WO2006085482A1

WO2006085482A1 - Self-replication factor and amplification method of hematopoietic stem cell

Info

Publication number: WO2006085482A1
Application number: PCT/JP2006/301838
Authority: WO
Inventors: Kazuo Todokoro
Original assignee: Riken
Priority date: 2005-02-10
Filing date: 2006-02-03
Publication date: 2006-08-17
Also published as: JPWO2006085482A1

Abstract

[PROBLEMS] A technique for amplifying a hematopoietic stem cell ex vivo is developed. A self-replication factor that amplifies a hematopoietic stem cell of a mammal, particularly human, and a technique for amplifying a hematopoietic stem cell ex vivo using this factor are provided. [MEANS FOR SOLVING PROBLEMS] The self-replication factor F1 of the invention that amplifies a hematopoietic stem cell comprises a four transmembrane protein EMP-3 (also called YMP, HNMP-1). By co-culturing a hematopoietic stem cell in the presence of F1, part thereof, a mutant thereof or a derivative thereof in a serum-free medium, co-culturing a hematopoietic stem cell with a feeder cell with the addition of F1, part thereof or the like, or co-culturing a hematopoietic stem cell with a feeder cell in which F1 or a derivative thereof is expressed, the hematopoietic stem cell can be amplified ex vivo efficiently and safely without differentiating it. By using the amplified hematopoietic stem cell or a stem cell of each of various tissues which can be amplified with F1, a transplantation therapy and a gene therapy for a patient with a variety of intractable hematologic diseases or a variety of organ diseases can be conducted.

Description

Specification

Self-renewal factor and amplification method of hematopoietic stem cells

Technical field

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.

Background art

[0002] Bone marrow transplantation is the most effective way to treat refractory blood diseases such as various leukemias and aplastic anemia. First, an HLA-compatible donor must be selected. Fortunately, 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. In this case, 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.

[0003] Conventionally, as a technique for culturing and amplifying hematopoietic stem cells in vitro, a method for culturing hematopoietic stem cells as floating cells using various combinations of hematopoietic factors and cytodynamic force is studied. It has been. For example, there has been a report that CD34 + CD38-human hematopoietic stem cells were cultured for 1 week and added twice by adding SCF, FL, IL3, IL6, and G-CSF (Non-patent Document 1). In addition, it has been reported that umbilical cord blood CD34-positive cells were cultured for 9 weeks by adding SCF, FL, IL6, and TPO and amplified 70-fold (Non-patent Document 2). In addition, there is a report that SCF, FL, TPO, IL6 and soluble IL6 receptor were calorized, and cord blood CD34 positive cells were cultured for 1 week and increased 4 times (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. In addition, 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). This is because mouse 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. First, the amplification efficiency is insufficient. Further, 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. In addition, ushi serum was added to the medium, and there was a risk of infectious diseases including BSE, which had a safety problem.

There may be new factors that allow hematopoietic stem cells to self-replicate, and various attempts have been made to search for their genes. In the past, Notch ligand (Jagged and Delta family), Tie-2 ligand (angiopoetin), BMP-4, etc. have been reported to play an important role in self-renewal of hematopoietic stem cells. Or, in combination, amplification was limited, and a solution was awaited as soon as possible. Recently, Weissman et al. Reported that Wnt was able to amplify hematopoietic stem cells. The guidance of HoxB4 and Notch reported by Daley et al. Is compelling. Nusse et al. 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). However, Wnt is expressed in hematopoietic stem cells themselves, and there is a contradiction in the function of self-replicating factors. In addition, 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). In addition, 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. Other factors involved in stem cell self-renewal other than Wnt were also reported one after another. Ueno et al. Reported that Kirre performs self-renewal of hematopoietic stem cells (Non-patent Document 7, Patent Document 1). However, Kirre alone has not been described as capable of expanding stem cells outside the body, and Walz et al. Report that the function as a self-replicating factor is suspicious, as reported by Walz et al. As a membrane protein with a completely different function. . Kirre has confirmed expression not only in bone marrow stromal cells but also in the AGM region at the time of hematopoiesis (unpublished data), but its detailed functions are for further study. There is also a report that ISF is involved in hematopoietic stem cell amplification (Non-patent Document 8), but it has not been confirmed.

According to the present invention, the self-replicating factor F1 that amplifies hematopoietic stem cells was identified. Was found to be identical to the 4-transmembrane protein EMP-3. This membrane protein itself is known (Patent Document 2), suggesting that this protein may have hematopoietic regulatory activity! (Patent Document 3).

[0005] 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

Disclosure of the invention

Problems to be solved by the invention

[0006] If 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. If umbilical cord blood can be amplified outside the body, treatment for adults is possible. Thus, the establishment of in vitro amplification technology of hematopoietic stem cells can save the lives of many patients with intractable blood diseases. In addition, stem cells necessary for gene therapy can be easily obtained, and safe gene transfer without using retrovirus becomes possible, and gene therapy can be spread. Development of in vitro amplification technology for hematopoietic stem cells It is an important technical issue to be solved urgently for transplantation therapy 'regenerative treatment and gene therapy.

