoietic stem cells
本発明の第一の態様によれば、 造血幹細胞を、 Wn t 2及び Wn t 5 aカ ら選 ばれる一種以上の蛋白質の存在下で培養することを特徴とする造血幹細胞の分化 抑制又は増殖方法 （以下、 「培養法」 ともいう） が提供される。 ここで、 「造血 幹細胞」 とは、 顆粒球系細胞 （mye 1 o i d) 、 リンパ球系細胞 （1 ymph o i d) , 赤血球系細胞 （e r y t h r o i d) 、 巨核球系細胞 （me g a k a r y o c y t e ) 等を含む全ての血球細胞へ分化する能力を有し、 且つ自己複製 能を有する細胞をいう。 造血幹細胞を体外で培養するためには、 何らかの造血因 子又は細胞刺激因子の共存が必要であり、 このような因子の非存在下では造血幹 細胞はすぐに死滅してしまう。 一方、 このような因子の存在下では造血幹細胞は 容易に分化してしまう。 造血幹細胞は分化してしまうと自己複製能を失う。 造血 幹細胞の分化を抑制するとは、 通常であれば造血幹細胞の分化が進むような条件 下で、 自己複製能を保持した造血幹細胞の存在割合および/または数を高めるこ とを意味する。 造血幹細胞の増殖 （または増幅とも言う） とは、 自己複製能を保 持した造血幹細胞が増えること即ち自己複製することを意味し、 造血幹細胞から 分化した細胞が増えることは造血幹細胞の増殖に該当しない。 According to a first aspect of the present invention, there is provided a method for inhibiting differentiation or expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a. (Hereinafter, also referred to as “culture method”). Here, “hematopoietic stem cells” include all cells including granulocyte cells (mye 1 oid), lymphocyte cells (1 ymph oid), erythroid cells (erythroid), megakaryocyte cells (me gakaryocyte), etc. Refers to cells that have the ability to differentiate into blood cells and have the ability to self-renew. Cultivation of hematopoietic stem cells in vitro requires the co-presence of some hematopoietic factors or cell stimulating factors, and in the absence of such factors hematopoietic stem cells Cells die quickly. On the other hand, in the presence of such factors, hematopoietic stem cells easily differentiate. Hematopoietic stem cells lose their ability to self-renew when differentiated. To suppress the differentiation of hematopoietic stem cells means to increase the abundance ratio and / or the number of hematopoietic stem cells having self-renewal ability under conditions that normally allow differentiation of hematopoietic stem cells to proceed. Proliferation (or expansion) of hematopoietic stem cells means that hematopoietic stem cells that retain self-renewal ability increase, that is, self-renewal. An increase in cells differentiated from hematopoietic stem cells corresponds to proliferation of hematopoietic stem cells. do not do.
本発明で用いる Wn t 2及び Wn t 5 aは公知の蛋白質であり、 そのアミノ酸 配列および該蛋白質をコードする遺伝子の塩基配列は以下の配列データベースか ら入手できる。 ヒト Wn t 2遺伝子及びヒト Wn t 5 a遺伝子は、 Ge nB an kに、 それぞれ、 Ac c e s s i on No. NM— 003391及び A c c e s s i on No. NM— 003392として登録されている。 ヒト Wn t 2夕 ンパク質及びヒト Wn t 5 aタンパク質は、 NCB I En t r e z P r o t e i n Da t ab a s eに、 それぞれ、 Ac c e s s i on No. NP— 0 03382及び Ac c e s s i on No. NP— 003383として登録され ている。 マウス Wn t 2遺伝子及びマウス Wn t 5 a遺伝子は、 Ge nB a n kに、 それぞれ、 Ac c e s s i on No. BC026373及び A c c e s s i on No. BC 018425として登録されている。 マウス Wn t 2夕 ンパク質及びマウス Wn t 5 aタンパク質は、 NCB I En t r e z P r o t e i n Da t ab a s eに、 それぞれ、 Ac c e s s i on No. P 21 552及び Ac c e s s i on No. A AH 18425として登録されている。 本発明で用いる Wn t 2及び Wn t 5 aは、 いずれの種由来のものでもよいが、 好ましくはヒト由来である。 また、 ヒト Wn t 2及び Wn t 5 aと同等の活性を 保持している限り、 ヒト蛋白質のアミノ酸配列に対して欠失、 置換、 挿入、 及び /または付加が生じたアミノ酸配列からなる変異蛋白質も Wn t 2及び Wn t 5 aと同様に使用しうる。 アミノ酸の変異部位および個数は、 変異蛋白質がヒト W n t 2及び Wn t 5 aと同等の活性を保持している限り特に制限はないが、 変異 個数は通常数十アミノ酸以内 （例えば、 60アミノ酸以内） 、 好ましくは 10ァ ミノ酸以内、 より好ましくは 1〜数個 （例えば、 1〜6個） 、 さらに好ましくは 1〜3個、 さらに好ましくは 1〜2個である。 本発明で用いる Wn t 2及び Wn t 5 aは、 該蛋白質を発現している細胞や組織から調製することができ、 またべ プチド合成機 （例えば、 ペプチドシンセサイザ一 433 A型、 アプライドバイオ システムズ ジャパン株式会社製） を使用した化学合成法でも、 また原核生物あ るいは真核生物から選択される適当な宿主細胞を用いた組換え方法によっても調 製することができる。 しかしながら、 その純度の面から遺伝子工学的な手法によ る生産ならびに組換え型蛋白質が好ましい。 本発明で用いる Wn t 2及び Wn t 5 aは、 それ単独の形態でも別種の蛋白質との融合蛋白質の形態でも使用するこ とができ、 また、 蛋白質を更に種々の形態へと変換させることも可能である。 例 えば、 蛋白質に対する種々の化学修飾、 ポリエチレングリコール等の高分子との 結合、 不溶性担体への結合、 糖鎖修飾、 脂質修飾等、 当業者に知られている多種 の手法による加工が考えられる。 Wnt2 and Wnt5a used in the present invention are known proteins, and their amino acid sequences and the nucleotide sequences of genes encoding the proteins can be obtained from the following sequence databases. The human Wnt2 gene and the human Wnt5a gene are registered in GenBank as Accession No. NM-003391 and Accession No. NM-003392, respectively. Human Wnt2 protein and human Wnt5a protein are registered as NC cession No. NP- 0 03382 and Ac cession No. NP- 003383 with NCB I Entrez Protein DataBase, respectively. It has been. The mouse Wnt2 gene and mouse Wnt5a gene are registered in GenBank as Accession No. BC026373 and Accession No. BC018425, respectively. Mouse Wnt 2 protein and mouse Wnt 5a protein were registered with NCB I Entrez Protein DataBase as Ac cession on No. P 21 552 and Ac cession on No. A AH 18425, respectively. ing. Wnt2 and Wnt5a used in the present invention may be derived from any species, but are preferably derived from humans. In addition, a mutant protein comprising an amino acid sequence in which a deletion, substitution, insertion, and / or addition has occurred to the amino acid sequence of a human protein, as long as the protein has the same activity as human Wnt 2 and Wnt 5a. Can be used in the same way as Wnt 2 and Wnt 5 a. The mutation site and number of amino acids are not particularly limited as long as the mutant protein retains the same activity as human Wnt2 and Wnt5a, but the number of mutations is usually within several tens of amino acids (for example, within 60 amino acids). ), Preferably within 10 amino acids, more preferably 1 to several (eg 1 to 6), even more preferably It is 1-3, more preferably 1-2. Wnt2 and Wnt5a used in the present invention can be prepared from cells or tissues expressing the protein, and can be prepared using a peptide synthesizer (for example, peptide synthesizer-1433A, Applied Biosystems Japan). (Produced by K.K.) or a recombinant method using an appropriate host cell selected from prokaryotes or eukaryotes. However, from the viewpoint of its purity, production by genetic engineering techniques and recombinant proteins are preferred. The Wnt2 and Wnt5a used in the present invention can be used in their own form or in the form of a fusion protein with another kind of protein, and can further convert the protein into various forms. It is possible. For example, processing by various methods known to those skilled in the art, such as various chemical modifications to proteins, binding to polymers such as polyethylene glycol, binding to insoluble carriers, sugar chain modification, lipid modification, and the like can be considered.
