WO2005056778A1 - Procede de regulation de differenciation des cellules souches hematopoietiques ou de leur proliferation - Google Patents

Procede de regulation de differenciation des cellules souches hematopoietiques ou de leur proliferation Download PDF

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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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Kazuo Todokoro
Hiroyuki Satofuka
Yoshikazu Kuwahara
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Riken
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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.

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Abstract

L'invention concerne un procédé de régulation des cellules souches hématopoïétiques à l'état non différencié ou de régulation de leur prolifération, un agent correspondant, etc. Plus précisément, on décrit un procédé de régulation des cellules souches hématopoïétiques à l'état non différencié ou de régulation de leur prolifération qui consiste à effectuer la culture des cellules en présence d'une ou plusieurs protéines choisies entre Wnt2 et Wnt5a. On décrit un système de culture qui contient (a) des cellules nourricières, (b) une ou plusieurs protéines choisies entre Wnt2 et Wnt5a et (c) un ou plusieurs facteurs hématopoïétiques ou facteurs de stimulation de cellule, mais dépourvus de sérum. On décrit enfin un agent de régulation des cellules souches hématopoïétiques à l'état non différencié ou de régulation de leur prolifération par contact avec une ou plusieurs protéines choisies entre Wnt2 et Wnt5a, etc.
PCT/JP2004/018860 2003-12-11 2004-12-10 Procede de regulation de differenciation des cellules souches hematopoietiques ou de leur proliferation WO2005056778A1 (fr)

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WO2011101468A1 (fr) * 2010-02-22 2011-08-25 Université Pierre Et Marie Curie (Paris 6) Milieu de culture de cellules pour la croissance et la différenciation de cellules du lignage hématopoïétique
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JP2015527083A (ja) * 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 造血幹細胞特異的レポーターマウスおよびその使用
US9701954B2 (en) 2010-06-22 2017-07-11 Lonza Cologne Gmbh Method and device for uniformly treating adherent cells

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012520671A (ja) * 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 Ｚｎｆ２８１を発現する臍帯血由来の万能幹細胞の分離方法
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 (fr) * 2010-02-22 2011-08-25 Université Pierre Et Marie Curie (Paris 6) Milieu de culture de cellules pour la croissance et la différenciation de cellules du lignage hématopoïétique
JP2016171811A (ja) * 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） 造血系列の細胞を増殖及び分化させる細胞培養培地
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 (ja) * 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 造血幹細胞特異的レポーターマウスおよびその使用
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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