WO1999022001A1 - Method for regulating the differentiation/proliferation of hematopoietic stem cells - Google Patents
Method for regulating the differentiation/proliferation of hematopoietic stem cells Download PDFInfo
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- WO1999022001A1 WO1999022001A1 PCT/JP1998/004884 JP9804884W WO9922001A1 WO 1999022001 A1 WO1999022001 A1 WO 1999022001A1 JP 9804884 W JP9804884 W JP 9804884W WO 9922001 A1 WO9922001 A1 WO 9922001A1
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- gene
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- differentiation
- hematopoietic stem
- stem cells
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Definitions
- the present invention relates to a method for regulating the differentiation and proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells, and to vectors and cells used in the method.
- Hematopoietic stem cells and hematopoietic progenitor cells can be used for blood cell transplantation instead of bone marrow transplantation and cord blood transplantation, or for gene therapy.
- BACKGROUND ART Mature blood cells flowing in the living body have a short life span (about 120 days for red blood cells and about 7 days for platelets in humans), and mature blood cells are differentiated and proliferated daily from hematopoietic progenitor cells. Maintains homeostasis of peripheral blood mature blood cells.
- the number of mature blood cells supplied to peripheral blood is 200 billion cells / day for red blood cells and 700 billion cells / day for neutrophils in humans.
- the progenitor cells of each of these differentiation lineages proliferate while differentiating from undifferentiated hematopoietic stem cells to form a system in which peripheral blood cells do not constantly die (Tsuda Suda, Blood Stem Cells Destiny, Sheepsha, 199 2).
- hematopoietic stem cells pluripotency
- hematopoietic stem cells pluripotency
- a transplantation experiment system using irradiated mice, and an in vitro (in vitro) colony formation method (Bradley, TR, J. Exp. Med., 44: 287-299). , 1966), and knowledge on the differentiation of hematopoietic stem cells and hematopoietic progenitor cells has been accumulated.
- the transplantation experiment system using irradiated mice is the most direct way to analyze the properties of hematopoietic stem cells.
- Transplantation of bone marrow cells isolated from another mouse (Donna 1) into a mouse (recipient) that has damaged the hematopoietic system by irradiation may reconstitute the donor-derived hematopoietic system in the recipient mouse. it can.
- Various differentiation antigens expressed on hematopoietic cells (Spangrude, GJ, Proc. Natl. Acad. Sci.
- Bone marrow cells were fractionated using substances (Rhl23, Hoechst 33342) (Wolf, NS, Exp. Hematol., 21:61 4-622, 1993) and the transplantation experiments described above were performed. Attempts have been made to identify hematopoietic stem cells.
- hematopoietic stem cells can differentiate into lymphoid cells and myeloid cells by transplanting a single cell, and hematopoietic stem cells can be transplanted for a long time It has also been demonstrated that systems can be constructed (Osawa, M., Science, 273: 242-245, 1996). Hematopoietic stem cells can survive over a long period of time in the recipient individual and supply differentiated blood cells. On the other hand, in transplantation experiments using hematopoietic progenitor cells, the cells transplanted from the recipient individual disappear in a short period of time and cannot undergo hematopoiesis for a long period of time (Osawa, M., Science, 278: 242- 245, 1996).
- hematopoietic progenitor cells can only differentiate into mature blood cells of a limited lineage even in the presence of many cytokins, hematopoietic stem cells that can build a long-term hematopoietic system in transplanted mice It is possible to differentiate into many cell lineages. From these results, the differentiation from hematopoietic stem cells to mature blood cells flowing in peripheral blood is interpreted as follows.
- Hematopoietic stem cells can be differentiated into various lineages It is capable of self-replication while maintaining this pluripotency property (pluripotency). Hematopoietic stem cells self-renew and partially differentiate, are affected by various site forces, gradually narrow the lineage of cells that can be differentiated, differentiate and proliferate into hematopoietic progenitor cells that can only differentiate into a limited number of cell types, It eventually becomes a mature blood cell (Hematopoietic Stem Cell, Levitt, D., Marcel Dekker, Inc., 1995). Bone marrow transplantation and umbilical cord blood transplantation are therapies to transplant hematopoietic cells to patients. It depends on what you can do.
- hematopoietic stem cells are considered to be an optimal target because they can survive for a long time in the transplant recipient as described above.
- gene therapy that complements the genetic deficiency of hematopoietic stem cells is considered to be a fundamental treatment for the disease.
- hematopoietic stem cells have been clinically recognized, and it has been an issue how to grow hematopoietic stem cells without differentiation.
- Attempts have been made to expand hematopoietic stem cells using cytokin-blood cell stimulating factors, but have not succeeded in efficiently expanding hematopoietic stem cells (Trevisan, M., Blood, 88: 4149, 1996).
- hematopoietic stem cells are differentiated and proliferation of hematopoietic progenitor cells is dominant. Therefore, if the differentiation of hematopoietic stem cells could be suppressed, hematopoietic stem cells could be expanded in the presence of cytokines and hematopoietic cell stimulating factors.
- Notch / Delta has been shown by genetic analysis to be involved in the formation of various organs such as nerves and wings during embryo development in Drosophila (Artavanis-T sakonas, S., Science, 268: 225, 1995).
- the ligand, Delta protein binds to the receptor, Notch protein, and transmits signals through Notch to suppress differentiation.
- homologous genes to the Notch / Delta gene family are known (Blaumueller, CM, Perspectives on Developmental Neurobiology, 4: 325, 1997).
- the regulation of differentiation by the Notch signaling system in neural cell differentiation is considered as follows (Artavanis-Tsakonas, S., Science, 268: 225, 1995; Simpson, P., Perspectives on Developmental Neurobiology, 4). : 297, 1997).
- the dorsal-ventral axis and anterior-posterior axis are determined during embryonic development, neuroblasts appear in the ectoderm and proneural clusters that can differentiate into epidermal cells appear.
- the Notch / Delta signaling system plays an important role in selecting cells capable of differentiating into neurons from this cell cluster.
- the cells present in this cell cluster express Notch equally, but some cells begin to express Notch ligand, which suppresses neighboring cells from differentiating into neural cells ( Lateral suppression). Cells that express Notch ligand differentiate into neuroblasts, and undergo terminal differentiation under various stimuli to achieve terminal differentiation into functional neurons. On the other hand, cells in which the Notch / Delta signaling system has been activated by binding to Notch ligand cannot differentiate into neurons, but differentiate into epidermal cells.
- Notch is activated by binding to Delta, Interacts with the nuclear localization protein RBP-J / c (CBF-1, also called KBF-2) through the region (Honjo, T., Genes to Cells, 1: 1, 1996).
- RBP-J / translocates into the nucleus following Notch activation and induces expression of the HLH (helix-loop-helix) transcription factor HES-1 (Ryuichiro Kageyama, Biochemistry, 67: 1093, 1995) I do.
- An HLH-type transcription factor is a transcription factor having a three-domain structure, that is, 1) a helix-one-loop part that forms a three-dimensional structure of one helix, which binds to itself or interacts with other HLH-type transcription factors. It comprises a binding portion for forming a homodimer or a heterodimer by binding, 2) a portion capable of binding to basic DNA, and 3) a portion having a transcription promoting activity.
- a characteristic of HLH-type transcription factors is that their DNA binding ability is regulated by the partner that forms the heterodimer.
- Mash-1 and MATH are known as HLH-type transcription factors that positively regulate the differentiation of nerve cells. Both Mash-1 and MATH are HLH-type transcription factors that form heterodimers with the ubiquitous HLH-type transcription factor E12 / E47, and recognize specific nucleotide sequences to promote neuronal cell differentiation. Activating genes (Johnson, JE, Proc. Natl. Acc. Sci. USA, 89: 3596, 1992).
- HES-1 binds to Mash-1 and MATH in a competitive manner with E12 / E47 and suppresses its DM binding activity, thereby inhibiting the expression of genes that promote differentiation into nerve cells, and It is thought to suppress the differentiation of cells (Sasai, Y., Genes Dev., 6: 2620, 1992).
- HES-1 plays an important role in regulating the differentiation of the Notch / Delta system by inhibiting the activity of transcription factors that positively regulate differentiation at the final stage. Similar regulatory mechanisms have been inferred for the HES-1 similar genes HES-3 and HES-5. (Ryuichiro Kageyama, Biochemistry, 67: 1093, 19995)
- the differentiation of the skeletal muscle system is regulated by the Notch / Delta system, but even if the HES-1 gene is directly introduced into and expressed in skeletal myoblasts, differentiation into skeletal muscle cells is suppressed.
- a different regulation mechanism from the signal transduction system from RBP-Jc to the HES family gene has been speculated (Shawber, C., Development, 122: 3765, 1996). In other words, differentiation control is not performed through HES-1 in all cell types.
- Notch-1 gene also known as TAN-1
- T cell leukemia Ellison, LW, Cell, 66: 649, 1991; Reynolds, TC, Cell, 50: 107, 1987.
- transgenic mice transfected with an activated Notch gene are involved in the control of T cell proliferation because leukemia with immature T cell properties is produced (Pear, WS, J. Exp. Med., 183: 2283, 1996).
- the Notch gene has been reported to be expressed in hematopoietic stem cells (Milner, LA, Blood, 83: 2057, 1994), but during the process of differentiating hematopoietic stem cells into various differentiated cells.
- the Notch / Delta feature has not been reported.
- Dll-1 Choitnis, A., Nature, 375: 761, 1995
- Dll-3 Dunwoodie, SL, Development, 124: 3065, 1997)
- Jagge d-1 Lidsell , CE, Cell, 80: 909, 1995
- Jagged-2 Jagged-2
- DLK also called SCP-1 or Pref-1
- DLK When DLK is expressed in stromal cells that support hematopoietic cells and cocultured with hematopoietic stem cell fractions, activity to support the proliferation of hematopoietic progenitor cells has been confirmed. However, no activity has been observed when DLK, a membrane protein, is solubilized and DLK acts directly on hematopoietic stem cells. Therefore, it is not clear whether DLK exerts its activity of inhibiting differentiation by directly acting on hematopoietic progenitor cells. Furthermore, because DLK has low homology to Delta, it is unclear at this time whether DLK signals through Notch.
- the present invention has been made in view of the above, and provides a method for suppressing the differentiation of hematopoietic stem cells and / or hematopoietic progenitor cells, and a method for allowing the cells to survive and preferably further proliferate in a state where the differentiation is suppressed. And to provide means used for these methods.
- Notch / Delta-based molecules could be used to control the differentiation of hematopoietic stem cells.
- Notch-l, Notch-2, Notch-3, Notch-4 and Notch-4 were actually used in hematopoietic cells including hematopoietic stem cells.
- HES-1, HES-3 and HES-5 were examined in detail.
- Notch and HES genes were widely expressed in hematopoietic cells at various stages of differentiation. From this expression status, it was speculated that the signaling system of the Notch / Delta system plays an important role also in cell differentiation from hematopoietic stem cells.
- the present invention provides a method for enhancing the expression of a differentiation-suppressing gene in mammalian hematopoietic stem cells and causing a blood cell stimulating factor to act thereon, whereby the hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells are activated. It is a method of regulating the differentiation and proliferation of the plant.
- the present invention also provides a gene transfer vector in which a differentiation-suppressing gene has been incorporated into a viral vector.
- the present invention further provides mammalian hematopoietic stem cells in which the expression of the differentiation inhibitory gene is enhanced.
- the present invention provides a hematopoietic stem cell characterized by culturing a mammalian hematopoietic stem cell in which the expression of a differentiation inhibitory gene is enhanced or a hematopoietic progenitor cell differentiated from the hematopoietic stem cell while allowing a blood cell stimulating factor to act. And / or provide a method for producing hematopoietic progenitor cells.
- a hematopoietic stem cell is a cell having a pluripotency capable of differentiating into all differentiation lineages of blood cells, and a cell capable of self-renewal while maintaining the pluripotency.
- CFU-Emix colony containing cell types of multiple differentiating lineages including erythrocytes in an in vitro Atsushi system. These cells are thought to contain cells that self-renew in vivo and survive for long periods of time.
- Hematopoietic progenitor cells are blood cells that have been slightly differentiated from hematopoietic stem cells, and have the ability to differentiate into single or two types of lineages.
- the differentiation inhibitory gene refers to a gene that encodes a transcription factor having an activity of suppressing the differentiation of a cell when expressed in a cell capable of differentiating.
- Blood cell stimulating factors are biological factors such as so-called site force in, inuichi leukin, growth stimulating factor, interferon, chemokine, etc.
- a stimulator that causes a change in activity is so-called site force in, inuichi leukin, growth stimulating factor, interferon, chemokine, etc.