本発明において用いる造血幹細胞は、 例えば、 ヒト及びマウス等の哺乳動物の 胎児肝臓、 骨髄、 末梢血、 臍帯血等から採取できるが、 造血幹細胞 が含まれる 限りその採取源を問うものではない。 またその調製、 単離は、 目的とする造血幹 細胞のマーカーを指標として通常の方法により行なうことができる。 例えば、 マ ウスでは、 細胞分化抗原 （L i n e g e) が陰性であり、 かつ c- kitならびに Sea- 1陽性の細胞の内、 CD34抗原が陰性から弱陽性の性質を示す細胞に造血幹 細胞の性質が見いだされている （Osawa, M.， Science, 27-3:242, 1996) 。 ヒト 造血幹細胞のマーカーとしては CD 34抗原が知られており、 特により未分化な マーカーとして CD 34抗原陽性、 CD 38抗原陰性、 細胞分化抗原陰性がある ことが知られている （Bha t i a e t a 1. , P r o c. Na t l . Ac a d. S c i. U. S. A. 94, 5320-5325, 1997) 。 また、 場合 によっては、 造血幹細胞を単離することなく、 ヒト又はマウス骨髄由来等の有核 細胞あるいは幹細胞分画をそのまま培養に供することもできる。 例えば、 マウス 骨髄由来等の幹細胞分画として SP (s i de p opu l a t i on) 細胞分 画 （造血幹細胞を約半分弱含み、 他の組織幹細胞も含むと考えられている） を用 いることもできる。 本発明によれば、 上記のようにして採取した造血幹細胞または造血幹細胞分画 を、 Wn t 2及び Wn t 5 aから選ばれる一種以上の蛋白質の存在下で培養する。 細胞の in vitro培養は、 公知の細胞培養技術を用いて行なうことができる （例 えば、 新生化学実験講座 18 細胞培養技術 （日本生化学会編、 東京化学同人発 行、 1989) 等参照） 。 造血幹細胞の培養は、 培養用のシャーレ、 フラスコ、 ある いは培地組成、 pHなどを機械的に制御できるバイオリアクターにおいて適当な 培地を用いて行なうことができる。 培養に用いる適当な培地は、 造血幹細胞の生 存 ·増殖が阻害されない限り特に制限されないが、 例えば、 SF—02培地 （三 光純薬） 、 〇p t i—MEM培地 （G I BCO BRL) 、 MEM培地 （G I B CO BRL) 、 DMEM培地 （G I BCO BRL) 、 I MDM培地 （G I B CThe hematopoietic stem cells used in the present invention can be collected from, for example, fetal liver, bone marrow, peripheral blood, umbilical cord blood, and the like of mammals such as humans and mice, but the source of collection is not limited as long as hematopoietic stem cells are included. The preparation and isolation can be carried out by a usual method using a marker of a target hematopoietic stem cell as an index. For example, in mice, of the cells that are negative for cell differentiation antigen (Linege) and positive for c-kit and Sea-1 and that show CD34 antigen negative to weakly positive, the characteristics of hematopoietic stem cells Have been found (Osawa, M., Science, 27-3: 242, 1996). The CD34 antigen is known as a marker for human hematopoietic stem cells. In particular, the more undifferentiated markers are known to be CD34 antigen positive, CD38 antigen negative, and cell differentiation antigen negative (Bhatiaeta 1. Sci. USA 94, 5320-5325, 1997). In some cases, a nucleated cell derived from human or mouse bone marrow or a stem cell fraction can be directly used for culture without isolating hematopoietic stem cells. For example, as a stem cell fraction derived from mouse bone marrow or the like, SP (si depopulation) cell fraction (which contains about half of hematopoietic stem cells and is considered to contain other tissue stem cells) can also be used. . According to the present invention, the hematopoietic stem cells or hematopoietic stem cell fraction collected as described above are cultured in the presence of one or more proteins selected from Wnt2 and Wnt5a. The cells can be cultured in vitro using known cell culture techniques (for example, see Shinsei Kagaku Kenkyusho 18 Cell Culture Techniques (edited by The Japanese Biochemical Society, published by Tokyo Chemical Dojin, 1989)). Hematopoietic stem cells can be cultured using an appropriate medium in a culture dish, flask, or bioreactor capable of mechanically controlling the composition and pH of the medium. The appropriate medium used for the culture is not particularly limited as long as the survival and proliferation of hematopoietic stem cells are not inhibited. For example, SF-02 medium (Sanko Junyaku), 〇pti-MEM medium (GI BCO BRL), MEM medium ( GIB CO BRL), DMEM medium (GI BCO BRL), I MDM medium (GIBC
0 BRL) 、 PRM I 1640培地 （G I BCO BRL) 、 RD培地 （RPM0 BRL), PRM I 1640 medium (GI BCO BRL), RD medium (RPM
1 1640： DMEM = 1 : 1 [V/V 混合培地） 等が挙げられる。 培養系に は、 通常、 添加される成分、 例えば、 インスリン、 トランスフェリン、 ラクトフ エリン、 2—メルカプトエタノール、 エタノールァミン、 亜セレン酸ナトリウム、 および HE PES、 血清 （例えば、 ゥシ胎児血清、 ヒト血清、 ゥマ血清） 、 モノ チォグリセロール、 ピルビン酸ナトリウム、 ポリエチレングリコール、 各種ビ夕 ミン、 各種アミノ酸、 各種増殖因子が必要に応じて添加される。 1 1640: DMEM = 1: 1 [V / V mixed medium]. Culture systems usually contain additional components such as insulin, transferrin, lactoferrin, 2-mercaptoethanol, ethanolamine, sodium selenite, and HE PES, serum (eg, fetal calf serum, human serum). , Poma serum), monothioglycerol, sodium pyruvate, polyethylene glycol, various vinamines, various amino acids, and various growth factors are added as needed.
本発明の培養法においては、 Wn t 2及び Wn t 5 aから選ばれる一種以上の 蛋白質に加え、 造血因子又は細胞刺激因子、 あるいはその両方を培養系に添加す ることによって、 より有効に造血幹細胞の分化抑制および Zまたは自己複製を行 なうことができる。 ここで、 「造血因子又は細胞刺激因子」 とは、 造血細胞に自 己複製、 増殖、 分化、 生存、 遊走などの刺激を与える因子を広義に意味する。 こ こで用いられる造血因子又は細胞刺激因子は、 造血幹細胞の生存 ·増殖を阻害し ない限り特に限定されない。 好ましく用いられる造血因子又は細胞刺激因子とし ては、 例えば、 S CF (幹細胞成長因子； s t em c e l l f a c t o r) , ΤΡ〇 （トロンポポェチン） 、 I L— 3 (インターロイキン一 3) 、 I L一 11 (インタ一ロイキン一 1 1) 、 GM— CSF (顆粒球マクロファージ 'コロニー 刺激因子； g r anu l o c y t e/mac r oph ag e c o l ony— s t imu l a t i ng f a c t o r) 、 G— CSF (顆粒球コロニー刺激因 子； g r anu l o c y t e c o l ony— s t imu l a t i ng f ac t o r) 、 TGF-/3 (トランスフォーミング成長因子一 /3) 、 MI P— 1 α、 F 1 t 3/F 1 k 2— 1 i g and、 EPO (エリスロポエチン） 、 No t c hリガ ンド （J agge dファミリー、 De l t aファミリ一） 、 T i e 2リガンド (アンジオボェチン） 、 BMP 4、 F 1 k 2/F 1 k 3リガンド、 FL、 b FG F、 オンコス夕チン M、 I L 6/s I L 6R、 E G Fおよび L I F等が挙げられ る。 本発明で使用される造血因子又は細胞刺激因子は、 遺伝子組換え技術、 ぺプ チド合成法、 細胞培養法等により製造されたヒト、 マウス等の哺乳動物由来の蛋 白質を含む。 さらにここで用いられる造血因子又は細胞刺激因子は、 アミノ酸配 列の 1部 （例えば、 1〜数個 （例えば、 1〜6個） ） 、 好ましくは 1〜 3個） が 置換、 挿入、 付加及び/または欠失した変異蛋白質でも、 活性を保持している限 りにおいて使用しうる。 または、 各因子を発現させたフィーダ一細胞を用いても よい。 In the culture method of the present invention, hematopoiesis can be more effectively achieved by adding a hematopoietic factor or a cell stimulating factor, or both to a culture system, in addition to one or more proteins selected from Wnt2 and Wnt5a. It can suppress stem cell differentiation and Z or self-renewal. Here, the term “hematopoietic factor or cell stimulating factor” broadly means a factor that stimulates hematopoietic cells to self-renew, proliferate, differentiate, survive, migrate, and the like. The hematopoietic factor or cell stimulating factor used here is not particularly limited as long as it does not inhibit the survival and proliferation of hematopoietic stem cells. Examples of preferably used hematopoietic factors or cell stimulating factors include, for example, SCF (stem cell growth factor), ΤΡ〇 (thrompopoetin), IL-3 (interleukin-13), and IL-11 (interleukin). 1 1), GM—CSF (granulocyte / macrophage colony stimulating factor; granulocyte / macrophage colony—st imu latin ng factor), G—CSF (granulocyte colony stimulating factor) Offspring; granulocyte colony—st imu lati ng f actor), TGF- / 3 (transforming growth factor 1/3), MIP-1α, F 1 t 3 / F 1 k 2—1 ig and, EPO (erythropoietin), Notch ligand (Jagge d family, Delta family), Tie2 ligand (angiobotin), BMP4, F1k2 / F1k3 ligand, FL, bFGF, Oncos Evening M, IL 6 / s IL 6R, EGF and LIF. The hematopoietic factor or cell stimulating factor used in the present invention includes proteins derived from mammals such as humans and mice produced by genetic recombination techniques, peptide synthesis methods, cell culture methods, and the like. Furthermore, the hematopoietic factor or the cell stimulating factor used herein is such that a part (for example, 1 to several (for example, 1 to 6), preferably 1 to 3) of the amino acid sequence is substituted, inserted, added and substituted. Even a mutant protein which has been deleted can be used as long as it retains its activity. Alternatively, a feeder cell expressing each factor may be used.
