WO2006085482A1 - Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique - Google Patents

Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique Download PDF

Info

Publication number
WO2006085482A1
WO2006085482A1 PCT/JP2006/301838 JP2006301838W WO2006085482A1 WO 2006085482 A1 WO2006085482 A1 WO 2006085482A1 JP 2006301838 W JP2006301838 W JP 2006301838W WO 2006085482 A1 WO2006085482 A1 WO 2006085482A1
Authority
WO
WIPO (PCT)
Prior art keywords
hematopoietic stem
stem cells
self
factor
seq
Prior art date
Application number
PCT/JP2006/301838
Other languages
English (en)
Japanese (ja)
Inventor
Kazuo Todokoro
Original Assignee
Riken
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Riken filed Critical Riken
Priority to JP2007502582A priority Critical patent/JPWO2006085482A1/ja
Publication of WO2006085482A1 publication Critical patent/WO2006085482A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to a self-replicating factor necessary for amplification of mammalian hematopoietic stem cells, and a technique for in vitro amplification of hematopoietic stem cells using this factor.
  • Bone marrow transplantation is the most effective way to treat refractory blood diseases such as various leukemias and aplastic anemia.
  • an HLA-compatible donor must be selected.
  • the transplantation treatment for adult patients requires the collection of large numbers of bone marrow cells, and there are significant risks associated with donors. Therefore, there are even cases where the consent of the donor cannot be obtained.
  • Peripheral blood stem cells can be used even if they have donor strength.
  • G-CSF is administered in large quantities to increase the number of stem cells. This is also a side effect of G-CSF for donors. There is a risk associated with apheresis.
  • Umbilical cord blood can also be used Forced blood cell recovery delay and the number of hematopoietic stem cells in umbilical cord blood are inadequate and are applicable for transplantation in children, but adults often have engraftment failure. It is difficult to apply. As described above, hematopoietic stem cells present in bone marrow and umbilical cord blood are useful for transplantation therapy and regenerative medicine, but cannot be applied immediately due to the limited number of hematopoietic stem cells and rejection.
  • Non-patent Document 3 Temporary extracorporeal amplification methods in the presence of these hematopoietic site force-ins are reportedly limited in force amplification and are not at a level applicable to medical treatment.
  • SCF, FL and TPO were added in the presence of Hess-5 mouse stromal cells separated by membranes, and cord blood CD34 positive In some cases, sex cells were cultured for 5 days and successfully amplified 10 times (Non-patent Document 4).
  • mice cells are co-cultured in the presence of serum, and there is a possibility of viral infection, so it cannot be applied to clinical practice.
  • Neither of these methods is an amplification method of hematopoietic stem cells suitable for transplantation treatment.
  • the amplification efficiency is insufficient.
  • this is an amplification method in which a small number of hematopoietic stem cells coexist in a large number of differentiated blood cells, not only amplifying hematopoietic stem cells, which is inefficient and not suitable for practical use.
  • ushi serum was added to the medium, and there was a risk of infectious diseases including BSE, which had a safety problem.
  • Non-patent Document 6 Succeeded in purifying Wnt3a as a soluble protein and reported that hematopoietic stem cells could be amplified about 6 times with Wnt3a alone (Non-patent Document 6).
  • Wnt is expressed in hematopoietic stem cells themselves, and there is a contradiction in the function of self-replicating factors.
  • Wnt3a is not expressed in the bone marrow, and the Wnt member mainly expressed is also different from the results of the present inventors (Japanese Patent Application 2003-41366 2).
  • amplification is far from practical use, and since it is not a serum-free medium, many differentiated cells are produced, which is problematic for practical use.
  • the self-replicating factor F1 that amplifies hematopoietic stem cells was identified.
  • This membrane protein itself is known (Patent Document 2), suggesting that this protein may have hematopoietic regulatory activity! (Patent Document 3).
  • Patent Document 1 Republished 03/018805 (WO03 / 018805)