- the source of hematopoietic stem cells used in the present invention may be, for example, umbilical cord blood, fetal liver, bone marrow, fetal bone marrow, peripheral blood, peripheral blood, cytokine and / or peripheral mobilized stem cells by administration of an anticancer agent in mammals such as humans and mice.
- Examples include a cell group derived from blood and peripheral blood, and any tissue may be used as long as it contains hematopoietic stem cells.
- Hematopoietic stem cells can be obtained from these tissues according to Seaberg, L.A., “Weir's Handbook of Experimental Imlunology, 5th edition, Blackwell Science Inc. 1997. That is, an anti-CD34 antibody It can be immunologically stained using an anti-CD33 antibody, an anti-CD38 antibody, or the like, and can be separated by the staining property of these antibodies using a cell sorter.
- the differentiation inhibitory gene used in the present invention examples include the HES-1, HES-3, and HES-5 genes. As shown in the Examples below, by enhancing the expression of the HES-1 gene in hematopoietic stem cells, the differentiation and proliferation of the hematopoietic stem cells and hematopoietic progenitor cells differentiated from the hematopoietic stem cells can be regulated. .
- the effect of HES-1 gene expression on hematopoietic stem cells is also similar to that of HES-1 similar proteins, HES-3 (Ryuichiro Kageyama, Biochemistry, 67: 1093, 1995), HES-5 (Ryuichiro Kageyama, Chemistry, 6 7: 1093, 1995).
- HES-1 gene human-derived one is also called HRY
- HES-3 gene and HES-5 gene are all known genes, and are described in Sasai et al. (Genes Dev., 6: 2620, 1992; mouse-derived HES- 1 and HES-3), disclosed by Akazawa et al. (J. Biol. Chem., 267: 21879, 1992; HES-5 derived from mouse) and Feder et al. (Genomics, 20: 56, 1994; HES-1 derived from human) It can be obtained by amplifying a DNA fragment containing each gene by PCR (polymerase chain, reaction) using oligo nucleotides prepared based on the given sequence.
- PCR polymerase chain, reaction
- to enhance the expression of a differentiation inhibitory gene in a hematopoietic stem cell means to operate the hematopoietic stem cell so that the expression level of the differentiation inhibitory gene in the hematopoietic stem cell is higher than the normal expression level for at least a certain period of time. That means.
- the expression level of the differentiation-suppressing gene does not need to be always high, but may be high as long as the present invention can increase the level of differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells.
- a differentiation inhibitory gene is incorporated in a form that can be expressed in a vector for mammalian cells, and the obtained recombinant vector is introduced into hematopoietic stem cells.
- the expression suppressor gene in an expressible form can be obtained by ligating an expression control factor such as a promoter upstream of the coding sequence of the differentiation suppressor gene.
- the expression of the differentiation inhibitory gene is regulatable.
- the differentiation inhibitory gene is expressed only during culture, and it is not preferable to express the cells even after transplanting the cells into a living body. Therefore, it is preferable that the expression can be regulated as needed.
- expression control include an expression control system using tetracycline (Gossen, M., Proc. Natl. Acad. Sci. USA, 89: 5547, 1992) and an expression control system using the insect hormone ecdysone (No. Natl. Acad. Sci.
- IPTG isoprovir 1 /?-D-thiogalactobilanoside
- the above vectors include retrovirus vector, adenovirus vector (Neering, SJ, Blood, 88: 1147, 1996), herpesvirus vector (Dilloo, D., Blood, 89: 119, 1997), and HIV vector.
- adenovirus vector Neering, SJ, Blood, 88: 1147, 1996)
- herpesvirus vector Dilloo, D., Blood, 89: 119, 1997)
- HIV vector HIV vector.
- the gene transfer vector of the present invention can be obtained.
- the transferred gene exists outside the chromosome and disappears after transient gene expression.
- the use of such a transient expression vector is advantageous in that the differentiation inhibitory gene can be expressed only during culture.
- the expression level of the differentiation inhibitory gene on the chromosomal DNA of hematopoietic stem cells may be increased.
- the expression control sequence such as bromo allyl specific to the differentiation control gene on the chromosomal DNA of hematopoietic stem cells
- the strong expression control sequence By inserting the gene upstream, the expression level of the gene can be increased.
- Replacement and insertion of the expression control sequence can be performed by homologous recombination or the like.
- hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells are increased.
- hematopoietic stem cells and / or hematopoietic progenitor cells can be expanded by culturing hematopoietic stem cells with enhanced expression of differentiation-suppressing genes or hematopoietic progenitor cells differentiated from the hematopoietic stem cells while allowing them to act on blood cell stimulating factors. Can be done.
- Blood cell stimulating factors are added to the culture medium to promote the growth of hematopoietic stem cells, and include so-called cytokines, interleukins, growth stimulating factors, interferon, chemokines, development-related gene products, etc. (See The Cytokine Factsbook, Callard, RE, Academic Press, 1994 for site power-in). Specific examples of blood cell stimulating factors include SCF (stem cell factor), IL-3 (interleukin-13), IL-6 (interleukin-16), and GM-CSF (requested).
- SCF stem cell factor
- IL-3 interleukin-13
- IL-6 interleukin-16
- GM-CSF GM-CSF
- Granulocyte macrophage colony stimulating factor Granulocyte macrophage colony stimulating factor
- TP0 thrombopoetin
- EP0 erythropoietin
- Wnt Thimoth, AW, Blood, 89: 3624-3635, 1997)
- culture conditions favorable for the growth of hematopoietic stem cells can be improved to be more effective.
- culture may be performed by adding a culture supernatant of stromal cells capable of maintaining hematopoietic stem cells.
- the culture medium used for the culture is not particularly limited as long as the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired.
- MEM-G medium GEBCO BRL
- SF-02 medium Sanko Pure Chemical
- Opti-MEM medium GIBCO BRL
- IMDM medium GIBCO BRL
- PRMI 1640 medium PRMI 1640 medium
- Materials added to the culture medium include fetal calf serum, human serum, poma serum, insulin, transferrin, lactoferrin, ethanolamine, sodium selenite, sodium monothioglycerol, Melka but-ethanol, ⁇ shea serum albumin, pyruvic Sanna door Li Umm, polyethylene grayed recall, various vitamins, various amino acids, C0 2 is, usually, is a 4 ⁇ , arbitrariness preferred 5%.
- the hematopoietic stem cells or hematopoietic progenitor cells produced as described above can be used as a transplant for blood cell transplantation instead of conventional bone marrow transplantation and umbilical cord blood transplantation.
- Construction Blood stem cell transplantation can improve conventional blood cell transplantation therapy because the transplant is semi-permanently engrafted.
- Hematopoietic stem cell transplantation can be used for various diseases in addition to these treatments when performing systemic X-ray therapy or advanced chemotherapy for leukemia.
- a treatment that causes bone marrow suppression as a side effect such as chemotherapy or radiation therapy
- bone marrow is collected before the operation, and hematopoietic stem cells and hematopoietic progenitor cells are expanded in vitro.
- hematopoietic disorders due to side effects can be recovered early, and stronger chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved.
- hematopoietic insufficiency caused by bone marrow hypoplasia exhibiting anemia such as aplastic anemia can be improved.
- Other diseases for which hematopoietic stem cell transplantation by the method of the present invention is effective include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, acquired immunodeficiency syndrome (AIDS), and the like.
- Immunodeficiency syndrome group thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage disease such as Gaucher disease and mucopolysaccharidosis, adrenal white matter degeneration, various cancers or tumors, etc.
- congenital anemia such as sickle cell disease
- lysosomal storage disease such as Gaucher disease and mucopolysaccharidosis
- adrenal white matter degeneration various cancers or tumors, etc.
- Transplantation of hematopoietic stem cells may be performed in the same manner as conventional bone marrow transplantation or umbilical cord blood transplantation, except for the cells used.
- hematopoietic stem cells that may be used for hematopoietic stem cell transplantation as described above is not limited to bone marrow, and stem cells can be obtained by administering fetal liver, fetal bone marrow, peripheral blood, cytokines, and / or anticancer drugs as described above. Mobilized peripheral blood, umbilical cord blood, and the like can be used.
- the transplant may be a composition containing a buffer solution or the like in addition to the hematopoietic stem cells and hematopoietic progenitor cells produced by the method of the present invention.
- hematopoietic stem cells or hematopoietic progenitor cells produced by the present invention can be used for ex vivo gene therapy.
- a foreign gene (therapeutic gene) is introduced into hematopoietic stem cells or hematopoietic progenitor cells, and the resulting transfected cells are used. Done.
- the foreign gene to be introduced is appropriately selected depending on the disease.
- Diseases targeted by gene therapy targeting blood cells include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, and acquired immune deficiency syndrome (AIDS).
- Examples include immunodeficiency syndrome, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage diseases such as Gaucher disease and mucopolysaccharidosis, adrenal leukemia, various cancers and tumors, etc. .
- retrovirus vector such as Moroni murine leukemia virus, adenovirus vector, adeno-associated virus (AAV) Vectors, simple herpes virus vectors, vector vectors for animal cells used in gene therapy for viruses derived from HIV vectors, etc.
- AAV adeno-associated virus
- retrovirus vectors see Verma, I.M. , Nature, 389: 239, 1997), calcium phosphate coprecipitation method, DEAE-dextran method, electoral poration method, liposome method, lipofection method, microinjection method, and the like.
- a retrovirus vector, an adeno-associated virus vector, or an HIV vector is preferable, since the gene can be expected to be permanently expressed by being integrated into the chromosomal DNA of the target cell.
- an adeno-associated virus (AAV) vector can be constructed as follows. First, 293 cells were transfected with a vector plasmid containing a therapeutic gene inserted between the ITRs (inverted termina 1 repeats) at both ends of the wild-type adeno-associated virus DNA, and helper plasmid for complementing the viral proteins. I do. Subsequently, when infected with an adenovirus of a helper virus, virus particles containing the AAV vector are produced. Alternatively, instead of adenovirus, a plasmid expressing an adenovirus gene responsible for helper function may be transfected.
- the obtained virus particles are used to infect hematopoietic stem cells or hematopoietic progenitor cells. It is preferable to insert an appropriate promoter and enhancer upstream of the target gene in the vector DNA, and to regulate the expression of the gene by these. Furthermore, when a marker gene such as a drug resistance gene is used in addition to the therapeutic gene, it becomes easy to select cells into which the therapeutic gene has been introduced. Therapeutic genes are sense sense It may be a gene or an antisense gene.
- the composition for gene therapy may be a composition containing a buffer, a novel active substance, and the like, in addition to the hematopoietic stem cells and hematopoietic progenitor cells produced by the method of the present invention.
- FIG. 1 is a diagram showing a construction procedure of pFLAG-CMV2-HES-1.1.
- FIG. 2 is a diagram showing a procedure for constructing pFLAG-CMV2-HES-1.2.
- FIG. 3 is a diagram showing a procedure for constructing pEGFP-CI-HES-1.
- FIG. 4 is a diagram showing a procedure for constructing pMSCV-EH. BEST MODE FOR CARRYING OUT THE INVENTION
- all antibodies used for cell separation were purchased from Pharmingen.
- All the restriction enzymes used for gene recombination were purchased from Behringer Mannheim. Separation of each blood cell was generally carried out by Sea, L.A., "Weir, s Handbook of Experimental Immunology, 5th edition", Blackwell Science Inc. 1997.
- Bone marrow cells, spleen cells, and thymocytes are separated from 6- to 8-week-old C57B1 / 6 mice (purchased from Nippon Charls River Co., Ltd.), and mononuclear cells and granulocytes are obtained using Celso overnight. , Mature T cells, immature T cells, mature B cells, hematopoietic progenitor cells, and hematopoietic stem cells.
- Anti-CD32 antibody was added to bone marrow cells removed from mouse femur bone marrow and placed on ice. After standing for 10 minutes, FITC-labeled anti-Mac-1 antibody and PE-labeled anti-Gr-1 antibody were added, and reacted on ice for 30 minutes.
- the cells were washed twice with a staining buffer (PBS (phosphate buffered saline), 5% FCS (CS fetal serum), 0.05% NaN 3 ), suspended in the staining buffer, and then suspended in a cell sorter (FACS Vantage, Mononuclear cells (Mac-1 positive 'Gr-1 negative cells) and granulocytes (Mac-1 positive and Gr-1 positive cells) were separated using Becton Dickinson.
- a staining buffer PBS (phosphate buffered saline), 5% FCS (CS fetal serum), 0.05% NaN 3
- FACS Vantage Mononuclear cells (Mac-1 positive 'Gr-1 negative cells) and granulocytes (Mac-1 positive and Gr-1 positive cells) were separated using Becton Dickinson.