培養系に添加される造血因子又は細胞刺激因子の量は、 一般的には因子特異的 であるが、 因子は、 造血幹細胞培養培地に、 通常、 約 1 n gZm 1〜約 100 n g/m 1の間の濃度で、 好ましくは約 5 n g/m 1〜約 50 n g/m 1の濃度で、 より好ましくは約 5 n gZm 1〜約 30 n gZm 1の間の濃度で添加される。 本発明の培養方法においては、 Wn t 2及び Wn t 5 aから選ばれる一種以上 のタンパク質、 および各種造血因子又は細胞刺激因子から選ばれる一種以上を培 養系に添加することもできるが、 それらタンパク質や因子を発現させたフィーダ 一細胞上で造血幹細胞を共培養してもよい。 ここで好ましく用いられるフィーダ 一細胞としては、 X線処理等で増殖能を欠損させた骨髄細胞または骨髄ストロー マ細胞、 A GM領域由来細胞、 線維芽細胞、 胎児肝臓由来細胞、 間葉系幹細胞、 血管内皮細胞、 および前脂肪細胞等を用いることができる。 本発明の特に好まし い態様によれば、 培養系に血清を添加しない無血清培地で培養を行なう。 無血清 培地で特に好ましく用いられるフィーダ一細胞としては、 C 127細胞が挙げら れる。 なお、 本発明で好ましく用いられる、 無血清培地で生存、 長期培養、 およ び/または増殖できる C 127細胞は、 例えば、 次のようにして得ることができ る。 まず、 C 127細胞を、 1 %FC S + 5 %KS R入りの DMEMで 1ヶ月ほ ど掛けて生き残る細胞を選択し、 更に 0. 1%FCS + 5%KSRを含むDME Mでも生き残る細胞を増殖する。 次いで完全無血清の DMEM+ 5 %KSRで増 幅する細胞を限界希釈法によってクローン化した細胞を、 無血清培地で生存、 長 期培養、 および Zまたは増殖可能な C 127細胞とする。 The amount of hematopoietic or cell stimulating factor added to the culture system is generally factor-specific, but the factor is usually in the hematopoietic stem cell culture medium at about 1 ng Zm1 to about 100 ng / m1. , Preferably at a concentration of about 5 ng / ml to about 50 ng / ml, more preferably at a concentration of about 5 ng / ml to about 30 ng / ml. In the culture method of the present invention, one or more proteins selected from Wnt2 and Wnt5a and one or more proteins selected from various hematopoietic factors or cell stimulating factors can be added to the culture system. Hematopoietic stem cells may be co-cultured on a single feeder expressing proteins and factors. The feeder cells preferably used here include bone marrow cells or bone marrow stromal cells whose growth ability has been lost by X-ray treatment or the like, cells derived from AGM regions, fibroblasts, cells derived from fetal liver, mesenchymal stem cells, Vascular endothelial cells, preadipocytes and the like can be used. According to a particularly preferred embodiment of the present invention, the culture is performed in a serum-free medium without adding serum to the culture system. A feeder cell that is particularly preferably used in a serum-free medium includes C127 cells. In addition, the C127 cells which can be used in the present invention and can survive, long-term culture, and / or proliferate in a serum-free medium can be obtained, for example, as follows. First, C127 cells were treated with DMEM containing 1% FCS + 5% KSR for 1 month. Select the cells that will survive, and then expand the cells that survive in DMEM containing 0.1% FCS + 5% KSR. C127 cells capable of surviving, long-term culture, and Z or proliferating in serum-free medium are then cloned by limiting dilution of cells that have been amplified with complete serum-free DMEM + 5% KSR.
フィーダ一細胞に Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質 や、 各種造血因子又は細胞刺激因子を発現させるには、 それらタンパク質や因子 のァミノ酸配列をコードする D N Aを含んでなる組換えベクターを作製し、 該組 換えべクタ一をフィーダ一細胞に導入すればよい。  Expression of one or more proteins selected from Wnt2 and Wnt5a in a feeder cell and various hematopoietic factors or cell stimulating factors include DNA encoding the amino acid sequence of those proteins or factors. A recombinant vector may be prepared and the recombinant vector may be introduced into a feeder cell.
なお、 培養は、 例えば、 33〜39で （好ましくは 37°C) の温度で、 3〜 6 % C O 2 (好ましくは 5%) 下、 5〜50日間行なう。 造血幹細胞の分化抑制 および/または自己複製の結果は、 Herzenberg, L. A. rweir' s Handbook of Experimental Immunology, 5th editionj ， Blackwell Science Inc. 1997」 、 Spangrude, G. J.， Proc. Natl. Acad. Sci.U. S. A., 87:7433-7437, 1990; Visser, J. M. W. , Flow cytometry in hematology] , Academic Press, p9-29, 1992等 を参考に細胞表面抗原を指標に確認することができる。 例えば、 ヒト造血幹細胞 のマーカーとしては、 少なくとも CD 34抗原陽性、 好ましくは CD 34抗原陽 性、 CD 38抗原陰性、 細胞分化抗原陰性であることをヒト造血幹細胞の指標と して用いることができる。 造血幹細胞の存在を ¾認する手段として放射線照射マ ウスを用いた移植実験系、 または、 in vitro (インビトロ） のコロニー形成法 (Bradley, T. R. , J. Exp. Med., 44: 287-299, 1966)を用いることも可能である。 放射線照射マウスを用いた移植実験系では、 放射線照射し造血系に障害を与えた マウス (レシピエント) に、 他のマウス （ドナ一） から分離した骨髄細胞や造血 幹細胞含有画分を移植する。 移植後、 レシピエント由来とドナー由来の造血系細 胞の割合 （キメリズム） を指標に、 長期骨髄再構築能を有する造血幹細胞の存在 を確認する（Osawa, M. , Science, 273: 242-245, 1996, B h a t i a e t a 1. , P r o c. Na t l . Ac ad. S c i. U. S. A. 94, 5320— 5325, 1997)。 コロニー形成法において、 造血幹細胞を種々の血液細胞 が出現できるように種々めサイ卜力インを添加した培地にて培養すると、 分化方 向の決定された造血前駆細胞は、 小数あるいは、 単一な分化系列の細胞しか含ま ないコロニーを形成するが、 多分化能を持つ造血幹細胞は、 複数の分化系列の血 液細胞を含むコロニーを形成することができる。 特に、 赤血球を含む混合コロニ 一 ( C F U - Em i x ) を形成することが、 ヒトでは造血幹細胞の指標とされて いる。 The cultivation is performed, for example, at a temperature of 33 to 39 (preferably 37 ° C.) under 3 to 6% CO 2 (preferably 5%) for 5 to 50 days. The results of the differentiation inhibition and / or self-renewal of hematopoietic stem cells are described in Herzenberg, LA rweir's Handbook of Experimental Immunology, 5th edition, Blackwell Science Inc. 1997, Spangrude, GJ, Proc. Natl. Acad. Sci. USA, 87 : 7433-7437, 1990; Visser, JMW, Flow cytometry in hematology], Academic Press, p9-29, 1992, etc., and can be confirmed using cell surface antigens as an index. For example, as a marker for human hematopoietic stem cells, at least CD34 antigen positive, preferably CD34 antigen positive, CD38 antigen negative, cell differentiation antigen negative can be used as an indicator of human hematopoietic stem cells. As a means to confirm the presence of hematopoietic stem cells, a transplantation experiment using an irradiated mouse or an in vitro (in vitro) colony formation method (Bradley, TR, J. Exp. Med., 44: 287-299, 1966) can also be used. In the transplantation experiment system using irradiated mice, a bone marrow cell or hematopoietic stem cell-containing fraction isolated from another mouse (Donna 1) is transplanted into a mouse (recipient) irradiated with radiation and damaging the hematopoietic system. After transplantation, the proportion of hematopoietic cells derived from recipients and donors (chimerism) is used as an indicator to confirm the presence of hematopoietic stem cells with long-term bone marrow remodeling ability (Osawa, M., Science, 273: 242-245). Natl. Ac ad. Sci. USA 94, 5320-5325, 1997). In the colony forming method, when hematopoietic stem cells are cultured in a medium supplemented with various cytokins so that various blood cells can appear, hematopoietic progenitor cells in which the differentiation direction has been determined can be a small number or a single cell. Contains only differentiated cells Hematopoietic stem cells that form no colonies but have pluripotency are able to form colonies that include blood cells of multiple differentiation lineages. In particular, the formation of a mixed colony containing red blood cells (CFU-Emix) is regarded as an indicator of hematopoietic stem cells in humans.