  • Patent Document 2 W098 / 11219
  • Patent Document 3 Special Table 2002-513280 (WO98 / 30696)
  • Non-patent literature l Conneally et al, Pro. Natl. Acad. Sci. 94, 9836-9841, 1997
  • Non-patent literature 2 Piacibello et al., Blood, 93, 3736-3749, 1999
  • Non-Patent Document 3 Ueda et al., J. Clin. Iinvest. 105, 1013-1021, 2000
  • Non-Patent Document 4 Kawada et al., Exp. HematoL, 27, 904-915, 1999
  • Non-Patent Document 5 Nature 423, 409-414, 2003
  • Non-Patent Document 6 Nature 423, 448-452, 2003
  • Non-Patent Document 7 Ueno et al., Nat. Immunol. 4, 457-463, 2003
  • Non-Patent Document 8 Tulin et al., J. Biol. Chem. 276, 27519-27526, 2001
  • hematopoietic stem cells can be cultured and amplified outside the body, the patient's own bone marrow (previously! /, Preserved umbilical cord blood at birth), etc. Stem cells can be produced, and safe medical treatment is possible without waiting for an HLA-compatible donor. In other words, autotransplantation is possible, the number of stem cells necessary for transplantation can be secured, and the problem of rejection can be solved. Even if autotransplantation is not possible, if amplification technology can be established, transplantation treatment can be performed simply by collecting a small amount of cells from the donor's bone marrow, eliminating the risk of donors and increasing the number of registered donors. We can expect a dramatic increase in opportunities to work.
  • an object of the present invention is to provide a self-replicating factor necessary for the amplification of mammalian hematopoietic stem cells and a technique for in vitro amplification of hematopoietic stem cells using this factor.
  • the present inventor prepared a cDNA library expressed from a mouse bone marrow stromal cell and mouse 'stromal cell line OP9, which is considered to contain a hematopoietic stem cell self-renewal factor, using pcDNA3.1 vector, and created a cDNA pool.
  • Transfected feeder cells (mouse fibroblasts C 127) were co-cultured with hematopoietic stem cells in a serum-free medium, assayed using the amplification ability as an index, and the candidate gene F1 of a self-replicating factor was isolated. From the analysis of the sequence, the present inventor found that F1 is identical to the 4-transmembrane protein EMP-3 (also known as YMP, HNMP-1).
  • the inventor further succeeded in efficiently amplifying hematopoietic stem cells in vitro without differentiation by co-culturing hematopoietic stem cells and a feeder cell expressing F1 in a serum-free medium, thereby completing the present invention. It came.
  • the present invention is a self-replicating factor that amplifies mammalian hematopoietic stem cells, which is composed of a four-transmembrane membrane protein EMP-3 (also known as YMP, HNMP-1).
  • EMP-3 also known as YMP, HNMP-1
  • the present invention also relates to a protein having the amino acid sequence ability shown in SEQ ID NO: 1 or SEQ ID NO: 3, or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, deleted, substituted or added. It is a self-replicating factor that amplifies mammalian hematopoietic stem cells with a protein power that has the ability to amplify mammalian hematopoietic stem cells.
  • the present invention relates to any force of a peptide in the extracellular domain of two strengths of the four-transmembrane membrane protein EMP-3, a mixture thereof, or a mixture of these two peptides via a spacer. It is a self-replicating factor that amplifies mammalian hematopoietic stem cells consisting of peptides bound in the above.
  • the present invention also relates to a method for amplifying hematopoietic stem cells, comprising culturing mammalian hematopoietic stem cells in a serum-free medium in the presence of the above self-replicating factor.
  • the present invention is also a hematopoietic stem cell amplified by the above method. Further, the present invention is a medium for culturing hematopoietic stem cells that contains the above self-replicating factor and does not contain serum.
  • the present invention is a feeder cell into which DNA encoding the above protein or peptide is introduced.
  • the present invention further provides a feeder cell into which DNA encoding at least one hematopoietic factor or cell stimulating factor is introduced.
  • the inventor has confirmed that hematopoietic stem cells are amplified by the self-replicating factor F1 of the present invention.