That is, 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. Means for solving the problem

[0007] 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.

[0008] That is, 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).

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.

Furthermore, 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.

Further, 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 invention's effect

[0009] The inventor has confirmed that hematopoietic stem cells are amplified by the self-replicating factor F1 of the present invention.

Using the self-replicating factor 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.

In addition, by separating the hematopoietic stem cells of a patient or another person into various blood cells and transferring them into the patient's body, patients with insufficient formation of various blood cells can be treated. Moreover, 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.

[0010] In addition, 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.

It is also considered that the self-replicating factor of the present invention can amplify tissue stem cells other than hematopoietic stem cells. In that case, the self-replicating factor F1 of the present invention can be applied to regenerative medicine of various tissues.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] The self-replicating factor of the present invention is a four-transmembrane membrane protein EMP-3 (also known as YMP, HN MP-1). In various databases, 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. In addition, as mouse 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.

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.

[0012] 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. Is 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.

[0013] In addition, since 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. In the case of the human membrane protein EMP-3 (SEQ ID NO: 1), 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.

Therefore, as the self-replicating factor of the present invention, 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!

[0014] 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. For example, in mice, the characteristics of hematopoietic stem cells are found in cells that are negative for the cytoplasmic antigen (Lineage) and positive for c_kit and Sca_l and that have negative to weakly positive properties for the CD34 antigen. . 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. In addition, 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. For example, as a stem cell fraction derived from human, mouse bone marrow, umbilical cord blood, peripheral blood, etc., SP (side population) cell fraction (contains about half of hematopoietic stem cells and other tissue stem cells! / ) May be used.

[0015] In the culture method for producing the hematopoietic stem cell of the present invention, 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.For example, SF-02 medium (Sanko Junyaku), Opti-MEM medium (GIBCO BRL), MEM medium (GIBCO BRL), DMEM medium (GIB CO BRL), IMDM medium (GIBCO BRL), PRMI1640 medium (GIBCO BRL), RD medium (RPMI1 640: DMEM = 1: 1 (V / V) mixed medium) and the like can be used. 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.

[0016] 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.

[0017] In the present invention, 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. For example, Wnt (wingless / int-l) family, 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 朿 [J granulocyte colony-stimulating factor), TGF-β (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, IL6 / sIL6R, EGF and LIF.

[0018] In particular, 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.

In the culture method for amplifying all the tissue stem cells and somatic cells that react with the hematopoietic stem cells and F1 of the present invention, 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.

In this feeder cell expressing this self-replicating factor F1, 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.

[0019] 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.

[0020] In order to produce the hematopoietic stem cells of the present invention, it is not essential, but it is preferable to co-culture hematopoietic stem cells with feeder cells. These 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. In addition, it is more preferable to use a primary cultured somatic cell derived from a patient such as a human oral epithelial cell as a human-derived cell that preferably uses a serum-free human-derived cell. In particular, it is most preferable to use 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.

2 2 for 5-50 days.

[0021] Amplification of hematopoietic stem cells can usually be confirmed using a cell surface antigen as an indicator. For example, human 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. As a means for confirming the presence of hematopoietic stem cells, a transplantation experiment system using irradiated mice or an in vitro colony formation method may be used. In the transplantation experiment system using irradiated mice, the bone marrow cells and hematopoietic stem cell-containing fractions isolated from other mice (donors) were transplanted into the mice (recipients) that had been irradiated and damaged the hematopoietic system. After transplantation, 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. In 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. In particular, the formation of mixed colonies containing red blood cells (CFU-Emix) is regarded as an indicator of hematopoietic stem cells in humans.

The following examples illustrate the invention but are not intended to limit the invention.

Example 1

In order to identify a new hematopoietic stem cell self-replicating factor, 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.

For hematopoietic stem cells, SP cells (Hoechst333 42-negative Rhodaminel23-negative cells) are sorted from the bone marrow of beta GAL transgenic mice using a fluorescence-activated cell sorter (FACS) (FACS Vantage SE manufactured by Betaton Deckson). Isolated. 90 to 400 SP cells per well were co-cultured with the above transfectant.

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.

Example 2

[0023] The F1 gene (SEQ ID NO: 4) introduced into the pcDNA3.1 vector was transfected into mouse fibroblasts C127 using lipofectamine 2000.

As mouse hematopoietic stem cells, 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.

As a control, when co-cultured with only one feeder cell (mouse fibroblast C127) that had not been transfected, the hematopoietic stem cells were vigorously proliferated. The number of hematopoietic stem cells amplified using the above F1-expressing feeder cell The background of only the feeder cell was inserted I to calculate the hematopoietic stem cell amplified by F1.

Since Lineage positive cells were not observed, it was thought that only hematopoietic stem cells increased. The amplification efficiency was estimated to be about 1000 times in 16 days. The rate of cell division 10 times in 2 weeks or so was considered to be a very reasonable number.