本発明は、 上述の本発明の培養方法を用いることを特徴とする、 造血幹細胞の 製造方法を提供する。 本発明の製造方法は、 （a ) 造血幹細胞を、 Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質の存在下で培養する工程を含んで なることを特徴とする。 本発明の製造方法は、 更に以下のような工程を含むこと ができるが、 これらに限定されるものではなく、 本発明の培養方法や培養系に準 じた工程を適宜含み得る； （b ) —種以上の造血因子又は細胞刺激因子の存在下、 かっ血清の非存在下で培養を行なう工程、 （c ) 造血幹細胞をフィーダ一細胞と 共培養する工程、 （d ) フィーダ一細胞に Wn t 2及び Wn t 5 aから選ばれる 一種以上のタンパク質を発現させる工程。 2 . 上記培養方法によって得られた造血幹細胞  The present invention provides a method for producing hematopoietic stem cells, characterized by using the above-described culture method of the present invention. The production method of the present invention is characterized by comprising the step of (a) culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a. The production method of the present invention may further include the following steps, but is not limited thereto, and may appropriately include the steps according to the culture method and culture system of the present invention; (b) -A step of culturing in the presence of at least one or more hematopoietic factors or cell stimulating factors, in the absence of serum, (c) a step of co-culturing hematopoietic stem cells with one feeder cell, and (d) a Wnt t A step of expressing one or more proteins selected from 2 and Wnt5a. 2. Hematopoietic stem cells obtained by the above culture method
上記本発明の方法によって得られた造血幹細胞は、 従来の骨髄移植や臍帯血移 植に代わる造血幹細胞移植用に用いることができる。 例えば、 自己免疫疾患等の 骨髄移植によってその改善の見られる疾患に対しては、 自己あるいは非自己の幹 細胞を増殖させるに際し、 本発明による幹細胞増殖技術を利用することができる。 具体的には、 本発明の方法により得られた造血幹細胞は、 様々な白血病に対する 全身 X線療法や高度化学療法を行う際に、 これらの治療と組み合わせて用いるこ とができる。 また、 造血幹細胞は、 例えば、 固形癌患者の化学療法、 放射線療法 等の骨髄抑制が副作用として生じる治療を実施する際に、 施術前に骨髄を採取し ておき、 造血幹細胞を試験管内で増幅し、 施術後に患者に戻すことで、 副作用に よる造血系の障害から早期に回復させることができ、 より強力な化学療法を行え るようになる。  The hematopoietic stem cells obtained by the above method of the present invention can be used for hematopoietic stem cell transplantation instead of conventional bone marrow transplantation or cord blood transplantation. For example, for a disease such as an autoimmune disease whose improvement is seen by bone marrow transplantation, the stem cell expansion technology according to the present invention can be used to expand autologous or non-autologous stem cells. Specifically, the hematopoietic stem cells obtained by the method of the present invention can be used in combination with systemic X-ray therapy or advanced chemotherapy for various leukemias. In addition, hematopoietic stem cells are obtained by collecting bone marrow prior to surgery and performing hematopoietic stem cell expansion in vitro, for example, when performing chemotherapy, radiation therapy, or other treatments that cause bone marrow suppression as a side effect in patients with solid cancer. By returning to the patient after the procedure, hematopoietic damage due to side effects can be recovered early, and stronger chemotherapy can be performed.
また、 本発明を利用して、 患者あるいは他人の造血幹細胞を各種血液細胞に分 化させ、 それらを患者の体内に移入することにより、 各種血液細胞の形成が不十 分な患者を治療することができる。 また、 本発明の培養方法によって得られる造 血幹細胞は、 再生不良性貧血などの貧血を呈する骨髄低形成に起因 る造血不全 症を改善することができる。 その他、 本発明の培養方法によって得られる造血幹 細胞の移植が有効な疾患としては、 慢性肉芽腫症、 重複免疫不全症候群、 無ガン マグロプリン血症、 Wi s ko t t— A l d r i c h症候群、 後天性免疫不全症 候群 （AI DS) 等の免疫不全症候群、 サラセミア、 酵素欠損による溶血性貧血、 鎌状赤血球症等の先天性貧血、 Gau c h e r病、 ムコ多糖症等のリソゾーム蓄 積症、 副腎白質変性症、 各種の癌または腫瘍等が挙げられる。 In addition, the present invention is used to treat a patient with insufficient formation of various blood cells by dividing a patient or another person's hematopoietic stem cells into various blood cells and transferring them into the patient's body. Can be. In addition, the culture obtained by the culture method of the present invention. Blood stem cells can ameliorate hematopoietic failure caused by bone marrow hypoplasia presenting anemia such as aplastic anemia. Other diseases for which hematopoietic stem cell transplantation obtained by the culture method of the present invention is effective include chronic granulomatous disease, double immunodeficiency syndrome, cancer-free magropurinemia, Wisko tt—Aldrich syndrome, acquired Immunodeficiency syndrome (AIDS) and other immune deficiency syndromes, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, Gaucher disease, lysosomal storage disease such as mucopolysaccharidosis, adrenal white matter Degeneration, various cancers or tumors, and the like.
造血幹細胞の移植は、 使用する細胞以外は、 従来行われている骨髄移植や臍帯 血移植と同様に行なうことができる。 なお、 本発明の移植片は、 本発明の方法に よって増殖した造血幹細胞を含む細胞成分の他に、 緩衝液等を含む組成物として 用いることもできる。  The transplantation of hematopoietic stem cells can be performed in the same manner as conventional bone marrow transplantation or cord blood transplantation, except for the cells used. The graft of the present invention can be used as a composition containing a buffer solution and the like in addition to the cell components containing hematopoietic stem cells grown by the method of the present invention.