  • all tissue stem cells and somatic cells that react with hematopoietic stem cells and F1 are amplified outside the body, and the amplified hematopoietic stem cells and all tissue stem cells and somatic cells that react with F1 are various. It can be used for transplantation therapy and gene therapy for patients with various intractable blood diseases and various tissue diseases.
  • Hematopoietic stem cells amplified using the self-replicating factor of the present invention and all tissue stem cells and somatic cells that react with F1 can be used for hematopoietic stem cell transplantation in place of conventional bone marrow transplantation or cord blood transplantation.
  • the hematopoietic stem cells obtained by the culture method of the present invention can improve hematopoietic insufficiency due to bone marrow hypoplasia that exhibits anemia such as aplastic anemia.
  • Other diseases for which transplantation of hematopoietic stem cells obtained by the culture method of the present invention is effective include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldric h syndrome, acquired immune deficiency syndrome (AIDS ) Immunodeficiency syndrome, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage diseases such as Gaucher's disease and mucopolysaccharidosis, adrenoleukodysplasia, various cancers or tumors, etc. Is mentioned.
  • the self-replicating factor of the present invention can be used for in vitro or hematopoietic stem cell culture / proliferation methods because hematopoietic stem cells can be propagated in vivo or in vitro without being differentiated.
  • Improvement of cytopenia with radiation therapy and chemotherapy drugs such as anticancer drugs, prevention of infection caused by lymphocyte depletion, treatment of bone marrow diseases such as myelodysplasia and myelosuppression, It can be used for the treatment of bone marrow diseases such as leukemia and advanced renal disorder's bone marrow suppression, the treatment of hypocytosis derived from genetic diseases, and the in vitro culture of recombinant stem cells at the time of gene therapy.
  • the self-replicating factor of the present invention can amplify tissue stem cells other than hematopoietic stem cells.
  • the self-replicating factor F1 of the present invention can be applied to regenerative medicine of various tissues.
  • the self-replicating factor of the present invention is a four-transmembrane membrane protein EMP-3 (also known as YMP, HN MP-1).
  • EMP-3 also known as YMP, HN MP-1
  • amino acid sequences such as NP_001416, NP-001415, NP-001414, CAGH09718, CAG33152, AAH09718, and P54852, and nucleotide sequences such as NM_001425, BC009718, and CR456871 are registered as human membrane protein EMP-3.
  • amino acid sequences such as NP_034259, AAH01999, 035912, and base sequences such as NM_010129 and BC001999 are registered. Any of these can be used in the present invention.
  • the origin of the membrane protein EMP-3 is not particularly limited as long as it amplifies all tissue stem cells and somatic cells that react with hematopoietic stem cells and F1 of mammals, particularly humans.
  • the self-replicating factor of the present invention is a protein having an amino acid sequence ability shown in SEQ ID NO: 1 (human) or SEQ ID NO: 3 (mouse). The homology between these amino acids is 92.6%.
  • the self-replicating factor of the present invention is a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably the amino acid sequence thereof.
  • a protein having an amino acid sequence with a homology of 90% or more and has the activity of amplifying mammalian hematopoietic stem cells. The ability of this protein to amplify hematopoietic stem cells can be measured by the method described later.
  • the mammal may be a mouse or a force L, but is preferably a human.
  • the self-replicating factor of the present invention is a four-transmembrane membrane protein, it is considered that the two extracellular domains also have the ability to amplify hematopoietic stem cells.
  • this extracellular domain has amino acid numbers 22 to 60.
  • Peptides and forces that are peptides with amino acid numbers 117-134 or 121-134 In the case of non-human membrane protein EMP-3, there are two corresponding peptides.
  • either one of the two extracellular domain peptides or a mixture thereof can be used. Furthermore, a peptide in which these two peptides are directly bound, or a peptide in which two peptides are bound through a spacer can be used.