- the thymocyte suspension is overlaid on a high-density cell separation solution (Nycomed, Lymphoprep), centrifuged at 1500 rpm for 30 minutes at 25 ° C, and cells collected at the interface between the suspension and Lymphoprep are removed. Collected. After the cells were washed twice with a staining buffer, an anti-CD32 antibody, a FITC-labeled anti-CD4 antibody, and a PE-labeled anti-CD8 antibody were added, and reacted on ice for 30 minutes. After the reaction, the cells were washed twice with a staining buffer and suspended in a staining buffer. Then, immature T cells (CD4 negative ⁇ CD8 negative cells and CD4 positive ⁇ CD8 positive cells) and mature T cells (CD4 positive ⁇ CD8-negative cells and CD4-negative and CD8-positive cells).
- a staining buffer an anti-CD32 antibody, a FITC-labeled anti-CD4 antibody, and
- the suspension of spleen cells was overlaid on Lymphoprep (Nycomed) and centrifuged at 1500 rpm at 25 ° C for 30 minutes to collect the cells collected at the interface. Wash the cells twice with staining buffer, add anti-CD32 antibody, and leave on ice for 10 minutes, then add FITC-labeled anti-B220 antibody and PE-labeled anti-mouse IgM antibody, and on ice for 30 minutes Reacted. After the reaction, the cells were washed twice with the staining buffer, suspended in the staining buffer, and then mature B cells (B220-positive-IgM-positive cells) were separated using a cell sorter.
- the bone marrow cell suspension was overlaid on Lymphoprep (Nycomed) and centrifuged at 1500 rpm at 25 C for 30 minutes to collect cells that had collected at the interface. After washing the cells twice with the staining buffer, the cells were washed twice with PBS and suspended in the staining buffer.
- An antibody against the biotinylated differentiation antigen marker ie, an anti-CD4 antibody, an anti-CD8 antibody, an anti-B220 antibody, an anti-Gr-1 antibody, and an anti-Tell19 antibody, were added to the cell suspension, and the mixture was left on ice for 30 minutes.
- avidin-coated magnetic beads (Avidinma Gnet beads (Perseptive)) and left on ice for 30 minutes.
- avidin magnet beads were collected using a magnet, and the cells presenting the differentiation antigen were removed to obtain differentiated antigen-negative cells (Lin-).
- a FITC-labeled anti-CD34 antibody, a PE-labeled anti-Sea-1 antibody, a Texas red-labeled avidin, and an APC-labeled anti-C-Kit antibody were added to the differentiation antigen-negative cell solution, and left on ice for 30 minutes.
- hematopoietic progenitor cells (Sea-1 negative, C-kit positive cells and CD34 positive, Sca-1 positive, C-kit positive cells) and hematopoietic stem cells (Selso overnight) CD34 negative to weak positive ⁇ Sea-1 positive ⁇ c-kit positive cells).
- RNA was obtained according to the instructions for use of the reagent.
- 5 units of DNase RNase free (GI BCO-BRL) were added, and the mixture was incubated at 37 ° C for 30 minutes to digest and degrade the mixed genomic DNA.
- RNA was obtained. From this MA, cDNA was synthesized using oligo dT as a primer. That, RNA corresponding to 10 5 cells to prepare a reaction liquid so as to correspond to 20 micro liters reaction.
- the composition of the reaction solution used was that recommended in the instruction manual for reverse transcriptase (Superscript IK GIBCO-BRL). The reaction was carried out at 42 ° C. for 60 minutes, and then the reverse transcriptase activity was inactivated by keeping the temperature at 72 ° C. for 10 minutes.
- sequences and annealing temperatures of the various primers used in the PCR reaction are as follows.
- Table 2 shows the results of evaluating the expression level based on the intensity of the amplified band.
- the symbols in the table are as follows.
- Notch- and Notch-2 were expressed in all of the examined hematopoietic cells.
- Notch-3 is confirmed to be cell type-specific expression in immature T cells, mature T cells, and B cells.
- Notch-4 was not expressed in hematopoietic cells.
- HES-1 HES-3 which is present downstream of the Notch / Delta signaling system, was expressed in all the cells examined.
- HES-5 was expressed in a cell type-specific manner in cells other than hematopoietic stem cells and hematopoietic progenitor cells.
- HES-1 Monitor expression and localization of HES-1 in cells transfected with retrovirus
- a vector was constructed so that HES-1 and a fluorescent protein, EGFP (Enhanced green fluorescent fluorescence protein, Clontech) were expressed as a fusion protein (see FIGS. 1 to 4).
- EGFP Enhanced green fluorescent fluorescence protein, Clontech
- PSV2CMVHES-1 (Kyoto University, distributed by Dr. Ryuichiro Kageyama, see Sasai, Y., Genes Dev., 6: 2620, 1992) is digested with EcoRI and EcoRI digested with pFLAG-CMV2 vector (Kodak)
- a subcloned PFLAG-CMV2-HES-1.1 (Fig. 1) was prepared in the same direction as the FLAG transfer direction.
- the translation initiation codon of HES-1 was changed, and the translation initiation codon of HES-1 was modified so that EGFP and HES-1 were expressed as a fusion protein, and changed to the Bglll site.
- a synthetic oligonucleotide (SEQ ID NO: 15) having a sequence that changes the translation start point ATG to Bglll, and an antisense oligonucleotide at a site located downstream from the Pstl site in the HES-1 gene. (SEQ ID NO: 16) was used as a primer, and PCR was performed using PSV2CMVHES-1 as type II. A fragment containing the HES-1 gene obtained by digesting this PCR product with Bglll and Pstl was cloned into Bglll and Pst-1 sites of pFLAG-CMV2-HES-1.1 described above. This brassmid was named PFLAG-CMV2-HES-1.2 (Fig. 2).
- a fragment containing HES-1 obtained by digesting PFLAG-CMV2-HES-1.2 with Bgll I and EcoRI was cloned into the Bglll / EcoRI site of pEGFP-CI (Clontech), and pEGFP-CI-HES- 1 ( Figure 3).
- a gene was constructed in which EGFP and HES-1 were on one transcription unit.
- a fragment containing HES-1 obtained by digesting pEGFP-CI-HES-1 with Eco47III and Sail was extracted from Hpal and Xhol using a PMSCV2.1 vector (obtained from Dr. R. Hawley, University of Toronto, Hawley , RG, Gene Ther., 1: 136, 199).
- This vector was named pMSCV-EH (Fig. 4). Thereafter, this plasmid was used for producing a retrovirus for HES-1 infection.
- a retrovirus vector into which only the EGFP gene was transferred was constructed.
- a fragment containing EGFP obtained by digesting pEGFP-CI with Eco47III and Sail was inserted into a PMSCV2.1 vector digested with Hpal and Xhol. This vector was pMSCV-E.
- mice Pregnant C57B1 / 6 mice were purchased from Nippon Charls River Inc. On the 14th day of pregnancy, the mouse was laparotomized and the fetus was aseptically removed. After carefully separating the fetal liver from contaminating other tissues, the cells were dispersed with a syringe fitted with a 21-gauge injection needle and layered on Lymphoprep (Nycomed). This was centrifuged at 1500 rpm at 25 ° C. for 30 minutes to collect cells collected at the interface. After washing the cells twice with PBS, the cells were suspended in a staining buffer (PBS, 5% FCS ⁇ 0.05% NaN 3 ).
- a staining buffer PBS, 5% FCS ⁇ 0.05% NaN 3
- the cell suspension was incubated with the differentiation antigen, which was biotinylated, that is, anti-CD8 antibody, anti-B220 antibody, anti-GII-l antibody, and anti-Terll9 antibody. And left on ice for 30 minutes. Then, after washing twice with a staining buffer, avidin magnet beads were added, and the mixture was left on ice for 30 minutes. After washing twice with the staining buffer again, remove the cells presenting the differentiation antigen using a magnet. Then, differentiated antigen-negative cells (Lin-) were obtained.
- the differentiation antigen which was biotinylated, that is, anti-CD8 antibody, anti-B220 antibody, anti-GII-l antibody, and anti-Terll9 antibody.
- FITC-labeled anti-CD34 antibody PE-labeled anti-Sca-1 antibody, Texas Red-labeled avidin, and APC-labeled anti-c-kit antibody were added to the differentiation antigen-negative cell solution, and left on ice for 30 minutes. After washing twice with the staining buffer, Lin-Sea-1 + c-kit + cells were selected on a cell saw.
- the cells were collected and examined for colony forming ability (the colony forming ability was determined by adding 0.9% methylcellulose, 30% FCS, 0.1% serum albumin, and 10 ng / ml SCF
- the culture was performed in the presence of I-3, IL-6, EP0, TP0, and 0.5 mg / ml G418.
- the morphology and number of emerging colonies were detected.
- the various hematopoietic factors used in the above are all recombinant and pure.
- Table 3 shows the results under the above conditions.
- the numbers in the table are the number of colonies per 2,000 infected hematopoietic stem cells.
- Table 3 Morphology of infected lettuce wiwi colonies (/ 2,000 hematopoietic stem cells)
- the number of colonies shown in the table is a colony resistant to G418, and indicates only the EGFP gene alone or only the cell into which the EGFP-HES-1 fusion gene has been introduced. Furthermore, the expression of the target gene in these colonies was also confirmed by observing the expression of EGFP under a fluorescence microscope. In cells transfected with the EGFP gene alone, the entire cytoplasm fluoresced, and no localization in the nucleus was confirmed. On the other hand, in the strain into which the EGFP-HES-1 fusion gene was introduced, it was confirmed by fluorescence microscopy that the nucleus emitted specific fluorescence, and this HES-1 was normally expressed and functioned in the nucleus. Was speculated to be.
- hematopoietic stem cells differentiate and only hematopoietic progenitor cells GM-CFC, G-CFC, and M-CFC appear.
- HES-1 hematopoietic progenitor cells
- undifferent hematopoietic cells were maintained, and the differentiation of hematopoietic stem cells was successfully regulated by HES-1 gene transfer.
- INDUSTRIAL APPLICABILITY According to the present invention, hematopoietic stem cells and / or hematopoietic progenitor cells can survive and proliferate in a state where differentiation is suppressed.
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Abstract
A method for regulating the differentiation and proliferation of mammalian hematopoietic stem cells and/or hematopoietic progenitor cells differentiated therefrom which comprises enhancing the expression of differentiation repressing genes in the hematopoietic stem cells by, for example, transferring thereinto a differentiation repressing gene integrated into a virus vector and, at the same time, treating the cells with blood cell stimulators.
Description
明細書 造血幹細胞の分化 ·増殖調節方法 技術分野 本発明は、 造血幹細胞及び/または造血前駆細胞の分化および増殖を調節する 方法、 及びその方法に用いるベクター及び細胞に関する。 造血幹細胞及び造血前 駆細胞は、 骨髄移植や臍帯血移植に代わる血液細胞移植、 あるいは遺伝子治療に 用いることができる。 背景技術 生体内を流れる成熟血液細胞は、 短期間の寿命 （ヒトでは赤血球で約 1 2 0日、 血小板で約 7日） しかなく、 成熟血液細胞は造血前駆細胞から毎日分化増殖する ことによって、 末梢血の成熟血液細胞の恒常性を保っている。 末梢血に供給され る成熟血液細胞の数は、 ヒ卜の場合赤血球で 2 0 0 0億個/日、 好中球で 7 0 0 億個/日にもなる。 これらの各分化系列の前駆細胞は、 さらに未分化な造血幹細 胞から分化しながら増殖することで恒常的に末梢血液細胞が枯渴しないようなシ ステムができている （須田年生、 血液幹細胞の運命、 羊土社、 1 9 9 2 ) 。 TECHNICAL FIELD The present invention relates to a method for regulating the differentiation and proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells, and to vectors and cells used in the method. Hematopoietic stem cells and hematopoietic progenitor cells can be used for blood cell transplantation instead of bone marrow transplantation and cord blood transplantation, or for gene therapy. BACKGROUND ART Mature blood cells flowing in the living body have a short life span (about 120 days for red blood cells and about 7 days for platelets in humans), and mature blood cells are differentiated and proliferated daily from hematopoietic progenitor cells. Maintains homeostasis of peripheral blood mature blood cells. The number of mature blood cells supplied to peripheral blood is 200 billion cells / day for red blood cells and 700 billion cells / day for neutrophils in humans. The progenitor cells of each of these differentiation lineages proliferate while differentiating from undifferentiated hematopoietic stem cells to form a system in which peripheral blood cells do not constantly die (Tsuda Suda, Blood Stem Cells Destiny, Sheepsha, 199 2).
このような血液細胞の分化系については、 マウスを用いた実験系においてその 性質が明らかにされてきた。 The nature of such a blood cell differentiation system has been clarified in an experimental system using mice.