3. 細胞培養系及びこれを用いるスクリーニング方法 3. Cell culture system and screening method using the same
本発明の他の態様によれば、 （a) フィーダ一細胞、 （1:) 11セ 2及び\¥11 t 5 aから選ばれる一種以上のタンパク質及び （c) 一種以上の造血因子又は細 胞刺激因子を含み、 かっ血清を含まないことを特徴とする造血幹細胞培養系を提 供する。 このような無血清培養系においては、 無血清下で少なくとも生存可能な、 好ましくは長期培養、 および/または増殖可能なフィーダ一細胞を用いる必要が ある。 好適なフィーダ一細胞は、 無血清下で生存可能なように調製した C 127 細胞である。 このような無血清培養系は、 造血幹細胞の分化または増殖を調節す る物質をスクリーニングする方法に利用することができる。 すなわち、 本発明の 造血幹細胞の増殖または分化を調節する物質をスクリーニングする方法は、 無血 清下で生存可能なフィーダ一細胞を用いることを特徴とし、 （a) フィーダ一細 胞および造血幹細胞を含み、 力つ血清を含まないことを特徴とする培養系に試験 物質を共存させる （添加あるいはフィーダ一細胞に発現させる） 工程、 （b) 試 験物質を共存させた （添加あるいはフィーダ一細胞に発現させた） 培養系で造血 幹細胞を培養する工程、 及び （c) 試験物質非共存下 （非添加または非発現） の 場合と比較して、 試験物質共存下 （添加または発現） の場合における造血幹細胞 の割合および/または数の変化を測定する工程を含む。 本発明のスクリーニング 方法において、 例えば、 造血幹細胞の増殖促進活性を有する物質を試験するに際 しては、 Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質を培養系に 含めておく （添加あるいはフィーダ一細胞に発現させる） ことにより、 幹細胞の 分化を抑制した状態で試験物質の増殖促進活性をより有効に試験することが可能 である。 また、 必要に応じて造血因子又は細胞刺激因子の一種以上を培養系に含 めてもよい。 増殖または分化の調節とは、 増殖抑制、 増殖促進、 分化抑制および 分化促進の何れかのことであり、 特に増殖促進が好ましい。 According to another embodiment of the present invention, (a) a feeder cell, (1 :) one or more proteins selected from 11 cells and 11c5a, and (c) one or more hematopoietic factors or cells. Provided is a hematopoietic stem cell culture system containing a stimulating factor and containing no serum. In such a serum-free culture system, it is necessary to use a feeder cell that is at least viable in serum-free, preferably long-term culture, and / or is capable of growing. A preferred feeder cell is a C 127 cell that has been prepared to survive in serum free. Such a serum-free culture system can be used for a method of screening for a substance that regulates differentiation or proliferation of hematopoietic stem cells. That is, the method of screening for a substance that regulates the proliferation or differentiation of hematopoietic stem cells of the present invention is characterized by using a feeder cell that can survive in serum-free, and (a) comprises a feeder cell and a hematopoietic stem cell. A step of coexisting (adding or expressing in a feeder cell) a test substance in a culture system characterized by the absence of virulent serum; (b) coexisting a test substance (expressing in an added or feeder cell) Culturing hematopoietic stem cells in a culture system, and (c) hematopoietic stem cells in the presence (addition or expression) of the test substance compared to the absence (non-addition or non-expression) of the test substance Measuring the change in the percentage and / or number of Screening of the present invention In the method, for example, when testing a substance having a hematopoietic stem cell proliferation promoting activity, one or more proteins selected from Wnt2 and Wnt5a are included in the culture system (addition or feeder). Thus, the proliferation promoting activity of the test substance can be more effectively tested in a state where the differentiation of the stem cells is suppressed. If necessary, one or more hematopoietic factors or cell stimulating factors may be included in the culture system. The regulation of proliferation or differentiation means any of growth suppression, growth promotion, differentiation suppression and differentiation promotion, and growth promotion is particularly preferred.
このスクリーニングにおいて用いる試験物質としては、 細胞の培養上清、 精製 タンパク質若しくはペプチド、 合成化合物、 微生物や植物に由来する天然物、 造 血幹細胞 の増殖あるいは分化を促進する因子を産生していることが予想される 支持細胞により産生される蛋白質や生体物質などが挙げられる。 試験物質の活性 は、 培養系において試験物質の存在下及び非存在下において、 処理前後の細胞に 含まれる造血幹細胞 の割合および Zまたは数を算出することで測定することが できる。 細胞に含まれる造血幹細胞 の割合および/または数は F A C Sによつ て求めることができる。 造血幹細胞の分化は、 分化した細胞に特異的な抗原に対 する抗体により、 F A C Sあるいは蛍光抗体法で測定することができる。 たとえ ば、 B細胞の特異抗原として B 2 2 0、 顆粒球の特異抗原として G r— 1、 赤芽 球系細胞の特異抗原として T E R 1 1 9が使用できる。 また、 C F U— GM、 C F U— M i x、 C F U—S等のコロニーアツセィにより測定することができる。 このようなスクリーニングの結果、 有意な造血幹細胞の自己複製 ·増殖、 分化抑 制、 または分化が検出されれば、 スクリーニングに用いた試験物質は、 造血幹細 胞の増殖または分化を調節する因子であると判定される。 このような因子は、 特 に血液系疾患の治療薬開発において有用である。 4. 造血幹細胞の分化抑 又は増殖剤  Test substances used in this screening include cell culture supernatants, purified proteins or peptides, synthetic compounds, natural products derived from microorganisms and plants, and factors that promote the growth or differentiation of hematopoietic stem cells. Examples include proteins and biological materials that are expected to be produced by supporting cells. The activity of the test substance can be measured by calculating the ratio and Z or number of hematopoietic stem cells contained in the cells before and after the treatment in the presence and absence of the test substance in the culture system. The proportion and / or number of hematopoietic stem cells contained in the cells can be determined by FACS. The differentiation of hematopoietic stem cells can be measured by FACS or a fluorescent antibody method using an antibody against an antigen specific to the differentiated cells. For example, B220 can be used as a specific antigen for B cells, Gr-1 as a specific antigen for granulocytes, and TER119 as a specific antigen for erythroid cells. In addition, it can be measured by colony assay such as CFU-GM, CFU-Mix, and CFU-S. As a result of such screening, if significant self-renewal / proliferation, proliferation suppression, or differentiation of hematopoietic stem cells is detected, the test substance used for screening is a factor that regulates the growth or differentiation of hematopoietic stem cells. It is determined that there is. Such factors are particularly useful in developing therapeutics for hematological disorders. 4. Hematopoietic stem cell differentiation inhibitor or proliferation agent
本発明の他の態様によれば、 Wn t 2及び Wn t 5 aから選ばれる一種以上の タンパク質を含有する造血幹細胞分化抑制又は増殖剤 （以下、 単に、 「造血幹細 胞増殖剤」 ともいう） が提供される。 本発明の増殖剤は、 好ましくは、 Wn t 2 及び Wn t 5 aから選ばれる一種以上のタンパク質に加えて、 上記した造血因子 又は細胞刺激因子の一種以上、 例えば、 S C F、 T P Oまたはその両方を含有す る。 According to another aspect of the present invention, a hematopoietic stem cell differentiation inhibitory or proliferating agent containing one or more proteins selected from Wnt2 and Wnt5a (hereinafter, also simply referred to as "hematopoietic stem cell proliferating agent") ) Is provided. The growth agent of the present invention preferably comprises, in addition to one or more proteins selected from Wnt2 and Wnt5a, Alternatively, it contains one or more cell stimulating factors, for example, SCF, TPO or both.
本発明の造血幹細胞増殖剤は、 造血幹細胞を未分化なまま、 体内または体外で 増殖させることができるので、 上記した i n V i t r oにおける造血幹細胞の 培養 ·増殖方法において用いることができる他、 放射線治療や制ガン剤等の化学 療法剤による血球減少症の改善、 リンパ球減少に起因する感染症の予防、 骨髄形 成不全症や骨髄抑制などの骨髄疾患の治療、 白血病、 高度腎障害 ·骨髄抑制など の骨髄疾患の治療、 遺伝的疾患に由来する低血球症の治療、 遺伝子治療時におけ る組換え幹細胞の体外培養等に用いることができる。  Since the hematopoietic stem cell proliferating agent of the present invention can proliferate hematopoietic stem cells in vivo or outside of the body while undifferentiated, it can be used in the above-mentioned method for culturing and expanding hematopoietic stem cells in vitro, and in addition to radiation therapy. Chemotherapeutic drugs such as anticancer drugs and anticancer drugs to prevent cytopenia caused by lymphopenia, treatment of bone marrow disorders such as bone marrow aplasia and bone marrow suppression, leukemia, severe renal disorders, bone marrow suppression, etc. It can be used for the treatment of bone marrow diseases, the treatment of hypocytosis caused by genetic diseases, the in vitro culture of recombinant stem cells during gene therapy, and the like.