  • the full-length gene of the self-replicating factor F1 of the present invention a variant thereof, a part of the extracellular membrane, a part or the whole of F1, and a fusion protein thereof, bacteria such as E. coli, yeast, animal cells, silkworms, etc. It is preferable to use it artificially produced by insects, cultured cells thereof, or individual mammals!
  • Hematopoietic stem cells are derived from umbilical cord blood, fetal liver, bone marrow, fetal bone marrow, peripheral blood, peripheral blood in which stem cells are mobilized by administration of anticancer agents and peripheral blood of mammals such as humans and mice, and terminal blood It is also possible to collect the cell group isotropic force. From these tissues, hematopoietic stem cells can be obtained by immunologically staining with anti-CD34 antibody, anti-CD133 antibody, anti-CD38 antibody, etc., and separating with a cell sorter according to the staining properties of these antibodies. it can.
  • CD34 antigen is known as a marker for human hematopoietic stem cells, and particularly unclear markers include CD34 positive, CD38 negative, CD133 positive, KDR positive, and cell differentiation antigen negative.
  • nucleated cells or stem cell fractions derived from human or mouse bone marrow without isolating hematopoietic stem cells can be used as they are for culturing.
  • SP side population cell fraction (contains about half of hematopoietic stem cells and other tissue stem cells! / ) May be used.
  • the hematopoietic stem cell or hematopoietic stem cell fraction is cultured in the presence of the self-replicating factor of the present invention.
  • Culture of hematopoietic stem cells can be performed using a suitable culture medium in a petri dish for culture, a flask, or a bioreactor capable of mechanically controlling the medium composition, pH, and the like.
  • Medium is hematopoietic stem It is not particularly limited as long as cell survival and growth are not inhibited.
  • SF-02 medium Sudo Junyaku
  • Opti-MEM medium GEBCO BRL
  • MEM medium GEBCO BRL
  • DMEM medium GIB CO BRL
  • IMDM medium GIBCO BRL
  • PRMI1640 medium GIBCO BRL
  • the medium further includes, for example, insulin, transferrin, ratatopherin, 2-mercaptoethanol, ethanolamine, sodium selenite, HEPES, monothioglycerol, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, various growths. Factors, various antibiotics, KSR (knockout serum replacement), etc. may be added as necessary.
  • the culture is preferably performed in the absence of serum.
  • the self-replicating factor of the present invention is added as a soluble protein (peptide) at the time of culturing hematopoietic stem cells, or as an insoluble protein (peptide) together with various compounds, or directly fixed to a culture vessel or various Can be covalently or non-covalently immobilized via a carrier such as a protein (peptide), and hematopoietic stem cells (various tissue stem cells and somatic cells that can be amplified with F1 factor) can be amplified outside the body in a serum-free medium. it can.
  • a carrier such as a protein (peptide)
  • hematopoietic stem cells variantous tissue stem cells and somatic cells that can be amplified with F1 factor
  • hematopoietic stem cells in amplifying hematopoietic stem cells, it is possible to improve the efficiency by coexisting at least one hematopoietic factor or cell stimulating factor in addition to using the self-replicating factor of the present invention alone. Hematopoietic stem cells can be amplified outside the body.
  • This hematopoietic factor or cell stimulating factor refers to a factor that gives a hematopoietic cell a stimulus such as self-renewal and proliferation.
  • Wnt wingless / int-l
  • SCF stem cell factor
  • TPO thrombopoietin
  • IL-3 interleukin 3
  • IL-11 interleukin-11
  • GM-CSF granulocyte / ma crophage colony-stimulating factor
  • G-CSF Condylar sphere colony ⁇ [J granulocyte colony-stimulating factor), TGF- ⁇ (transforming growth factor 1 j8), MIP-1a, Flt3 / Flk2-ligand (FL), Flk2 / Flk3 ligand, EPO (erythropoietin ), Notch ligand (Jagged family, Delta family), Tie2 ligand (angiopoetin), BMP4, bFG F, oncostatin M,
  • Wnt such as Wnt2 and Wnt5a, which have already been discovered by the present inventors, seems to function as a factor that suppresses cell differentiation and plays a role in synergistic amplification of proliferation.