The results are shown in Fig. 1.

[0024] The amplification ability was confirmed not only using FACS but also under a microscope. Beta GAL positive, GFP positive, or hematopoietic stem cells to which a fluorescent substance was added and F1-expressing C127 cells were co-cultured and observed with a fluorescence microscope after passage after 2 days to 2 weeks. It was observed that hematopoietic stem cells were submerged under C127 cells and amplified. Figure 2 shows the amplified mouse hematopoietic stem cells taken by forcing the feeder cells to detach with forceps. The presence of feeder cells may be necessary for hematopoietic stem cells to proliferate Suggests sex.

Example 3

[0025] As hematopoietic stem cells, 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).

Example 4

[0026] 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.

Using a C127 feeder cell expressing this human F1 gene (SEQ ID NO: 2), amplification experiments were performed in the same manner as in Example 2 using human umbilical cord blood-derived CD34 positive CD38 weakly positive cells and mouse KSL cells as hematopoietic stem cells. However, the same amplification ability as that of a mouse F1-expressing feeder cell was observed (results omitted).

Mouse F1 and human F1 were able to amplify mouse and human hematopoietic stem cells across species.

Example 5

[0027] Since 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!

Brief Description of Drawings

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.

Claims

The scope of the claims

[I] A self-replicating factor that amplifies mammalian hematopoietic stem cells consisting of the four-transmembrane protein EMP-3.

[2] From a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or 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. A self-replicating factor that amplifies mammalian hematopoietic stem cells, consisting of a protein having the ability to amplify mammalian hematopoietic stem cells.

[3] Four-transmembrane protein EMP-3, the force of any of the peptides in the extracellular domain of the two strengths, or a mixture of these, or these two peptides via a spacer. Self-replicating factor that amplifies mammalian hematopoietic stem cells consisting of conjugated peptides.

[4] The membrane protein EMP-3 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or one or several amino acids are deleted in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, 4. The self-replicating factor according to claim 3, wherein the self-replicating factor is a protein that also has a substituted or added amino acid sequence ability and has an ability to amplify mammalian hematopoietic stem cells.

[5] The peptide in the extracellular domain of the two strengths is a peptide of amino acid numbers 22 to 60 and a peptide of amino acid numbers 117 to 134 or 121 to 134 of human membrane protein EMP-3 (SEQ ID NO: 1), or The self-replicating factor according to claim 3 or 4, which is two peptides corresponding to these non-human membrane protein EMP-3.

6. The self-replicating factor according to any one of claims 1 to 5, wherein the mammal is a human.

[7] A method for amplifying hematopoietic stem cells, comprising culturing mammalian hematopoietic stem cells in a serum-free medium in the presence of the self-replicating factor according to any one of claims 1 to 6.

8. The method according to claim 7, wherein the culture is performed in the presence of at least one hematopoietic factor or cell stimulating factor and in the absence of serum.

9. The method according to claim 7 or 8, wherein the mammal is a human.

[10] The method according to any one of [7] to [9], wherein the self-replicating factor is added to a medium.

[II] The self-replicating factor is added to a medium, and the hematopoietic stem cells are cultured with a feeder cell and serum-free. The method according to any one of claims 7 to 9, wherein the co-culture is performed on the ground.

12. The method according to any one of claims 7 to 9, wherein the hematopoietic stem cells are co-cultured with a feeder cell expressing the self-replicating factor in a serum-free medium.

[13] The method according to [11] or [12], wherein the feeder cell is a human-derived cell that can survive under serum-free condition.

[14] A hematopoietic stem cell amplified by the method according to any one of claims 7 to 13.

[15] A medium for culturing hematopoietic stem cells containing the self-replicating factor according to any one of claims 1 to 6 and not containing serum.

16. The medium according to claim 15, further comprising a feeder cell.

17. The medium according to claim 15 or 16, further comprising at least one hematopoietic factor or cell stimulating factor.

18. The medium according to claim 16, wherein the feeder cell is a human-derived cell that can survive under serum-free conditions.

[19] A feeder cell into which DNA encoding a protein or peptide of any one of (1) to (4) is introduced.

(1) Four-time transmembrane protein EMP-3

(2) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. , A protein capable of amplifying mammalian hematopoietic stem cells

(3) Four-transmembrane membrane protein EMP-3, one of the two peptides in the extracellular domain, or a mixture of these, or these two peptides via a spacer. Peptide bound with

(4) The peptide in the extracellular region of (3) is a peptide of amino acid number 22 to 60 of human membrane protein EMP-3 (SEQ ID NO: 1) and a peptide of amino acid number 117 to 134 or 121 to 134, Or, in the case of non-human membrane protein EMP-3, two corresponding peptides,

[20] Furthermore, a request for introducing DNA encoding at least one hematopoietic factor or cell stimulating factor Item 20. A feeder cell according to Item 19.