本発明の造血幹細胞増殖剤は、 経口、 非経口、 骨髄等の局所その他の適当な経 路で投与することができる。 造血幹細胞増殖剤の望ましい投与量は、 その有効成 分 （上記 Wn tタンパク質、 及び必要に応じて造血因子又は細胞刺激因子それぞ れを 「有効成分」 という） として、 1日あたり 1〜1 0 0 0 i GZ k g体重程度 で、 好ましくは 5〜5 0 0 ^ 71^ 8体重程度であるが、 患者の体重および症状 や個々の投与経路によって当然変動する。 場合によっては前記範囲の下限より低 い投与量が適当なこともあるし、 前記範囲より投与量を多くしてもそれを 1日に 何回にも分けて少量ずつ投与すれば有害な副作用を生じない場合もある。  The hematopoietic stem cell proliferating agent of the present invention can be administered orally, parenterally, locally, such as bone marrow, or any other suitable route. The desired dose of the hematopoietic stem cell proliferating agent is 1 to 10 per day as its active ingredient (the Wnt protein and, if necessary, each hematopoietic factor or cell stimulating factor is referred to as “active ingredient”). It is about 0 i GZ kg body weight, preferably about 5 to 500 ^ 71 ^ 8 body weight, but naturally varies depending on the weight and symptoms of the patient and individual administration route. In some cases, a dose lower than the lower limit of the above range may be appropriate, and even if the dose is higher than the above range, if it is divided into several times a day and administered in small amounts, adverse side effects may occur. Sometimes it does not happen.
本発明で用いられる上記有効成分は、 前記 3つの投与経路のいずれをとつても 単独または薬学的あるいは薬剤学的に許容される担体または希釈剤と共に投与す ることができ、 またその投与は 1回または数回に分けて行うことができる。 より 具体的に述べると、 本発明の増殖剤は様々な種類の投与形態で投与することがで き、 たとえば各種の薬剤学的に許容される不活性担体と併用して錠剤、 カプセル、 薬用ドロップ、 トローチ、 硬質キャンディ、 粉末剤、 噴霧剤、 クリーム、 膏薬、 坐薬、 ゼリー、 ジエル、 ペースト、 ローション、 軟膏、 水性懸濁液、 注射液、 ェ リキシル、 シロップ等の形態とすることができる。 これらの担体には、 固体希釈 剤または賦形剤、 無菌水性媒体、 各種の非毒性有機溶媒等が含まれる。 実 施 例 The active ingredient used in the present invention can be administered alone or together with a pharmaceutically or pharmaceutically acceptable carrier or diluent by any of the above three administration routes. It can be performed once or in several times. More specifically, the proliferating agent of the present invention can be administered in various types of administration forms, for example, tablets, capsules, medicinal drops in combination with various pharmaceutically acceptable inert carriers. Troches, hard candy, powders, sprays, creams, salves, suppositories, jellies, jewels, pastes, lotions, ointments, aqueous suspensions, injections, elixirs, syrups and the like. These carriers include solid diluents or excipients, sterile aqueous media, various non-toxic organic solvents, and the like. Example
以下、 本発明を実施例に基づいてより具体的に説明するが、 本発明はこれに限 定されるものではない。 (実施例 1 )  Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto. (Example 1)
1 . マウス及びヒト成体骨髄ストローマ細胞で発現している Wnt遺伝子の同定 1 . 1 骨髄幹細胞の調製  1. Identification of Wnt gene expressed in mouse and human adult bone marrow stromal cells 1.1 Preparation of bone marrow stem cells
非働化したゥシ胎児血清 (FCS)を 2%含むリン酸緩衝化生理食塩水 (PBS)を、 26 ゲージの針で骨髄腔に一気に注ぐことにより、 マウス骨髄細胞を 8週令の C57BL/6マウス（Charles River)から調製した。 1成人ヒト骨髄細胞を、 インフォ —ムドコンセントと、 施設内倫理委員会の承認に基づいて正常ドナ一の後腸骨稜 (pos terior i l iac cres t)から得た。 細胞懸濁液を Ly即 hop rep (Nycomed)上に置 き、 800 gで 20分間遠心した。 単核細胞を回収し、 2% の FCSを含む PBSで洗浄 した。 そして、 接着細胞を 10% の FCSを含む IMD培養液 (Gibco- BRL)で 10日間 培養した。 全 RNAは AGPC法で単離した後、 degenerat ive RT- PCRで発現してい る W n tの種類を同定した。 Mouse bone marrow cells were injected into C57BL / 6 mice at 8 weeks of age by rapidly pouring phosphate-buffered saline (PBS) containing 2% inactivated fetal calf serum (FCS) into the marrow cavity with a 26-gauge needle. Prepared from mice (Charles River). 1 Adult human bone marrow cells were obtained from the posterior iliac crest (pos terior il iac crest) of normal donors based on informed consent and institutional ethics committee approval. The cell suspension was placed on Ly hop rep (Nycomed) and centrifuged at 800 g for 20 minutes. Mononuclear cells were collected and washed with PBS containing 2% FCS. Then, the adherent cells were cultured for 10 days in an IMD culture solution (Gibco-BRL) containing 10% FCS. After isolating total RNA by the AGPC method, the type of Wnt expressed was identified by degenerative RT-PCR.
1 . 2 Degenerat ive RT-PCR解析 1.2 Degenerative RT-PCR analysis
逆転写ポリメ一ラーゼ連鎖反応 (RT- PCR)を以下のように行った。 第一鎖 (f irs t s t rand) cDNAを Superscript I I (Gibco- BRL)を用いて合成した。 PCR の増幅は LA Tag polymerase (Takara)とデジエネレートプライマ一  Reverse transcription polymerase chain reaction (RT-PCR) was performed as follows. First strand (firststrand) cDNA was synthesized using Superscript II (Gibco-BRL). PCR amplification is performed with LA Tag polymerase (Takara) and
5' - TG (C/T) AA (G/A) TGCCA (C/T) GG (G/C) -3' (配列番号 1 )  5 '-TG (C / T) AA (G / A) TGCCA (C / T) GG (G / C) -3' (SEQ ID NO: 1)
5 ' - (G/A) CAGCACCAGTGGAA-3 ' (配列番号 2 )  5 '-(G / A) CAGCACCAGTGGAA-3' (SEQ ID NO: 2)
を用いて、 94° Cで 1 分を 1サイクル、 94° Cで 1 分、 54° Cで 30秒； 72° C で 45 秒を 60サイクル、 そして 72° Cで 5 分を 1サイクル行った。 精製した PCR産物を、 pBluescr iptにサブクローニングし、 そして核酸配列を ABI 3700 ジェネティック ·アナライザ一 （Appl ied Biosys tems)を用いて決定した。 この 方法によって、 マウスでは Wn t 2、 Wn 1 5a, ヒトでは Wn t 2、 Wn t 5a、 W n 1 5bが発現していることを見いだした。 両者とも Wn t 2が最も多く発現され ていた。 1 cycle at 94 ° C for 1 minute, 1 minute at 94 ° C, 30 seconds at 54 ° C; 60 cycles of 45 seconds at 72 ° C and 1 cycle of 5 minutes at 72 ° C . The purified PCR product was subcloned into pBluescript and the nucleic acid sequence was determined using an ABI 3700 Genetic Analyzer (Applied Biosystems). By this method, Wnt2, Wnt15a in mice and Wnt2, Wnt5a, W in humans It was found that n15b was expressed. In both cases, Wnt 2 was most frequently expressed.
2 . Wntを用いた幹細胞分画の増殖アツセィ 2. Proliferation of stem cell fraction using Wnt
2 . 1 造血幹細胞の調製 2.1 Preparation of hematopoietic stem cells
有核細胞を 7- 8週令の lacZ トランスジエニックマウス （B6 ; S129- Gt (R0SA26) Sorの骨髄細胞から、 Lymphoprepを用いた遠心によって調製した。 2% FCSを含む PBS中に懸濁した細胞（1 X 106細胞/ ml)を Hoechs t33342 (l. Nucleated cells were prepared from bone marrow cells of 7-8 week old lacZ transgenic mice (B6; S129-Gt (R0SA26) Sor) by centrifugation using Lymphoprep and suspended in PBS containing 2% FCS. Cells (1 × 10 6 cells / ml) were transferred to Hoechs t33342 (l.
と、 Rhodamme l23 (0. 1 g/ml)で染色した。 造血幹細胞に富んだ分画である、 s ide populat ion (SP)細胞を FACS Vantage SEセルソ一ター (商標： Beckton Dickinson Bioscience)で単離した。 一匹のマウスから約 4000個の SP細胞を単 離した。 lacZ トランスジエニックマウスから、 マウス骨髄造血幹細胞である Lin 陰性 c—Ki t陽性 Seal陽性 CD34陰性または lowの細胞分画を FACSを用いて単 離した。 一匹のマウスから約 10000個の細胞を単離した。 And Rhodamme l23 (0.1 g / ml). Side population (SP) cells, a fraction rich in hematopoietic stem cells, were isolated on a FACS Vantage SE cell sorter (trademark: Beckton Dickinson Bioscience). Approximately 4000 SP cells were isolated from one mouse. From lacZ transgenic mice, Lin-negative c-Kit-positive Seal-positive CD34-negative or low cell fractions, mouse bone marrow hematopoietic stem cells, were isolated using FACS. Approximately 10,000 cells were isolated from one mouse.