  • Patent Application 2003-413662 which is considered to be an effective cell stimulating factor.
  • the self-replicating factor F1 of the present invention may be added to the medium, or the self-replicating factor of the present invention.
  • F1 may be added to the medium and co-cultured with a single feeder cell, or the self-replicating factor F1 of the present invention may be attached or covalently attached to a petri dish or other incubator (device) with or without various carriers.
  • the cells may be cultured or co-cultured with a feeder cell expressing the self-replicating factor F1 of the present invention.
  • a DNA encoding the amino acid sequence of the self-replicating factor F1 of the present invention (eg, SEQ ID NO: 2) is introduced into an expression vector, and this recombinant is transferred to the feeder cell. It can be obtained by feeding. Any vector that can be expressed in animal cells, such as pcDNA3.1, can be used as this vector.
  • the concentration of the hematopoietic factor or cell stimulating factor added to the medium is usually about lng / ml to about lOOng / ml, preferably about 5 ng / ml to about 50 ng / ml, more preferably about 5 ng / ml to about 30 ng. / ml.
  • feeder cells include fibroblasts that can be serum-free cultured (C127, NN3T3, P3, etc.), other cell lines that can be cultured without serum (JCT-19, JCT-12, COS7, etc.), fetuses, etc.
  • the derived cells, mesenchymal stem cells, vascular endothelial cells, preadipocytes and the like may be used, and various human cells and various animal cells and insect cells established may be used.
  • a primary cultured somatic cell derived from a patient such as a human oral epithelial cell
  • a human-derived cell that preferably uses a serum-free human-derived cell.
  • a cell line that can be cultured in a serum-free medium or a primary culture cell collected from a patient in a serum-free medium.
  • Cultivation is usually performed at 33-39 ° C, preferably 37 ° C, 3-6% CO, preferably 5% CO.
  • hematopoietic stem cell markers should be at least CD34 antigen positive, preferably CD34 antigen positive, CD38 antigen negative, CD133 positive, KDR positive, cytoplasmic antigen negative, etc. It can be used as an index of hematopoietic stem cells.
  • a transplantation experiment system using irradiated mice or an in vitro colony formation method may be used.
  • the bone marrow cells and hematopoietic stem cell-containing fractions isolated from other mice were transplanted into the mice (recipients) that had been irradiated and damaged the hematopoietic system.
  • the presence of hematopoietic stem cells having long-term bone marrow reconstitution ability is confirmed using the ratio of recipient-derived and donor-derived hematopoietic cells (chimerism) as an index.
  • the mouth-one formation method when hematopoietic stem cells are cultured in a medium supplemented with various site strengths so that various blood cells can appear, the number of hematopoietic progenitor cells whose orientation has been determined is small or The ability to form a colony that does not contain a single split-line cell and possibly a hematopoietic stem cell that has the potential to form a colony that includes a plurality of split-lineage blood cells.
  • CFU-Emix mixed colonies containing red blood cells
  • the present inventor created one cDNA library expressed from mouse bone marrow stromal cell mRNA and mouse stromal cell line OP9 mRNA in a pcDNA3.1 vector.
  • the pooled cDNA (100, 200, 500, etc.) was transfected into mouse fibroblasts C127 using lipofectamine 2000, and G418 resistant cells were obtained in a 24-well or 96-well plate one week later.
  • the amount of DNA used ranged from 0.02 to lmg under various conditions.
  • SP cells Hoechst333 42-negative Rhodaminel23-negative cells
  • FACS fluorescence-activated cell sorter
  • the medium was a serum-free medium containing only 5% Knockout serum replacement (KSR) and penicillin'streptomycin in DMEM. After 5-7 days, the cells were fixed and the number of beta GAL positive cells was counted. From pools with positive cells higher than knock ground (0-50), 10 One-half (or one-fifth) sized cDNA pools were re-transfected into C127 cells and repeated. When the number of pools became 8 or less, 5 times the number of cDNA inserts were sequenced to search for a novel factor that encodes a full-length membrane protein and searched for potential candidate genes. As a result, an isolated single candidate gene (SEQ ID NO: 4) was obtained, which was designated F1. This F1 is the same gene as mouse EMP-3, whose function is unclear, and encoded an 18 kDa four-transmembrane membrane protein.