末梢血を流れる種々の成熟血液細胞が、 骨髄に存在する造血幹細胞に由来する ことが明らかにされたのは、 Ti l lと McCul lochの研究による（Ti l l , J . E.， Rad. Res . , 14 : 213-222, 1961 )。 放射線を照射して骨髄の造血系を破壊したマウスに他 のマウス由来の骨髄細胞を移植したところ、 脾臓に血液細胞からなるコロニーの 形成（CFU-S ; colony- forming- unit spleen)が確認された。 このコロニーの数は移 植する骨髄細胞の数に比例すること、 さらに一個の細胞に由来するコロニーの中 に多くの血球系の細胞の存在が確認されることから、 多くの血液細胞種に分化で きる多分化能を持つ造血幹細胞 （多能性幹細胞） が骨髄中に存在することが明ら かにされたのである。
その後、 造血細胞の性質を解析する手段として放射線照射マウスを用いた移植 実験系、 さらには、 in vitro (インビトロ） のコロニー形成法（Bradley, T.R., J. Exp. Med., 44: 287-299, 1966)が開発され、 造血幹細胞および、 造血前駆細 胞の分化に関する知見が蓄積されてきた。 Research by Till and McCulloch revealed that various mature blood cells flowing in peripheral blood were derived from hematopoietic stem cells present in the bone marrow (Till, JE, Rad. Res. , 14: 213-222, 1961). When bone marrow cells from other mice were transplanted into mice whose bone marrow hematopoietic system was destroyed by irradiation, colony formation of blood cells in the spleen (CFU-S; colony-forming-unit spleen) was confirmed. Was. The number of these colonies is proportional to the number of bone marrow cells to be transplanted.Moreover, the presence of many hematopoietic cells in a colony derived from a single cell confirms the differentiation into many blood cell types. It has been revealed that hematopoietic stem cells (pluripotent stem cells) with the potential for pluripotency exist in the bone marrow. Then, as a means of analyzing the properties of hematopoietic cells, a transplantation experiment system using irradiated mice, and an in vitro (in vitro) colony formation method (Bradley, TR, J. Exp. Med., 44: 287-299). , 1966), and knowledge on the differentiation of hematopoietic stem cells and hematopoietic progenitor cells has been accumulated.
放射線照射マウスを用いた移植実験系は、 最も直接的に造血幹細胞の性質を解 析する手法である。 放射線照射し造血系に障害を与えたマウス （レシピエント） に、 他のマウス （ドナ一） から分離した骨髄細胞を移植すると、 レシピエントマ ウス中にドナー由来の造血系を再構築することができる。 造血細胞に発現される 各種分化抗原（Spangrude, G.J., Proc.Natl.Acad.Sci.U.S.A., 87:7433-7437, 1 990; Visser, J.M.W. , 「Flow cytometry in hematology」， Academic Press, p 9-29， 1992)を利用して、 あるいは、 造血細胞の大きさ（Jones, R.J., Nature, 3 47: 188-189, 1990)を利用して、 あるいは、 細胞の性質、 状態によって染色性の 異なる蛍光物質等（Rhl23， Hoechst 33342) (Wolf, N.S., Exp. Hematol., 21:61 4-622, 1993)を用いて、 骨髄細胞を分画し上記の移植実験を行うことで、 骨髄細 胞中の造血幹細胞を同定する試みがなされてきた。 これまでの知見から、 造血幹 細胞は一個の細胞を移植しただけでも、 リンパ球系の細胞にも骨髄球系の細胞に も分化することができ、 かつ、 長期にわたり移植先個体の中で造血系を構築する ことができることも明らかにされている （Osawa, M., Science, 273: 242-245, 1996)。 造血幹細胞は、 移植先の個体で長期間にわたり生着し、 分化血球を供給す ることができるのである。 一方、 造血前駆細胞を用いた移植実験では、 短期間に 移植先個体から移植した細胞群が消失してゆき長期にわたり造血をになうことが できない (Osawa, M.， Science, 278: 242-245, 1996) 。 The transplantation experiment system using irradiated mice is the most direct way to analyze the properties of hematopoietic stem cells. Transplantation of bone marrow cells isolated from another mouse (Donna 1) into a mouse (recipient) that has damaged the hematopoietic system by irradiation may reconstitute the donor-derived hematopoietic system in the recipient mouse. it can. Various differentiation antigens expressed on hematopoietic cells (Spangrude, GJ, Proc. Natl. Acad. Sci. USA, 87: 7433-7437, 1990; Visser, JMW, "Flow cytometry in hematology", Academic Press, p 9- 29, 1992), or the size of hematopoietic cells (Jones, RJ, Nature, 347: 188-189, 1990), or the fluorescence that differs in staining depending on the nature and state of the cells. Bone marrow cells were fractionated using substances (Rhl23, Hoechst 33342) (Wolf, NS, Exp. Hematol., 21:61 4-622, 1993) and the transplantation experiments described above were performed. Attempts have been made to identify hematopoietic stem cells. Based on the findings so far, hematopoietic stem cells can differentiate into lymphoid cells and myeloid cells by transplanting a single cell, and hematopoietic stem cells can be transplanted for a long time It has also been demonstrated that systems can be constructed (Osawa, M., Science, 273: 242-245, 1996). Hematopoietic stem cells can survive over a long period of time in the recipient individual and supply differentiated blood cells. On the other hand, in transplantation experiments using hematopoietic progenitor cells, the cells transplanted from the recipient individual disappear in a short period of time and cannot undergo hematopoiesis for a long period of time (Osawa, M., Science, 278: 242- 245, 1996).
近年、 種々のサイ トカインが取得され、 in vitroで形成されるコロニー形態か ら、 血液細胞の性質を解析できるようになった （Ogawa, M.， Blood, 81:2844-28 53, 1993)。 造血前駆細胞は、 多くのサイ ト力インが存在しても、 限られた分化系 列の成熟血液細胞にしか分化することはできないが、 移植マウスで長期の造血系 を構築できる造血幹細胞は、 多くの細胞分化系列に分化することが可能である。 これらの結果から、 造血幹細胞から末梢血を流れる成熟血液細胞までの分化は、 以下のように解釈されている。 造血幹細胞は、 各種分化系列に分化可能な多分化
能を有しており、 かつ、 この多分化能の性質 （分化多能性） を保持したまま自己 複製することが可能である。 造血幹細胞は、 自己複製するとともに一部は分化し、 各種サイ ト力インの作用を受け、 次第に分化できる細胞系列が狭まり、 限られた 細胞種へしか分化できない造血前駆細胞へと分化増殖し、 最終的に成熟血液細胞 になる (Hematopoietic Stem Cel ls, Levitt, D . , Marcel Dekker, Inc. , 1995 ) 。 骨髄移植療法、 臍帯血移植療法は患者に造血細胞を移植する治療法であるが、 移植後長期に治療効果が持続するかは、 前述のように移植した造血幹細胞が移植 先の患者に生着できるかに関わる。 In recent years, various cytokines have been obtained, and the properties of blood cells can be analyzed from the morphology of colonies formed in vitro (Ogawa, M., Blood, 81: 2844-2853, 1993). Although hematopoietic progenitor cells can only differentiate into mature blood cells of a limited lineage even in the presence of many cytokins, hematopoietic stem cells that can build a long-term hematopoietic system in transplanted mice It is possible to differentiate into many cell lineages. From these results, the differentiation from hematopoietic stem cells to mature blood cells flowing in peripheral blood is interpreted as follows. Hematopoietic stem cells can be differentiated into various lineages It is capable of self-replication while maintaining this pluripotency property (pluripotency). Hematopoietic stem cells self-renew and partially differentiate, are affected by various site forces, gradually narrow the lineage of cells that can be differentiated, differentiate and proliferate into hematopoietic progenitor cells that can only differentiate into a limited number of cell types, It eventually becomes a mature blood cell (Hematopoietic Stem Cell, Levitt, D., Marcel Dekker, Inc., 1995). Bone marrow transplantation and umbilical cord blood transplantation are therapies to transplant hematopoietic cells to patients. It depends on what you can do.
先天性の遺伝子疾患患者に対し、 欠失あるいは、 変異遺伝子を相補する遺伝子 治療の試みがなされている （大橋十也、 実験医学、 1 2 : 3 3 3、 1 9 9 4 ) 。 このような遺伝子治療においては、 造血幹細胞は、 前述のように移植先個体で長 期に生存しうることから、 最適な標的として考えられている。 すなわち、 造血幹 細胞の遺伝的な欠損を補完する遺伝子治療は、 疾患の根本的な治療になると考え られている。 Attempts have been made in gene therapy to complement deletion or mutation genes in patients with congenital genetic diseases (Johya Ohashi, Experimental Medicine, 12: 3333, 1994). In such gene therapy, hematopoietic stem cells are considered to be an optimal target because they can survive for a long time in the transplant recipient as described above. In other words, gene therapy that complements the genetic deficiency of hematopoietic stem cells is considered to be a fundamental treatment for the disease.
このように、 造血幹細胞の有用性が臨床的に認識されており、 造血幹細胞をい かに未分化のままで増殖させるかが課題となっている。 造血幹細胞をサイ トカイ ンゃ血液細胞刺激因子を用いて増殖させる試みがなされてきたが、 効率的に造血 幹細胞を増殖させることに成功するに至っていない （Trevisan, M. , Blood, 88 : 4149 , 1996 ) 。 これらの培養系では造血幹細胞が分化し、 造血前駆細胞の増殖が 優位になっている。 そこで、 造血幹細胞の分化を抑制することができれば、 サイ トカイン、 造血細胞刺激因子の存在下で造血幹細胞を増殖させることも可能にな ると思われる。 Thus, the usefulness of hematopoietic stem cells has been clinically recognized, and it has been an issue how to grow hematopoietic stem cells without differentiation. Attempts have been made to expand hematopoietic stem cells using cytokin-blood cell stimulating factors, but have not succeeded in efficiently expanding hematopoietic stem cells (Trevisan, M., Blood, 88: 4149, 1996). In these culture systems, hematopoietic stem cells are differentiated and proliferation of hematopoietic progenitor cells is dominant. Therefore, if the differentiation of hematopoietic stem cells could be suppressed, hematopoietic stem cells could be expanded in the presence of cytokines and hematopoietic cell stimulating factors.
造血系の細胞の分化を人為的に調節しょうとするとき、 造血系以外の細胞の分 化の制御様式を調べることも重要と考えられる （ Morrison, S . J . , Cel l , 88 :28 7, 1997)。 近年、 ショウジヨウバエの発生分化の研究を通して、 分化の調節を行 う分子群が存在することが明らかにされてきた。 Notch/Del ta (ノッチ/デル夕） を介したシグナル伝達系はこれら分化調節をになうシグナル伝達系の 1つである。 When trying to artificially regulate the differentiation of hematopoietic cells, it is also important to examine the mode of differentiation of non-hematopoietic cells (Morrison, S.J., Cell, 88: 287. , 1997). In recent years, studies on the development and differentiation of Drosophila have revealed the existence of a group of molecules that regulate differentiation. The signaling system via Notch / Delta is one of these signaling systems that regulate differentiation.
Notch/Deltaは、 ショウジヨウバエの胚発生時の神経、 翅などの種々の器官の形 成に関わっていることが遺伝学的な解析により明らかにされてきた（Artavanis-T
sakonas, S.， Science, 268 : 225 , 1995 )。 リガンドである Delta (デルタ） 蛋白質 は受容体である Notch (ノッチ） 蛋白質と結合し、 Notchを通して分化を抑制する ようなシグナルを伝達する。 また、 哺乳類においても、 Notch/Delta遺伝子フアミ リーの相同遺伝子が知られている （ Blaumuel ler, C . M. , Perspectives on Dev elopmental Neurobiology, 4 : 325 , 1997) 。 さらには、 Notch/Deltaのシグナル伝 達を細胞内で担う分子についても、 ショウジヨウバエの相同遺伝子が哺乳類にお いても見出されており （Artavanis- Tsakonas, S.， Science, 268 : 225 , 1995) 、 Notch/Delta系の分化調節機構は、 基本的には昆虫から哺乳類までほぼ似通った経 路で行われるものと考えられる。 Notch / Delta has been shown by genetic analysis to be involved in the formation of various organs such as nerves and wings during embryo development in Drosophila (Artavanis-T sakonas, S., Science, 268: 225, 1995). The ligand, Delta protein, binds to the receptor, Notch protein, and transmits signals through Notch to suppress differentiation. Also, in mammals, homologous genes to the Notch / Delta gene family are known (Blaumueller, CM, Perspectives on Developmental Neurobiology, 4: 325, 1997). Furthermore, a homologous gene of Drosophila has also been found in mammals for the molecule responsible for Notch / Delta signal transmission in cells (Artavanis-Tsakonas, S., Science, 268: 225, 1995), it is thought that the mechanism of regulating the differentiation of the Notch / Delta system is basically performed by a similar route from insects to mammals.