2 . 2 in vi troでの幹細胞の自己複製アツセィ 2.2 Self-renewal of stem cells in vitro
マウス又はヒ卜の全長 Wnt2及び Wnt5aの cDNAを K0D+ DNA polymeraseを用い た RT-PCRで単離し、 Wnt cDNAの完全な核酸配列がデ一夕べ一スに登録されてい る配列と同一であることを確認した。 PCDNA3. 1発現べクタ一にマウスまたはヒ トの各 Wntの c D NAを組換え、 C127細胞に Lipofect Amine 2000 (invi trogen) を用いてトランスフエクトし、 G418 (l mg/ml)に耐性の安定発現細胞を無血清培 地 （DMEM+5%の KSR (Knockout serum replacement) (Gibco) ) 中に維持した。 Wnt の発現は RT- PCRでは確認済みであり、 一部抗体が利用できる Wnt5aに関しては ウェス夕ン解析で発現を確認した。  The full-length mouse or human Wnt2 and Wnt5a cDNAs were isolated by RT-PCR using K0D + DNA polymerase, and the complete nucleic acid sequence of the Wnt cDNA was identical to the sequence registered in the database overnight. confirmed. Recombinant mouse or human Wnt cDNAs in PCDNA3.1 expression vector, transfect C127 cells using Lipofect Amine 2000 (invitrogen), resistant to G418 (l mg / ml) The stably expressing cells were maintained in a serum-free medium (DMEM + 5% Knockout serum replacement (KSR) (Gibco)). The expression of Wnt has been confirmed by RT-PCR, and expression of Wnt5a, for which some antibodies can be used, was confirmed by Western blot analysis.
組み換え Wntを発現する C127 トランスフエクタントまたは組み換え Wnt非発 現 C127細胞を 2 4穴プレートに播種した後、 単離した lacZ トランスジエニック マウス由来骨髄造血幹細胞を 5 0 0 0個/ゥエルで植え込み、 共培養した。 培地 は無血清培地または T P 0および S C F添加無血清培地を用いた。 2日おきに培 地を交換し 6日目に細胞をトリプシン消化して回収し、 /3 - Gal 陽性細胞即ち lacZ トランスジエニックマウス由来の細胞を f luorescein di- jQ - galactopyranos ideを基質に用いて蛍光発色によって分画し、 その中の Lin陰性 c一 Ki t陽性 Seal陽性細胞 （造血幹細胞） の割合を算出した。 After seeding C127 transfectants expressing recombinant Wnt or C127 cells not expressing recombinant Wnt in a 24-well plate, the isolated lacZ transgenic mouse-derived bone marrow hematopoietic stem cells were implanted at 500 / well, and Co-cultured. The medium used was a serum-free medium or a serum-free medium supplemented with TP0 and SCF. The medium was changed every two days, and the cells were collected by trypsin digestion on the sixth day. Cells derived from lacZ transgenic mice are fractionated by fluorescence using fluoroscein di-jQ-galactopyranoside as a substrate, and the percentage of Lin-negative c-Kit-positive Seal-positive cells (hematopoietic stem cells) is calculated. did.
組み換え Wnt発現 C127 トランスフェクタントまたは組み換え Wnt非発現 C127 細胞と、 マウス骨髄造血幹細胞の共培養において、 T P Oおよび S C Fを添加し ていない無血清培地で培養した場合、 3日後には lacZトランスジエニックマウ ス由来細胞は生存しておらず、 造血幹細胞は維持も増幅もできなかった。 組み換 え Wnt非発現 C127細胞との共培養において、 T P〇および S C F添加無血清培 地で培養した場合、 回収した jS _Gal 陽性細胞のうち 8 8 %は i n陽性細胞で あり分化した細胞であった。 1 2 %は Lin陰性 c -Ki t陽性 Seal陽性細胞であ り幹細胞であると考えられた。 これは従来の幹細胞のサイトカインを用いた増幅 結果とほぼ一致したものである。 一方、 組み換え Wnt発現 C127 トランスフエク タントとの共培養において、 T P Oおよび S C F添加無血清培地で培養した場合、 回収した ]3 - Gal 陽性細胞のうち 2 3 %〜2 7 % (マウス Wn t 2において 2 7 %、 マウス Wn t 5 aにおいて 2 5 %、 ヒト Wn t 5 aにおいて 2 3 %) は造 血幹細胞であった。 組み換え Wn t発現 C127 トランスフエクタントとの共培養 の場合、 共培養処理後生存している lacZ トランスジエニックマウス由来細胞に おける造血幹細胞の割合は、 組み換え Wn t非発現 C127細胞との共培養の場合 と比べて、 マウス Wn t 2において 2 . 3倍、 マウス Wn t 5 aにおいて 2 . 1 倍、 ヒト Wn t 5 aにおいて 1 . 9倍を示した。 即ち、 Wn t 2および Wn t 5 aは、 T P Oや S C Fのような造血幹細胞の分化を促す因子の 在下において、 造血幹細胞の分化を抑制し、 未分化な状態を維持して生存させるのに有効である ことが分かった。 産業上の利用可能性  When co-cultured with recombinant Wnt-expressing C127 transfectants or non-recombinant Wnt-expressing C127 cells and mouse bone marrow hematopoietic stem cells, lacZ transgenic after 3 days when cultured in serum-free medium without TPO and SCF Mouse-derived cells were not viable, and hematopoietic stem cells could not be maintained or expanded. When co-cultured with C127 cells that did not express recombinant Wnt, when cultured in serum-free medium supplemented with TPII and SCF, 88% of the recovered jS_Gal-positive cells were in-positive and differentiated cells. Was. 12% were Lin-negative c-Kit-positive Seal-positive cells and were considered to be stem cells. This is almost consistent with the results of conventional stem cell amplification using cytokines. On the other hand, when co-cultured with recombinant Wnt-expressing C127 transfectant and cultured in a serum-free medium supplemented with TPO and SCF, 23% to 27% of the recovered 3-gal-positive cells (mouse Wnt 2 (27%, 25% in mouse Wnt5a, and 23% in human Wnt5a) were hematopoietic stem cells. In the case of co-culture with recombinant Wnt-expressing C127 transfectants, the percentage of hematopoietic stem cells in lacZ transgenic mouse-derived cells surviving the co-culture treatment was higher than that of co-culture with recombinant Wnt-non-expressing C127 cells. As compared to the case, mouse Wnt2 showed 2.3 times, mouse Wnt5a showed 2.1 times, and human Wnt5a showed 1.9 times. In other words, Wnt 2 and Wnt 5a are effective in suppressing the differentiation of hematopoietic stem cells and maintaining their undifferentiated state in the presence of factors that promote hematopoietic stem cell differentiation, such as TPO and SCF. It turned out that. Industrial applicability
本発明の造血幹細胞の分化抑制又は増殖方法、 造血幹細胞の製造方法、 並びに、 本発明の造血幹細胞の分化抑制又は増殖剤を用いることにより、 造血幹細胞を未 分化なまま、 体内または体外で増殖させることができるので、 化学療法剤による 血球減少症の改善、 リンパ球減少に起因する感染症の予防、 骨髄疾患の治療、 白 血病 ·高度腎障害 ·骨髄抑制等の骨髄疾患の治療などに用いることができる。 また、 本発明のスクリーニング法によれば、 造血幹細胞の増殖または分化を調 節する物質をスクリーニングすることができるので、 造血幹細胞レベルの疾患の 解明や新たな治療法 ·治療剤の開発に有用である。 Hematopoietic stem cells are differentiated or proliferated in vivo or in vitro without undifferentiation by using the method for suppressing or proliferating hematopoietic stem cells of the present invention, the method for producing hematopoietic stem cells, and the agent for suppressing or proliferating hematopoietic stem cells of the present invention Can be because of It can be used to improve cytopenia, prevent infectious diseases caused by lymphopenia, treat bone marrow disease, and treat bone marrow diseases such as leukemia, severe renal impairment, and bone marrow suppression. In addition, according to the screening method of the present invention, a substance that regulates the proliferation or differentiation of hematopoietic stem cells can be screened, which is useful for elucidating diseases at the level of hematopoietic stem cells and for developing new therapeutic methods and therapeutic agents. is there.