  • SEQ ID NO: 4 an isolated single candidate gene
  • the F1 gene (SEQ ID NO: 4) introduced into the pcDNA3.1 vector was transfected into mouse fibroblasts C127 using lipofectamine 2000.
  • mice normal mice (8 to 10 weeks old, 578/6/6) and bone marrow strength were also KSL cells (c- Kit positive Seal positive Lineage negative CD34 negative or weak positive).
  • This C127 transfectant was used as a feeder cell and co-cultured with mouse hematopoietic stem cells in the above-mentioned serum-free medium for 1 to 16 days. After incubation, c-Kit positive Seal positive cells were counted with FACS.
  • CD34-positive CD38 weakly-positive cells from human umbilical cord blood were collected using FACS, and mouse hematopoietic stem cells (KSL cells) were used as mouse F1-expressing feeder cells in the same manner as in Example 2. After co-culture, human CD34 positive cells were observed to be amplified (results omitted).
  • the human homologue of the mouse F1 gene (SEQ ID NO: 4) was isolated from human bone marrow cells by RT-PCR and introduced into pcDNA3.1.
  • the human F1 gene (SEQ ID NO: 2) was 92.6% identical in amino acid sequence with the mouse.
  • Mouse F1 and human F1 were able to amplify mouse and human hematopoietic stem cells across species.
  • F1 is a four-transmembrane membrane protein
  • the ability to amplify hematopoietic stem cells was examined using only the F1 extracellular domain.
  • a peptide of the extracellular domain of human F1 (SEQ ID NO: 1) (the two strengths of amino acid 22-60 peptide and amino acid 117-134 or 121-134 peptide) was prepared and assembled. C127 cells coexist with each peptide mix or fusion peptide of each amino acid (directly bound type and bound with Gly-Gly-Ser-Gly-Gly-Ser (SEQ ID NO: 5) spacer) Below, when added and cultured, hematopoietic stem cell amplification was weak!
  • FIG. 1 is a graph showing the amplification efficiency of hematopoietic stem cells.
  • the horizontal axis indicates the culture time.
  • Mouse hematopoietic stem cells (KSL cells c-Kit + Scal + Lin—CD34 1 ⁇ > W / —cells) were co-cultured with mouse Fl-expressing feeder cells, and C- Kit + Scal + cells amplified by FACS were counted. Amplification efficiency is before culture It is expressed as a ratio of the number of cells later.
  • FIG. 2 is a photograph of mouse hematopoietic stem cells amplified under one F1-expressing feeder cell. The feeder cells were physically removed and photographed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Le problème à résoudre dans le cadre de cette invention réside dans le développement d’une technique d’amplification d’une cellule souche hématopoïétique ex vivo. Un facteur d’autoréplication amplifiant une cellule souche hématopoïétique issue d'un mammifère, particulièrement d'un humain, et une technique d’amplification d'une cellule souche hématopoïétique ex-vivo utilisant ce facteur sont proposés. La solution proposée dans l’invention concerne le facteur d’autoréplication F1 qui amplifie une cellule souche hématopoïétique comprenant une protéine 4 transmembranaire EMP-3 (également appelée YMP, HNMP-1). En co-cultivant une cellule souche hématopoïétique en présence de F1, de sa partie, de son mutant ou de son dérivé dans un milieu exempt de sérum, en co-cultivant une cellule souche hématopoïétique et une cellule nourricière en ajoutant du F1, de sa partie ou similaire, ou en co-cultivant une cellule souche hématopoïétique et une cellule nourricière dans laquelle F1 ou un dérivé de celui-ci est exprimé, la cellule souche hématopoïétique peut être efficacement et sûrement amplifiée ex-vivo sans se différencier. En utilisant la cellule souche hématopoïétique amplifiée ou une cellule souche de chacun des divers tissus pouvant être amplifiés par F1, une transplantation et une thérapie génique pour un patient présentant diverses maladies hématologiques chroniques ou diverses maladies organiques peuvent être menées.
PCT/JP2006/301838 2005-02-10 2006-02-03 Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique WO2006085482A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2007502582A JPWO2006085482A1 (ja) 2005-02-10 2006-02-03 造血幹細胞の自己複製因子及び増幅方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005-033909 2005-02-10
JP2005033909 2005-02-10