神経細胞分化における Notchシグナル伝達系による分化制御は、 以下のように考 えられている (Artavanis - Tsakonas, S.， Sci ence, 268 : 225 , 1995 ; Simpson, P . , Perspectives on Developmental Neurobiology, 4 : 297, 1997) 。 胚発生時に 背腹軸、 前後軸が決定されてくると、 外胚葉に神経芽細胞、 及び表皮細胞に分化 が可能な細胞集塊 （proneural cluster) が出現する。 この細胞集塊の中から神経 細胞に分化可能な細胞が選別される際に、 Notch/Delta系のシグナル伝達系が重要 な役割を果たす。 この細胞集塊に存在する細胞は等しく Notchを発現しているが、 一部の細胞が Notchリガンドを発現するようになり、 その細胞は隣接する細胞が神 経細胞に分化することを抑制する （側方抑制） 。 Notchリガンドを発現する細胞は、 神経芽細胞へと分化し、 さらに種々の刺激を受けて分化を続けることで機能的な 神経細胞へ終末分化を遂げる。 一方、 Notchリガンドと結合することで Notch/Del taシグナル伝達系が活性化された細胞は、 神経細胞に分化することができず、 表 皮細胞へと分化していく。 The regulation of differentiation by the Notch signaling system in neural cell differentiation is considered as follows (Artavanis-Tsakonas, S., Science, 268: 225, 1995; Simpson, P., Perspectives on Developmental Neurobiology, 4). : 297, 1997). When the dorsal-ventral axis and anterior-posterior axis are determined during embryonic development, neuroblasts appear in the ectoderm and proneural clusters that can differentiate into epidermal cells appear. The Notch / Delta signaling system plays an important role in selecting cells capable of differentiating into neurons from this cell cluster. The cells present in this cell cluster express Notch equally, but some cells begin to express Notch ligand, which suppresses neighboring cells from differentiating into neural cells ( Lateral suppression). Cells that express Notch ligand differentiate into neuroblasts, and undergo terminal differentiation under various stimuli to achieve terminal differentiation into functional neurons. On the other hand, cells in which the Notch / Delta signaling system has been activated by binding to Notch ligand cannot differentiate into neurons, but differentiate into epidermal cells.
Notch/Deltaシグナル伝達系が機能できない変異を持つ個体では、 神経細胞への 分化が抑制されないため、 神経細胞が過形成されることになる。 逆に、 細胞外領 域がほとんど欠失している、 あるいは細胞領域内のみが夕ンパクとして発現する ようになった活性化型 Notch/Deltaを発現する個体では神経細胞への分化が抑制さ れている （Artavanis-Tsakonas, S.， Sci ence , 268 : 225 , 1995) 。 In individuals with mutations in which the Notch / Delta signal transduction system cannot function, neuronal cell hyperplasia occurs because differentiation into neuronal cells is not suppressed. Conversely, in an individual expressing an activated Notch / Delta in which the extracellular region is almost completely deleted or only the intracellular region is expressed as protein, differentiation into neural cells is suppressed. (Artavanis-Tsakonas, S., Science, 268: 225, 1995).
さらに、 細胞内における Notch/Delta系のシグナル伝達については以下のように 考えられている。 Notchは、 Deltaと結合することで活性化され、 Notchの細胞内領
域を介して、 核移行タンパクである RBP- J/c(CBF- 1、 KBF-2ともいう） と相互作用 する （Honjo, T.， Genes to Cells, 1:1， 1996)。 RBP- J/ は、 Notch活性化にした がい核内へ移行し、 HLH (ヘリックス—ループ一ヘリックス） 転写因子である HES- 1 (影山 龍一郎、 生化学、 67 : 1093、 1995) の発現を誘導する。 HLH 型の転写因 子は、 3つのドメイン構造からなる転写因子で、 つまり、 1 ) ヘリ ックス一ループ 一ヘリックスの立体構造を形成する部分で自身との結合あるいは他の HLH型の転写 因子との結合により、 ホモダイマ一あるいはヘテロダイマ一を形成するための結 合部分、 2 ) 塩基性の DNAとの結合能を有する部分、 3 ) 転写促進活性を有する部 分、 よりなる。 HLH型転写因子の特徴は、 DNA結合能がヘテロダイマ一を形成する 相手によって調節されることにある。 Furthermore, the signaling of the Notch / Delta system in cells is considered as follows. Notch is activated by binding to Delta, Interacts with the nuclear localization protein RBP-J / c (CBF-1, also called KBF-2) through the region (Honjo, T., Genes to Cells, 1: 1, 1996). RBP-J / translocates into the nucleus following Notch activation and induces expression of the HLH (helix-loop-helix) transcription factor HES-1 (Ryuichiro Kageyama, Biochemistry, 67: 1093, 1995) I do. An HLH-type transcription factor is a transcription factor having a three-domain structure, that is, 1) a helix-one-loop part that forms a three-dimensional structure of one helix, which binds to itself or interacts with other HLH-type transcription factors. It comprises a binding portion for forming a homodimer or a heterodimer by binding, 2) a portion capable of binding to basic DNA, and 3) a portion having a transcription promoting activity. A characteristic of HLH-type transcription factors is that their DNA binding ability is regulated by the partner that forms the heterodimer.
一方、 神経細胞の分化を正に調節する HLH型転写因子として、 Mash- 1、 MATHが知 られている。 これら Mash-1、 MATHはともに HLH型の転写因子で、 普遍的に存在する HLH型転写因子 E 12/E47とへテロダイマーを形成し、 特異な塩基配列を認識して神 経細胞の分化を促進する遺伝子群を活性化する （Johnson, J.E., Proc. Natl. Ac ad. Sci. U.S.A., 89:3596, 1992)。 HES- 1は、 E12/E47と拮抗的に Mash-1、 MATHと 結合しその DM結合活性を抑制することで、 神経細胞への分化を促進する遺伝子の 発現を阻害することによって、 神経細胞への分化を抑制するものと考えられてい る （Sasai, Y.， Genes Dev., 6:2620, 1992)。 On the other hand, Mash-1 and MATH are known as HLH-type transcription factors that positively regulate the differentiation of nerve cells. Both Mash-1 and MATH are HLH-type transcription factors that form heterodimers with the ubiquitous HLH-type transcription factor E12 / E47, and recognize specific nucleotide sequences to promote neuronal cell differentiation. Activating genes (Johnson, JE, Proc. Natl. Acc. Sci. USA, 89: 3596, 1992). HES-1 binds to Mash-1 and MATH in a competitive manner with E12 / E47 and suppresses its DM binding activity, thereby inhibiting the expression of genes that promote differentiation into nerve cells, and It is thought to suppress the differentiation of cells (Sasai, Y., Genes Dev., 6: 2620, 1992).
上記のように、 HES-1は、 Notch/Delta系の分化調節において最終段階で分化を 正の方向に調節する転写因子の活性を阻害するという重要な役割を果たしている。 HES- 1類似遺伝子である HES-3、 HES-5にも同様な調節機構が類推されている。 （影 山 龍一郎、 生化学、 67： 1093、 1 99 5 ) As described above, HES-1 plays an important role in regulating the differentiation of the Notch / Delta system by inhibiting the activity of transcription factors that positively regulate differentiation at the final stage. Similar regulatory mechanisms have been inferred for the HES-1 similar genes HES-3 and HES-5. (Ryuichiro Kageyama, Biochemistry, 67: 1093, 19995)
一方、 骨格筋系の分化では Notch/Delta系による分化の調節がなされているが、 HES-1遺伝子を骨格筋芽細胞に直接導入し発現させても、 骨格筋細胞への分化は抑 制されず、 RBP-J cから HESフアミ リ一遺伝子へのシグナル伝達系とは異なった分 化調節機構が推測されている (Shawber, C.， Development, 122： 3765, 1996)。 つまり、 全ての細胞種において、 HES-1を介して分化制御が行われているのではな い。 On the other hand, the differentiation of the skeletal muscle system is regulated by the Notch / Delta system, but even if the HES-1 gene is directly introduced into and expressed in skeletal myoblasts, differentiation into skeletal muscle cells is suppressed. However, a different regulation mechanism from the signal transduction system from RBP-Jc to the HES family gene has been speculated (Shawber, C., Development, 122: 3765, 1996). In other words, differentiation control is not performed through HES-1 in all cell types.
血液細胞における Notch遺伝子の機能の解析は、 Notch- 1遺伝子の転座により細
胞外領域のほとんどが欠失した Notch-1遺伝子 （TAN-1ともいう） が T細胞の白血 病化を引き起こすことが明らかとなったことに始まる （Ellison, L.W., Cell, 6 6:649, 1991; Reynolds, T. C, Cell, 50:107, 1987) 。 そして、 活性化型の No tchを導入されたトランスジエニックマウスでは、 未熟な T細胞の性質を持つ白血 病が生じることから、 T細胞の増殖制御に関与することが報告されている （Pear, W. S., J. Exp. Med. ,183:2283, 1996)。 その後 T細胞の分化の過程で、 実際に Notchが CD4+CD8—、 CD4— CD8+の表現形を持つ成熟 T細胞の分化決定に関わることも 明らかにされた （Robey, E.， Cell, 87:483, 1996)。 さらに、 前骨髄球細胞株の 分化を Notchシグナル伝達系が阻害することが報告されている （Milner, L.， Pro c. Natl. Acad. Sci. USA, 93:13014, 1997) 。 Analysis of the function of the Notch gene in blood cells is more detailed by translocation of the Notch-1 gene. It begins with the discovery that the Notch-1 gene (also known as TAN-1), in which most of the extracellular region has been deleted, causes T cell leukemia (Ellison, LW, Cell, 66: 649, 1991; Reynolds, TC, Cell, 50: 107, 1987). It has been reported that transgenic mice transfected with an activated Notch gene are involved in the control of T cell proliferation because leukemia with immature T cell properties is produced (Pear, WS, J. Exp. Med., 183: 2283, 1996). In the course of differentiation of subsequent T cells, actually Notch is CD4 + CD8-, were also revealed be involved in the differentiation determination of mature T cells with CD4- CD8 + phenotype (Robey, E., Cell, 87 : 483, 1996). Furthermore, it has been reported that the differentiation of promyelocytic cell lines is inhibited by the Notch signaling system (Milner, L., Proc. Natl. Acad. Sci. USA, 93: 13014, 1997).
一方、 造血幹細胞では、 Notch遺伝子が造血幹細胞で発現されていることは報告 されているが（Milner, L. A.， Blood, 83:2057, 1994)、 造血幹細胞から各種の分 化細胞に分化する過程での Notch/Deltaの機能については今まで報告はない。 On the other hand, in hematopoietic stem cells, the Notch gene has been reported to be expressed in hematopoietic stem cells (Milner, LA, Blood, 83: 2057, 1994), but during the process of differentiating hematopoietic stem cells into various differentiated cells. The Notch / Delta feature has not been reported.