Claims

請求の範囲 The scope of the claims
I. 造血幹細胞を、 Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク 質の存在下で培養することを特徴とする造血幹細胞の分化抑制又は増殖方法。 I. A method for inhibiting differentiation or expanding hematopoietic stem cells, comprising culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
2. 一種以上の造血因子又は細胞刺激因子の存在下、 かっ血清の非存在下で培 養を行なう前記請求項 1に記載の方法。  2. The method according to claim 1, wherein the culturing is performed in the presence of one or more hematopoietic factors or cell stimulating factors and in the absence of serum.
3. SC F及び T P 0から選ばれる造血因子の一種以上の存在下で培養を行な う前記請求項 2に記載の方法。  3. The method according to claim 2, wherein the culture is performed in the presence of one or more hematopoietic factors selected from SCF and TP0.
4. Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質を培養系に添 加し、 あるいはそのタンパク質を発現させたフィーダ一細胞上で培養する前記請 求項 3に記載の方法。  4. The method according to claim 3, wherein one or more proteins selected from Wnt2 and Wnt5a are added to a culture system, or cultured on a feeder cell expressing the protein.
5. 前記請求項 1〜 4のいずれかの方法によって得られる造血幹細胞。  5. A hematopoietic stem cell obtained by the method according to any one of claims 1 to 4.
6. 造血幹細胞を、 Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク 質の存在下で培養する工程を含んでなる、 造血幹細胞の製造方法。  6. A method for producing hematopoietic stem cells, comprising a step of culturing hematopoietic stem cells in the presence of one or more proteins selected from Wnt2 and Wnt5a.
7. (a) フィーダ一細胞、 （b) Wn t 2及び Wn t 5 aから選ばれる一種 以上のタンパク質及び （c) 一種以上の造血因子又は細胞刺激因子を含み、 かつ 血清を含まないことを特徴とする造血幹細胞培養系。 ·  7. (a) Feeder cells, (b) one or more proteins selected from Wnt2 and Wnt5a, and (c) one or more hematopoietic or cell stimulating factors and no serum. Characteristic hematopoietic stem cell culture system. ·
8. フィーダ一細胞として、 無血清下で生存可能な C 127細胞を用いること を特徴とする前記請求項 7に記載の培養系。  8. The culture system according to claim 7, wherein C127 cells that can survive without serum are used as one feeder cell.
9. 無血清下で生存可能なフィーダ一細胞を用いることを特徴とし、 （a) フ ィーダ一細胞および造血幹細胞を含み、 かっ血清を含まないことを特徴とする培 養系に試験物質を共存させる工程、 （b) 試験物質が共存した培養系で造血幹細 胞を培養する工程、 及び （c) 試験物質非共存下の場合と比較して、 試験物質共 存下の場合における造血幹細胞の割合および/または数の変化を測定する工程を 含む、 造血幹細胞の増殖または分化を調節する物質をスクリーニングする方法。  9. Use of feeder cells that can survive in serum-free condition. (A) Coexist with test substance in culture system containing feeder cells and hematopoietic stem cells and free of serum. (B) culturing the hematopoietic stem cells in a culture system in which the test substance is present; and (c) comparing the hematopoietic stem cells in the presence of the test substance in comparison with the absence of the test substance. A method for screening a substance that regulates proliferation or differentiation of hematopoietic stem cells, comprising a step of measuring a change in the ratio and / or the number.
10. Wn t 2及び Wn t 5 aから選ばれる一種以上のタンパク質を含有する 造血幹細胞分化抑制又は増殖剤。  10. An agent for inhibiting or proliferating hematopoietic stem cell differentiation, comprising one or more proteins selected from Wnt2 and Wnt5a.
I I. さらに、 造血因子又は細胞刺激因子を含む前記請求項 10に記載の造血 幹細胞分化抑制又は増殖剤。 O 2005/056778 I I. The agent for suppressing or proliferating hematopoietic stem cell differentiation according to claim 10, further comprising a hematopoietic factor or a cell stimulating factor. O 2005/056778
19 19
12. 前記造血因子として、 SCF及び TP 0から選ばれる一種以上の造血因 子を含む前記請求項 11に記載の造血幹細胞分化抑制又は増殖剤。  12. The agent for suppressing or proliferating hematopoietic stem cell differentiation according to claim 11, wherein the agent includes one or more hematopoietic factors selected from SCF and TPO as the hematopoietic factor.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011101468A1 (en) * 2010-02-22 2011-08-25 Université Pierre Et Marie Curie (Paris 6) Cell culture medium for the growth and differentiation of cells of the hematopoietic lineage
JP2012520671A (en) * 2009-03-20 2012-09-10 エスエヌユー アールアンドディービー ファウンデーション Method for separating umbilical cord blood-derived universal stem cells expressing ZNF281
US20120258538A1 (en) * 2009-09-10 2012-10-11 Keio University Culture method for hematopoietic stem cells
JP2015527083A (en) * 2012-09-07 2015-09-17 チルドレンズ メディカル センター コーポレーション Hematopoietic stem cell specific reporter mouse and use thereof
US9701954B2 (en) 2010-06-22 2017-07-11 Lonza Cologne Gmbh Method and device for uniformly treating adherent cells

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DUNCAN A. ET AL: "Regulation of stem cell development by the Wnt signaling pathway.", DAI 26 KAI THE MOLECULAR BIOLOGY SOCIETY OF JAPAN NENKAI PROGRAM. KOEN YOSHISHU., 25 November 2003 (2003-11-25), pages 398, XP002991217 *
MURDOCH B. ET AL: "Wnt-5A augments repopulating capacity and primitive hematopoietic development of human blood stem cells in vivo.", PROC.NATL.ACAD.SCIUSA, vol. 100, no. 6, March 2003 (2003-03-01), pages 3422 - 3427, XP002987165 *
REYA T. ET AL: "A role for Wnt signaling in self-renewal of haematopoietic stem cells.", NATURE, vol. 423, no. 6938, 22 May 2003 (2003-05-22), pages 409 - 414, XP002975077 *
SATOFUKA H. ET AL: "Kotsuzuichu no Taiseikan Saibo wa Wnt Dakede Jiko Fukusei suruka?", DAI 26 KAI THE MOLECULAR BIOLOGY SOCIETY OF JAPAN NENKAI PROGRAM. KOEN YOSHISHU., 25 November 2003 (2003-11-25), pages 500, XP002991216 *
WILLERT K. ET AL: "Wnt proteins are lipidmodifield and can act as stem cell growth factors.", NATURE, vol. 423, no. 6938, 22 May 2003 (2003-05-22), pages 448 - 452, XP002279471 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012520671A (en) * 2009-03-20 2012-09-10 エスエヌユー アールアンドディービー ファウンデーション Method for separating umbilical cord blood-derived universal stem cells expressing ZNF281
US10584312B2 (en) 2009-03-20 2020-03-10 Snu R&Db Foundation Isolating method for umbilical cord blood-derived pluripotent stem cells expressing ZNF281
US20120258538A1 (en) * 2009-09-10 2012-10-11 Keio University Culture method for hematopoietic stem cells
WO2011101468A1 (en) * 2010-02-22 2011-08-25 Université Pierre Et Marie Curie (Paris 6) Cell culture medium for the growth and differentiation of cells of the hematopoietic lineage
JP2016171811A (en) * 2010-02-22 2016-09-29 ユニベルシテ ピエール エ マリー キュリー（パリ シズエム） Cell culture medium for growth and differentiation of cells of hematopoietic lineage
US9701954B2 (en) 2010-06-22 2017-07-11 Lonza Cologne Gmbh Method and device for uniformly treating adherent cells
US11021698B2 (en) 2010-06-22 2021-06-01 Lonza Cologne Gmbh Method and device for uniformly treating adherent cells
JP2015527083A (en) * 2012-09-07 2015-09-17 チルドレンズ メディカル センター コーポレーション Hematopoietic stem cell specific reporter mouse and use thereof
US10080354B2 (en) 2012-09-07 2018-09-25 Children's Medical Center Corporation Hematopoietic stem cell specific reporter mouse and uses thereof

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