Publications (1)

Publication Number Publication Date
WO2006085482A1 true WO2006085482A1 (fr) 2006-08-17

Family

ID=36793056

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/301838 WO2006085482A1 (fr) 2005-02-10 2006-02-03 Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique

Country Status (2)

Country Link
JP (1) JPWO2006085482A1 (fr)
WO (1) WO2006085482A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020095029A1 (fr) * 2018-11-05 2020-05-14 Nhs Blood & Transplant Procédé de production de cellules érythroïdes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011219A1 (fr) * 1996-09-11 1998-03-19 Incyte Pharmaceuticals, Inc. Proteines membranaires associees a des maladies (damp)
WO2000035473A2 (fr) * 1998-12-18 2000-06-22 Scios Inc. Methodes de detection et d'utilisation de genes exprimes de façon differentielle dans des maladies
JP2002513280A (ja) * 1997-01-13 2002-05-08 ジェネティックス・インスチチュート・インコーポレーテッド 分泌蛋白およびそれらをコードするポリヌクレオチド

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998011219A1 (fr) * 1996-09-11 1998-03-19 Incyte Pharmaceuticals, Inc. Proteines membranaires associees a des maladies (damp)
JP2002513280A (ja) * 1997-01-13 2002-05-08 ジェネティックス・インスチチュート・インコーポレーテッド 分泌蛋白およびそれらをコードするポリヌクレオチド
WO2000035473A2 (fr) * 1998-12-18 2000-06-22 Scios Inc. Methodes de detection et d'utilisation de genes exprimes de façon differentielle dans des maladies

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BEN-PORATH I. ET AL.: "Characterization of a tumor-associated gene, a member of a novel family of genes encoding membrane glycoproteins", GENE, vol. 183, 1996, pages 69 - 75, XP004062729 *
BOLIN L.M. ET AL.: "HNMP-1: A novel hematopoietic and neural membrane protein differentially regulated in neural development and injury", J. NEUROSCI., vol. 17, no. 14, 1997, pages 5493 - 5502, XP002048248 *
REYA T. ET AL.: "A role for Wnt signalling in self-renewal of haematopoietic stem cells", NATURE, vol. 423, 2003, pages 409 - 414, XP002975077 *
TAYLOR V. ET AL.: "Epithelial membrane protein-2 and epithelial membrane protein-3: two novel members of the peripheral myelin protein 22 gene family", GENE, vol. 175, 1996, pages 115 - 120, XP004043302 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020095029A1 (fr) * 2018-11-05 2020-05-14 Nhs Blood & Transplant Procédé de production de cellules érythroïdes

Also Published As

Publication number Publication date
JPWO2006085482A1 (ja) 2008-06-26

Similar Documents

Publication Publication Date Title
US8143059B2 (en) Method for identification of cell growth or differentiation factors
KR102510368B1 (ko) 혈관 콜로니 형성 세포
US20190002828A1 (en) Methods for producing enucleated erythroid cells derived from pluripotent stem cells
US5914268A (en) Embryonic cell populations and methods to isolate such populations
KR101445337B1 (ko) 지방 흡인 후의 지방흡인물로부터 조혈모세포의 분리 및 정제
US20050221482A1 (en) Methods and compositions for obtaining hematopoietic stem cells derived from embryonic stem cells and uses thereof
KR20040023724A (ko) 조혈 간세포의 제조법
CA3109546A1 (fr) Generation de cellules progenitrices hematopoietiques a partir de cellules souches pluripotentes humaines
WO2020227885A1 (fr) Globules rouges génétiquement modifiés portant un anticorps à chaîne unique anti-pd -1 et leur procédé de préparation
JP2014036651A (ja) ヒト赤血球前駆細胞株及びヒト脱核赤血球の製造方法
WO2012133948A1 (fr) Composition pour thérapie cellulaire par allogreffe, ladite composition contenant une cellule souche pluripotente positive pour ssea-3 pouvant être isolée de tissu corporel
JP5511039B1 (ja) Ｎｋ細胞の調製方法
Perlingeiro et al. A role for thrombopoietin in hemangioblast development
JP2022172252A (ja) 造血移植片の改善方法
WO2019127664A1 (fr) Cellules souches multipotentes et lymphocytes t différenciés et leur utilisation
JP6164650B2 (ja) Ｎｋ細胞の調製方法
Xie et al. Marrow mesenchymal stem cells transduced with TPO/FL genes as support for ex vivo expansion of hematopoietic stem/progenitor cells
KR101027288B1 (ko) 스탓3 활성화 줄기세포
WO2021200901A1 (fr) Procédé de production de progéniteurs des lymphocytes t
WO2006085482A1 (fr) Facteur d'autoreplication et procede d’amplification d’une cellule souche hematopoietique
JP2007238473A (ja) 造血幹細胞の維持増幅を促進する因子及び造血幹細胞の増幅方法
JPWO2005056778A1 (ja) 造血幹細胞の分化抑制又は増殖方法
CN117866110B (zh) 一种靶向flt-3的***嵌合抗原受体及其应用
JP2024518632A (ja) 体液免疫系の再生方法およびその使用
WO2024073665A1 (fr) Expansion évolutive de cellules progénitrices érythroïdes cd71+ pour thérapie cellulaire

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2007502582

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06712981

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 6712981

Country of ref document: EP