Deltaの哺乳類の相同遺伝子には Dll-1 (Chitnis, A., Nature, 375: 761, 199 5) 、 Dll-3 (Dunwoodie, S.L., Development, 124: 3065, 1997) ならびに Jagge d-1 (Lindsell, C.E., Cell, 80: 909， 1995) 、 Jagged-2 (Shauber, C.， Dev. Biolo., 180: 370, 1996) が報告されているが、 その造血幹細胞に対する作用の 報告はない。 一方、 Deltaに低いながら相同性を有する分子として、 Delta-like protein； DLK (SCP - 1、 Pref - 1ともいう) (Laborda, J., Jounal of Biological Chemistry, 268:3817, 1993)が造血系の細胞に作用するという報告がある （Moor e, K. A.， Proc Natl. Acad. Sci. U.S.A., 94:4011, 1997; W097/31647) 。 DLK を造血細胞を支持するス トローマ細胞に発現させ、 造血幹細胞分画と共培養を行 なうと、 造血前駆細胞の増殖を支持する活性が確認されている。 しかし、 膜タン パクである DLKを可溶化型にして、 DLKを直接造血幹細胞に作用させても活性は認 められない。 したがって、 DLKが造血前駆細胞に直接作用して分化を抑制する活性 を発揮しているのかは明らかでない。 さらに、 DLKは Deltaとの相同性が低いこと から、 DLKが Notchを介してシグナル伝達を行なうのかについても、 今のところ不 明である。
発明の開示 本発明は、 上記観点からなされたものであり、 造血幹細胞及び/または造血前 駆細胞の分化を抑制する方法、 ならびに分化を抑制した状態で生存させ、 好まし くはさらに増殖させる方法及びこれらの方法に用いる手段を提供することを課題 とする。 Dll-1 (Chitnis, A., Nature, 375: 761, 1995), Dll-3 (Dunwoodie, SL, Development, 124: 3065, 1997) and Jagge d-1 (Lindsell , CE, Cell, 80: 909, 1995) and Jagged-2 (Shauber, C., Dev. Biolo., 180: 370, 1996), but there is no report on its effects on hematopoietic stem cells. On the other hand, as a molecule having low homology to Delta, Delta-like protein; DLK (also called SCP-1 or Pref-1) (Laborda, J., Jounal of Biological Chemistry, 268: 3817, 1993) (Moore, KA, Proc Natl. Acad. Sci. USA, 94: 4011, 1997; W097 / 31647). When DLK is expressed in stromal cells that support hematopoietic cells and cocultured with hematopoietic stem cell fractions, activity to support the proliferation of hematopoietic progenitor cells has been confirmed. However, no activity has been observed when DLK, a membrane protein, is solubilized and DLK acts directly on hematopoietic stem cells. Therefore, it is not clear whether DLK exerts its activity of inhibiting differentiation by directly acting on hematopoietic progenitor cells. Furthermore, because DLK has low homology to Delta, it is unclear at this time whether DLK signals through Notch. DISCLOSURE OF THE INVENTION The present invention has been made in view of the above, and provides a method for suppressing the differentiation of hematopoietic stem cells and / or hematopoietic progenitor cells, and a method for allowing the cells to survive and preferably further proliferate in a state where the differentiation is suppressed. And to provide means used for these methods.
本発明者は、 造血幹細胞の分化制御に Notch/Delta系の分子を利用できないかと 考え、 まず、 実際に造血幹細胞を含む造血細胞で Notch-l、 Notch-2, Notch-3、 Notch- 4及び、 HES- 1、 HES-3、 HES- 5が発現しているかを詳細に調べた。 その結果、 Notch, HES遺伝子が、 種々の分化段階における造血細胞で広汎に発現しているこ とを世界で初めて確認した。 そして、 この発現状況から Notch/Del ta系のシグナル 伝達系が造血幹細胞からの細胞分化にも重要な役割を果たしていると推測した。 そこで、 HES-1遺伝子を造血幹細胞に導入することで造血幹細胞を未分化な状態に 留めて置くことも可能と考え、 造血幹細胞の分化調節に HES- 1を利用することを 検討した。 その結果、 HES- 1を造血幹細胞に強制発現させることで造血幹細胞の 分化を抑制することに成功し、 本発明を完成させるに至った。 The present inventors wondered whether Notch / Delta-based molecules could be used to control the differentiation of hematopoietic stem cells.First, Notch-l, Notch-2, Notch-3, Notch-4 and Notch-4 were actually used in hematopoietic cells including hematopoietic stem cells. , HES-1, HES-3 and HES-5 were examined in detail. As a result, it was confirmed for the first time in the world that Notch and HES genes were widely expressed in hematopoietic cells at various stages of differentiation. From this expression status, it was speculated that the signaling system of the Notch / Delta system plays an important role also in cell differentiation from hematopoietic stem cells. Therefore, it was considered possible to keep the hematopoietic stem cells in an undifferentiated state by introducing the HES-1 gene into the hematopoietic stem cells, and the use of HES-1 to regulate the differentiation of hematopoietic stem cells was studied. As a result, the differentiation of hematopoietic stem cells was successfully suppressed by forcibly expressing HES-1 in hematopoietic stem cells, and the present invention was completed.
すなわち本発明は、 哺乳動物の造血幹細胞中で分化抑制遺伝子の発現を強化す ると共に、 血液細胞刺激因子を作用させることにより、 前記造血幹細胞及び/ま たは前記造血幹細胞から分化した造血前駆細胞の分化および増殖を調節する方法 である。 That is, the present invention provides a method for enhancing the expression of a differentiation-suppressing gene in mammalian hematopoietic stem cells and causing a blood cell stimulating factor to act thereon, whereby the hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells are activated. It is a method of regulating the differentiation and proliferation of the plant.
本発明はまた、 分化抑制遺伝子がウィルスベクターに組込まれた、 遺伝子導入 ベクターを提供する。 The present invention also provides a gene transfer vector in which a differentiation-suppressing gene has been incorporated into a viral vector.
本発明はさらに、 分化抑制遺伝子の発現が強化された哺乳類の造血幹細胞を提 供する。 The present invention further provides mammalian hematopoietic stem cells in which the expression of the differentiation inhibitory gene is enhanced.
また本発明は、 分化抑制遺伝子の発現が強化された哺乳類の造血幹細胞又はこ の造血幹細胞から分化した造血前駆細胞を、 血液細胞刺激因子を作用させつつ培 養することを特徴とする造血幹細胞及び/または造血前駆細胞の生産方法を提供 する。 Further, the present invention provides a hematopoietic stem cell characterized by culturing a mammalian hematopoietic stem cell in which the expression of a differentiation inhibitory gene is enhanced or a hematopoietic progenitor cell differentiated from the hematopoietic stem cell while allowing a blood cell stimulating factor to act. And / or provide a method for producing hematopoietic progenitor cells.
本明細書において用いる用語につき、 以下の通り定義する。
造血幹細胞とは、 血球の全ての分化系列に分化し得る多分化能を有する細胞で あり、 かつ、 その多分化能を維持したまま自己複製ができる細胞である。 造血幹 細胞の評価系では、 in vitroのアツセィ系で赤血球を含む複数の分化系統の細胞 種を含むコロニー （CFU- Emix) を形成し得る細胞として識別される。 これらの細 胞は生体内で自己複製し、 長期にわたり生存する細胞を含むと考えられる。 Terms used in the present specification are defined as follows. A hematopoietic stem cell is a cell having a pluripotency capable of differentiating into all differentiation lineages of blood cells, and a cell capable of self-renewal while maintaining the pluripotency. In the hematopoietic stem cell evaluation system, it is identified as a cell that can form a colony (CFU-Emix) containing cell types of multiple differentiating lineages including erythrocytes in an in vitro Atsushi system. These cells are thought to contain cells that self-renew in vivo and survive for long periods of time.
造血前駆細胞とは、 造血幹細胞よりやや分化した血液細胞であり、 単一あるい は、 2種類の分化系列に分化する能力を持つ。 Hematopoietic progenitor cells are blood cells that have been slightly differentiated from hematopoietic stem cells, and have the ability to differentiate into single or two types of lineages.
分化抑制遺伝子とは、 分化能を持つ細胞内において発現させた時に、 その細胞 の分化を抑制する活性を持つ転写因子をコードする遺伝子をいう。 The differentiation inhibitory gene refers to a gene that encodes a transcription factor having an activity of suppressing the differentiation of a cell when expressed in a cell capable of differentiating.
血液細胞刺激因子とは、 いわゆるサイ ト力イン、 イン夕一ロイキン、 増殖刺激 因子、 インターフェロン、 ケモカインなど造血細胞に対し増殖能、 サイ ト力イン 産生能、 分化能、 遊走能などの生物学的活性の変化をもたらす刺激因子のことを 指す。 Blood cell stimulating factors are biological factors such as so-called site force in, inuichi leukin, growth stimulating factor, interferon, chemokine, etc. A stimulator that causes a change in activity.
以下、 本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明で用いる造血幹細胞の採取源としては、 ヒト及びマウス等の哺乳動物の 臍帯血、 胎児肝臓、 骨髄、 胎児骨髄、 末梢血、 サイ トカインおよび/または抗癌 剤の投与によって幹細胞を動員した末梢血、 及び末梢血由来の細胞群等が挙げら れ、 造血幹細胞を含む組織であればいずれであってもよい。 これらの組織からの 造血幹細胞の取得は、 Herzenberg, L . A. 「Wei r' s Handbook of Experimental Im lunology, 5th editionj, Blackwel l Science Inc . 1997に従い実施することがで きる。 すなわち、 抗 CD34抗体、 抗 CD33抗体、 抗 CD38抗体などを用いて免疫学的に 染色し、 セルソ一ターを用いてこれらの抗体の染色性により分離することができ る。 The source of hematopoietic stem cells used in the present invention may be, for example, umbilical cord blood, fetal liver, bone marrow, fetal bone marrow, peripheral blood, peripheral blood, cytokine and / or peripheral mobilized stem cells by administration of an anticancer agent in mammals such as humans and mice. Examples include a cell group derived from blood and peripheral blood, and any tissue may be used as long as it contains hematopoietic stem cells. Hematopoietic stem cells can be obtained from these tissues according to Herzenberg, L.A., “Weir's Handbook of Experimental Imlunology, 5th edition, Blackwell Science Inc. 1997. That is, an anti-CD34 antibody It can be immunologically stained using an anti-CD33 antibody, an anti-CD38 antibody, or the like, and can be separated by the staining property of these antibodies using a cell sorter.
本発明に用いる分化抑制遺伝子として、 具体的には HES- 1遺伝子、 HES- 3遺伝子、 HES-5遺伝子等が挙げられる。 後述の実施例に示されるように、 造血幹細胞中で H ES-1遺伝子の発現を強化することによって、 この造血幹細胞及びこの造血幹細胞 から分化した造血前駆細胞の分化および増殖を調節することができる。 造血幹細 胞における HES-1遺伝子発現の効果は、 同様に HES- 1類似の蛋白質である HES-3 (影 山 龍一郎、 生化学、 67 : 1093、 1995 ) 、 H E S - 5 (影山 龍一郎、 生化学、 6
7 : 1093、 1995) をコードする遺伝子を発現させることによつても得られると推測 される。 Specific examples of the differentiation inhibitory gene used in the present invention include the HES-1, HES-3, and HES-5 genes. As shown in the Examples below, by enhancing the expression of the HES-1 gene in hematopoietic stem cells, the differentiation and proliferation of the hematopoietic stem cells and hematopoietic progenitor cells differentiated from the hematopoietic stem cells can be regulated. . The effect of HES-1 gene expression on hematopoietic stem cells is also similar to that of HES-1 similar proteins, HES-3 (Ryuichiro Kageyama, Biochemistry, 67: 1093, 1995), HES-5 (Ryuichiro Kageyama, Chemistry, 6 7: 1093, 1995).
HES-1遺伝子 （ヒト由来のものは HRYともいう） 、 HES-3遺伝子及び HES-5遺伝子 はいずれも公知の遺伝子であり、 Sasaiら （Genes Dev., 6: 2620, 1992； マウス 由来 HES-1と HES-3) 、 Akazawaら (J. Biol. Chem., 267:21879, 1992 ;マウス由 来 HES- 5) および Federら （Genomics, 20: 56, 1994； ヒト由来 HES- 1) に開示され ている配列に基づいて作製したォリゴヌクレオチドを用いた PCR (ポリメラ一ゼ · チェイン ， リアクション） により各遺伝子を含む DNA断片を増幅することによって、 取得することができる。 The HES-1 gene (human-derived one is also called HRY), HES-3 gene and HES-5 gene are all known genes, and are described in Sasai et al. (Genes Dev., 6: 2620, 1992; mouse-derived HES- 1 and HES-3), disclosed by Akazawa et al. (J. Biol. Chem., 267: 21879, 1992; HES-5 derived from mouse) and Feder et al. (Genomics, 20: 56, 1994; HES-1 derived from human) It can be obtained by amplifying a DNA fragment containing each gene by PCR (polymerase chain, reaction) using oligo nucleotides prepared based on the given sequence.
本発明において、 造血幹細胞中で分化抑制遺伝子の発現を強化するとは、 造血 幹細胞中の分化抑制遺伝子の発現量を、 少なく とも一定期間、 通常の発現量より も高くなるように造血幹細胞を操作することをいう。 尚、 分化抑制遺伝子の発現 量は常に高い必要はなく、 本発明により造血幹細胞および造血前駆細胞の分化お よび増殖を調節する際に高くすることができればよい。 In the present invention, to enhance the expression of a differentiation inhibitory gene in a hematopoietic stem cell means to operate the hematopoietic stem cell so that the expression level of the differentiation inhibitory gene in the hematopoietic stem cell is higher than the normal expression level for at least a certain period of time. That means. The expression level of the differentiation-suppressing gene does not need to be always high, but may be high as long as the present invention can increase the level of differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells.
造血幹細胞中で分化抑制遺伝子の発現を強化する方法として具体的には、 哺乳 動物細胞用のベクタ一に発現可能な形態で分化抑制遺伝子を組み込み、 得られる 組換えべクタ一を造血幹細胞に導入する方法が挙げられる。 発現可能な形態の分 化抑制遺伝子は、 プロモーター等の発現調節因子を分化抑制遺伝子のコード配列 の上流に連結することにより得られる。 分化抑制遺伝子の発現は、 調節可能であ ることが好ましい。 すなわち、 本発明を利用して増殖された造血幹細胞又は造血 前駆細胞を移植に用いる場合には、 分化抑制遺伝子は培養時にのみ発現し、 生体 に細胞を移植してからも発現することは好ましくないため、 必要に応じて発現を 調節できることが好ましい。 そのような発現調節としては、 テトラサイクリンに よる発現調節系 (Gossen, M.， Proc. Natl. Acad. Sci. U.S.A., 89:5547, 1992) 、 昆虫ホルモン .ェクダイソンを用いる発現調節系 （No， D.， Proc. Natl. Acad. Sci. U.S.A., 93:3346, 1996) 、 IPTG (イソプロビル一/?— D—チォガラク トビ ラノシド） を用いる発現調節系 （Biard, D.S., Biochem. Biophys. Acta., 1130 :68, 1992) 等が挙げられる。 As a method for enhancing the expression of the differentiation inhibitory gene in hematopoietic stem cells, specifically, a differentiation inhibitory gene is incorporated in a form that can be expressed in a vector for mammalian cells, and the obtained recombinant vector is introduced into hematopoietic stem cells. Method. The expression suppressor gene in an expressible form can be obtained by ligating an expression control factor such as a promoter upstream of the coding sequence of the differentiation suppressor gene. Preferably, the expression of the differentiation inhibitory gene is regulatable. That is, when hematopoietic stem cells or hematopoietic progenitor cells grown using the present invention are used for transplantation, the differentiation inhibitory gene is expressed only during culture, and it is not preferable to express the cells even after transplanting the cells into a living body. Therefore, it is preferable that the expression can be regulated as needed. Examples of such expression control include an expression control system using tetracycline (Gossen, M., Proc. Natl. Acad. Sci. USA, 89: 5547, 1992) and an expression control system using the insect hormone ecdysone (No. Natl. Acad. Sci. USA, 93: 3346, 1996) and an expression control system using IPTG (isoprovir 1 /?-D-thiogalactobilanoside) (Biard, DS, Biochem. Biophys. Acta., 1130: 68, 1992).
上記ベクターとしては、 レトロウイルスベクタ一、 アデノウイルスベクタ一
(Neering, S.J., Blood, 88:1147, 1996) 、ヘルぺスウィルスベクタ一 （Dilloo, D.， Blood, 89:119, 1997) 、 HIVベクターが挙げられる。 このようなベクターに 分化抑制遺伝子を組み込むことにより、 本発明の遺伝子導入ベクターが得られる。 特にアデノウィルスベクター、ヘルぺスウィルスベクタ一は、 移入された遺伝子は 染色体外に存在し、 一過性に遺伝子の発現を行なった後消失してゆく。 このよう な一過性発現ベクターを用いることは、 分化抑制遺伝子を培養時にのみ発現させ ることができる点で、 優位性がある。 The above vectors include retrovirus vector, adenovirus vector (Neering, SJ, Blood, 88: 1147, 1996), herpesvirus vector (Dilloo, D., Blood, 89: 119, 1997), and HIV vector. By incorporating the differentiation-suppressing gene into such a vector, the gene transfer vector of the present invention can be obtained. In particular, in the case of adenovirus vectors and herpes virus vectors, the transferred gene exists outside the chromosome and disappears after transient gene expression. The use of such a transient expression vector is advantageous in that the differentiation inhibitory gene can be expressed only during culture.
また、 造血幹細胞中で分化抑制遺伝子の発現を強化するには、 造血幹細胞の染 色体 DNA上の分化抑制遺伝子の発現量を増加させてもよい。 例えば、 造血幹細胞の 染色体 DNA上の分化抑制遺伝子固有のブロモ一夕一等の発現調節配列を、 これより も強力な発現調節配列で置換することによって、 あるいは強力な発現調節配列を 分化抑制遺伝子の上流に挿入することによって、 該遺伝子の発現量を増加させる ことができる。 発現調節配列の置換、 挿入は、 相同組換え等によって行うことが できる。 To enhance the expression of the differentiation inhibitory gene in hematopoietic stem cells, the expression level of the differentiation inhibitory gene on the chromosomal DNA of hematopoietic stem cells may be increased. For example, by replacing the expression control sequence such as bromo allyl specific to the differentiation control gene on the chromosomal DNA of hematopoietic stem cells with a stronger expression control sequence, or by replacing the strong expression control sequence with the By inserting the gene upstream, the expression level of the gene can be increased. Replacement and insertion of the expression control sequence can be performed by homologous recombination or the like.
上記のようにして造血幹細胞中で分化抑制遺伝子の発現を強化すると共に、 血 液細胞刺激因子を作用させることにより、 前記造血幹細胞及び/または前記造血 幹細胞から分化した造血前駆細胞の分化および増殖を調節することができる。 例 えば、 分化抑制遺伝子の発現が強化された造血幹細胞又はこの造血幹細胞から分 化した造血前駆細胞を、 血液細胞刺激因子を作用させつつ培養することにより、 造血幹細胞及び/または造血前駆細胞を増殖させることができる。 By enhancing the expression of the differentiation inhibitory gene in the hematopoietic stem cells as described above, and by causing a blood cell stimulating factor to act thereon, the differentiation and proliferation of the hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells are increased. Can be adjusted. For example, hematopoietic stem cells and / or hematopoietic progenitor cells can be expanded by culturing hematopoietic stem cells with enhanced expression of differentiation-suppressing genes or hematopoietic progenitor cells differentiated from the hematopoietic stem cells while allowing them to act on blood cell stimulating factors. Can be done.
血液細胞刺激因子は、 造血幹細胞の増殖を促進するために培地に添加されるも のであり、 いわゆるサイ トカイン、 インタ一ロイキン、 増殖刺激因子、 インタ一 フエロン、 ケモカイン、 発生関連遺伝子産物などが挙げられる （サイ ト力インに ついては、 The Cytokine Factsbook, Callard, R.E. , Academic Press, 1994参 照） 。 血液細胞刺激因子として具体的には、 S CF (幹細胞成長因子（stem cell factor)) 、 IL-3(インターロイキン一 3 ) 、 IL-6 (インタ一ロイキン一 6) 、 G M-CSF (頼粒球マクロファージコロニー刺激因子） 、 TP0 (スロンボポェチン） 、 EP0 (エリスロポエチン） 、 Wnt(Thimoth， A. W., Blood, 89:3624-3635, 1997)や Notch/Delta系の遺伝子産物である Dll-1、 Dll-3、 Jagged-1, Jagged - 2、 および D
LKなどが挙げられる。 Blood cell stimulating factors are added to the culture medium to promote the growth of hematopoietic stem cells, and include so-called cytokines, interleukins, growth stimulating factors, interferon, chemokines, development-related gene products, etc. (See The Cytokine Factsbook, Callard, RE, Academic Press, 1994 for site power-in). Specific examples of blood cell stimulating factors include SCF (stem cell factor), IL-3 (interleukin-13), IL-6 (interleukin-16), and GM-CSF (requested). Granulocyte macrophage colony stimulating factor), TP0 (thrombopoetin), EP0 (erythropoietin), Wnt (Thimoth, AW, Blood, 89: 3624-3635, 1997), and Notch / Delta gene products Dll-1, Dll- 3, Jagged-1, Jagged-2, and D LK and the like.
上記血液細胞刺激因子を培地に添加する際に、 その濃度を調節することで、 造 血幹細胞の生育に良い培養条件をさらに有効なものに改善できる。 さらに、 造血 幹細胞を維持できるストローマ細胞の培養上清を添加して培養してもよい。 By adjusting the concentration of the blood cell stimulating factor when it is added to the medium, culture conditions favorable for the growth of hematopoietic stem cells can be improved to be more effective. Furthermore, culture may be performed by adding a culture supernatant of stromal cells capable of maintaining hematopoietic stem cells.
造血幹細胞または造血前駆細胞を培養するにあたっては、 いわゆる培養用のシ ヤーレ、 フラスコを用いた培養法が可能であるが、 培地組成、 pHなどを機械的に 制御し、 高密度での培養が可能なバイオリアクタ一によって、 その培養系を改善 することもできる（Schwartz, Proc . Natl . Acad. Sci . U. S. A. , 88 : 6760, 1991 ; Kol le r， M. R. , Bio/Technology, 11 : 358, 1993 ; Kol ler, M丄， Blood, 82 : 378, 1993 ； Palsson, B . O . , Bio/Technology, 11 : 368, 1993 )。 When culturing hematopoietic stem cells or hematopoietic progenitor cells, culture methods using so-called culture dishes and flasks are possible, but high-density culture is possible by mechanically controlling the medium composition, pH, etc. Natl. Acad. Sci. USA, 88: 6760, 1991; Koller, MR, Bio / Technology, 11: 358, 1993; Schwartz, Proc. Natl. Acad. Sci. Koller, M 丄, Blood, 82: 378, 1993; Palsson, B.O., Bio / Technology, 11: 368, 1993).
培養に用いる培地としては、 造血幹細胞または造血前駆細胞の増殖、 生存が害 されない限り特に制限されないが、 例えば MEM-ひ培地 （GIBCO BRL) 、 SF-02培地 (三光純薬） 、 Opti- MEM培地 （GIBCO BRL ) 、 IMDM培地 （GIBCO BRL) 、 PRMI 1640 培地 （GIBCO BRL) 、 が好ましいものとして挙げられる。 培養温度は、 通常 25〜3 9°C、 好ましくは 33〜39°Cである。 また、 培地に添加する物質としては、 ゥシ胎児 血清、 ヒ ト血清、 ゥマ血清、 インシュリン、 トランスフェリ ン、 ラク トフエリン、 エタノールァミン、 亜セレン酸ナ ト リ ウム、 モノチォグリセロール、 2—メルカ ブトエタノール、 ゥシ血清アルブミ ン、 ビルビン酸ナ ト リ ウム、 ポリエチレング リコール、 各種ビタミン、 各種アミノ酸、 C02は、 通常、 4〜^であり、 5%が好ま しい。 The culture medium used for the culture is not particularly limited as long as the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired. For example, MEM-G medium (GIBCO BRL), SF-02 medium (Sanko Pure Chemical), Opti-MEM medium (GIBCO BRL), IMDM medium (GIBCO BRL) and PRMI 1640 medium (GIBCO BRL) are preferred. Culture temperature is usually 25 to 39 ° C, preferably 33 to 39 ° C. Materials added to the culture medium include fetal calf serum, human serum, poma serum, insulin, transferrin, lactoferrin, ethanolamine, sodium selenite, sodium monothioglycerol, Melka but-ethanol, © shea serum albumin, pyruvic Sanna door Li Umm, polyethylene grayed recall, various vitamins, various amino acids, C0 2 is, usually, is a 4 ^, arbitrariness preferred 5%.
本発明において、 「造血幹細胞及び前記造血幹細胞から分化した造血前駆細胞 の分化および増殖を調節する」 とは、 具体的には例えば、 造血幹細胞及び/又は この造血幹細胞から分化した造血前駆細胞を、 少なく とも一部が多分化能を維持 したまま生存、 好ましくは増殖させることをいう。 多分化能を維持したまま増殖 させることにより、 造血幹細胞又は造血前駆細胞を生産することができる。 さら に、 これら造血幹細胞又は造血前駆細胞の分化を誘導することにより、 各種血液 細胞を生産することができる。 In the present invention, `` regulating the differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells differentiated from the hematopoietic stem cells '' specifically, for example, hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells, It means that at least a part of the cells survive while maintaining pluripotency and is preferably expanded. By proliferating while maintaining pluripotency, hematopoietic stem cells or hematopoietic progenitor cells can be produced. Furthermore, various blood cells can be produced by inducing the differentiation of these hematopoietic stem cells or hematopoietic progenitor cells.
上記のようにして産生される造血幹細胞または造血前駆細胞は、 従来の骨髄移 植ゃ臍帯血移植に代わる血液細胞移植用の移植片として用いることができる。 造
血幹細胞の移植は、 移植片が半永久的に生着させられることから、 従来の血液細 胞移植治療を改善することができる。 The hematopoietic stem cells or hematopoietic progenitor cells produced as described above can be used as a transplant for blood cell transplantation instead of conventional bone marrow transplantation and umbilical cord blood transplantation. Construction Blood stem cell transplantation can improve conventional blood cell transplantation therapy because the transplant is semi-permanently engrafted.
造血幹細胞の移植は、 白血病に対する全身 X線療法や高度化学療法を行う際に、 これらの治療と組み合わせる他、 種々の疾患に用いることができる。 例えば、 固 形癌患者の化学療法、 放射線療法等の骨髄抑制が副作用として生じる治療を実施 する際に、 施術前に骨髄を採取しておき、 造血幹細胞、 造血前駆細胞を試験管内 で増幅し、 施術後に患者に戻すことで、 副作用による造血系の障害から早期に回 復させることができ、 より強力な化学療法を行えるようになり、 化学療法の治療 効果を改善する事ができる。 また、 本発明により得られる造血幹細胞ならびに造 血前駆細胞を各種血液細胞に分化させ、 それらを患者の体内に移入することによ り、 各種血液細胞の低形成により不全な状況を呈している患者の改善を図ること ができる。 また、 再生不良性貧血などの貧血を呈する骨髄低形成に起因する造血 不全症を改善することができる。 その他、 本発明の方法による造血幹細胞の移植 が有効な疾患としては、 慢性肉芽腫症、 重複免疫不全症候群、 無ガンマグロプリ ン血症、 Wiskott-Aldri ch症候群、 後天性免疫不全症候群 （AIDS) 等の免疫不全症 候群、 サラセミア、 酵素欠損による溶血性貧血、 鎌状赤血球症等の先天性貧血、 Gaucher病、 ムコ多糖症等のリソゾーム蓄積症、 副腎白質変性症、 各種の癌または 腫瘍等が挙げられる。 Hematopoietic stem cell transplantation can be used for various diseases in addition to these treatments when performing systemic X-ray therapy or advanced chemotherapy for leukemia. For example, when performing a treatment that causes bone marrow suppression as a side effect, such as chemotherapy or radiation therapy, for solid cancer patients, bone marrow is collected before the operation, and hematopoietic stem cells and hematopoietic progenitor cells are expanded in vitro. By returning to the patient after the procedure, hematopoietic disorders due to side effects can be recovered early, and stronger chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved. In addition, a patient who is in a poor state due to hypoplasia of various blood cells by differentiating the hematopoietic stem cells and hematopoietic progenitor cells obtained by the present invention into various blood cells and transferring them into the patient's body. Can be improved. In addition, hematopoietic insufficiency caused by bone marrow hypoplasia exhibiting anemia such as aplastic anemia can be improved. Other diseases for which hematopoietic stem cell transplantation by the method of the present invention is effective include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, acquired immunodeficiency syndrome (AIDS), and the like. Immunodeficiency syndrome group, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage disease such as Gaucher disease and mucopolysaccharidosis, adrenal white matter degeneration, various cancers or tumors, etc. Can be
造血幹細胞の移植は、 用いる細胞以外は、 従来行われている骨髄移植や臍帯血 移植と同様に行えばよい。 Transplantation of hematopoietic stem cells may be performed in the same manner as conventional bone marrow transplantation or umbilical cord blood transplantation, except for the cells used.
上記のような造血幹細胞移植に用いられる可能性のある造血幹細胞の由来は、 骨髄に限られず、 前述したような胎児肝臓、 胎児骨髄、 末梢血、 サイ トカインぉ よび/または抗癌剤の投与によって幹細胞を動員した末梢血、 及び臍帯血等を用 いることができる。 The origin of hematopoietic stem cells that may be used for hematopoietic stem cell transplantation as described above is not limited to bone marrow, and stem cells can be obtained by administering fetal liver, fetal bone marrow, peripheral blood, cytokines, and / or anticancer drugs as described above. Mobilized peripheral blood, umbilical cord blood, and the like can be used.
移植片は、 本発明の方法によって産生した造血幹細胞及び造血前駆細胞の他に、 緩衝液等を含む組成物としてもよい。 The transplant may be a composition containing a buffer solution or the like in addition to the hematopoietic stem cells and hematopoietic progenitor cells produced by the method of the present invention.
また、 本発明により産生される造血幹細胞または造血前駆細胞は、 ex vivoの遺 伝子治療に用いることができる。 この遺伝子治療には、 造血幹細胞または造血前 駆細胞に外来遺伝子 （治療用遺伝子） を導入し、 得られる遺伝子導入細胞を用い
て行われる。 導入される外来遺伝子は、 疾患によって適宜選択される。 血液細胞 を標的細胞とする遺伝子治療の対象となる疾患としては、 慢性肉芽腫症、 重複免 疫不全症候群、 無ガンマグロブリン血症、 Wiskott-Aldrich症候群、 後天性免疫不 全症候群 （AIDS) 等の免疫不全症候群、 サラセミア、 酵素欠損による溶血性貧血、 鎌状赤血球症等の先天性貧血、 Gaucher病、 ムコ多糖症等のリソゾーム蓄積症、 副 腎白質変性症、 各種の癌または腫瘍等が挙げられる。 In addition, hematopoietic stem cells or hematopoietic progenitor cells produced by the present invention can be used for ex vivo gene therapy. In this gene therapy, a foreign gene (therapeutic gene) is introduced into hematopoietic stem cells or hematopoietic progenitor cells, and the resulting transfected cells are used. Done. The foreign gene to be introduced is appropriately selected depending on the disease. Diseases targeted by gene therapy targeting blood cells include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, and acquired immune deficiency syndrome (AIDS). Examples include immunodeficiency syndrome, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage diseases such as Gaucher disease and mucopolysaccharidosis, adrenal leukemia, various cancers and tumors, etc. .
造血幹細胞または造血前駆細胞に治療用遺伝子を導入するには、 通常動物細胞 の遺伝子導入に用いられる方法、 例えば、 モロニ一マウス白血病ウィルス等のレ トロウィルスベクタ一、 アデノウイルスベクター、 アデノ随伴ウィルス （AAV) ベ クタ一、 単純へルぺスウィルスベクタ一、 HIVベクター等のウィルス由来の遺伝子 治療に用いられる動物細胞用べクタ一 （遺伝子治療用べクタ一については、 Verm a, I . M. , Nature, 389 : 239, 1997 参照） を用いる方法、 リン酸カルシウム共沈法、 DEAE-デキス トラン法、 エレク ト口ポレーシヨン法、 リポソ一ム法、 リポフエクシ ヨン法、 マイクロインジェクション法等を用いることができる。 これらの中では、 標的細胞の染色体 DNAに組み込まれて恒久的に遺伝子の発現が期待できるという点 から、 レトロウイルスベクタ一、 アデノ随伴ウィルスベクタ一または HIVベクタ一 が好ましい。 In order to introduce a therapeutic gene into hematopoietic stem cells or hematopoietic progenitor cells, a method usually used for gene transfer into animal cells, for example, retrovirus vector such as Moroni murine leukemia virus, adenovirus vector, adeno-associated virus ( (AAV) Vectors, simple herpes virus vectors, vector vectors for animal cells used in gene therapy for viruses derived from HIV vectors, etc. (For gene therapy vectors, see Verma, I.M. , Nature, 389: 239, 1997), calcium phosphate coprecipitation method, DEAE-dextran method, electoral poration method, liposome method, lipofection method, microinjection method, and the like. Among them, a retrovirus vector, an adeno-associated virus vector, or an HIV vector is preferable, since the gene can be expected to be permanently expressed by being integrated into the chromosomal DNA of the target cell.
例えば、 アデノ随伴ウィルス （AAV) ベクタ一は、 次のようにして作製すること ができる。 まず、 野生型アデノ随伴ウィルス DNAの両端の ITR ( inverted termina 1 repeat) の間に治療用遺伝子を挿入したベクターブラスミ ドと、 ウィルスタン パク質を補うためのへルパープラスミ ドを 293細胞にトランスフエクシヨンする。 続いてヘルパーウィルスのアデノウィルスを感染させると、 AAVベクタ一を含むゥ ィルス粒子が産生される。 あるいは、 アデノウイルスの代わりに、 ヘルパー機能 を担うアデノウイルス遺伝子を発現するプラスミ ドを トランスフエクシヨンして もよい。 次に、 得られるウィルス粒子を造血幹細胞または造血前駆細胞に感染さ せる。 ベクター DNA中において、 目的遺伝子の上流には、 適当なプロモ一夕一及び ェンハンサーを挿入し、 これらによって遺伝子の発現を調節することが好ましい。 さらに、 治療用遺伝子に加えて薬剤耐性遺伝子等のマーカー遺伝子を用いると、 治療用遺伝子が導入された細胞の選択が容易となる。 治療用遺伝子は、 センス遺
伝子であってもアンチセンス遺伝子であってもよい。 For example, an adeno-associated virus (AAV) vector can be constructed as follows. First, 293 cells were transfected with a vector plasmid containing a therapeutic gene inserted between the ITRs (inverted termina 1 repeats) at both ends of the wild-type adeno-associated virus DNA, and helper plasmid for complementing the viral proteins. I do. Subsequently, when infected with an adenovirus of a helper virus, virus particles containing the AAV vector are produced. Alternatively, instead of adenovirus, a plasmid expressing an adenovirus gene responsible for helper function may be transfected. Next, the obtained virus particles are used to infect hematopoietic stem cells or hematopoietic progenitor cells. It is preferable to insert an appropriate promoter and enhancer upstream of the target gene in the vector DNA, and to regulate the expression of the gene by these. Furthermore, when a marker gene such as a drug resistance gene is used in addition to the therapeutic gene, it becomes easy to select cells into which the therapeutic gene has been introduced. Therapeutic genes are sense sense It may be a gene or an antisense gene.
遺伝子治療用組成物は、 本発明の方法によって産生された造血幹細胞及び造血 前駆細胞の他に、 緩衝液、 新規の活性物質等を含む組成物としてもよい。 図面の簡単な説明 図 1は、 pFLAG- CMV2-HES- 1. 1の構築手順を示す図である。 The composition for gene therapy may be a composition containing a buffer, a novel active substance, and the like, in addition to the hematopoietic stem cells and hematopoietic progenitor cells produced by the method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a construction procedure of pFLAG-CMV2-HES-1.1.
図 2は、 pFLAG- CMV2-HES- 1.2の構築手順を示す図である。 FIG. 2 is a diagram showing a procedure for constructing pFLAG-CMV2-HES-1.2.
図 3は、 pEGFP-CI - HES- 1の構築手順を示す図である。 FIG. 3 is a diagram showing a procedure for constructing pEGFP-CI-HES-1.
図 4は、 pMSCV-EHの構築手順を示す図である。 発明を実施するための最良の形態 以下に、 実施例により本発明をさらに具体的に説明する。 尚、 以下の実施例に おいて、 細胞分離に用いた抗体は全て Pharmingen社より購入した。 遺伝子組み換 えに用いた制限酵素は、 全てべ一リンガーマンハイム社より購入した。 また、 各 血球細胞の分離は、 おおむね、 Herzenberg, L .A. 「Weir，s Handbook of Experim ental Immunology, 5th edition」， Blackwel l Science Inc . 1997 【こ従 、実施し た。 実施例 1 Notch/Del ta関連遺伝子の造血細胞における発現 造血細胞における Notch/Delta関連遺伝子の果たす役割を知る目的で、 RT-PCR法 により Notch、 HES遺伝子の造血細胞における発現を確認した。 FIG. 4 is a diagram showing a procedure for constructing pMSCV-EH. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to examples. In the following examples, all antibodies used for cell separation were purchased from Pharmingen. All the restriction enzymes used for gene recombination were purchased from Behringer Mannheim. Separation of each blood cell was generally carried out by Herzenberg, L.A., "Weir, s Handbook of Experimental Immunology, 5th edition", Blackwell Science Inc. 1997. Example 1 Expression of Notch / Delta-Related Genes in Hematopoietic Cells In order to understand the role of Notch / Delta-related genes in hematopoietic cells, the expression of Notch and HES genes in hematopoietic cells was confirmed by RT-PCR.
< 1 >造血細胞の分離 <1> Separation of hematopoietic cells
6- 8週齢の C57B1/6マウス（日本チヤ一ルスリバ一株式会社より購入） より骨髄細 胞、 脾臓細胞、 胸腺細胞をそれそれ分離し、 セルソ一夕一を用いて単核球、 顆粒 球、 成熟 T細胞、 未熟 T細胞、 成熟 B細胞、 造血前駆細胞、 および造血幹細胞を 選別した。 Bone marrow cells, spleen cells, and thymocytes are separated from 6- to 8-week-old C57B1 / 6 mice (purchased from Nippon Charls River Co., Ltd.), and mononuclear cells and granulocytes are obtained using Celso overnight. , Mature T cells, immature T cells, mature B cells, hematopoietic progenitor cells, and hematopoietic stem cells.
( 1 ) 単核球、 頼粒球細胞の分離法 (1) Method for separating mononuclear cells and granulocytes
マウス大腿骨骨髄より取り出した骨髄細胞に対し、 抗 CD32抗体を添加し氷中に
て 10分放置した後、 FITC標識抗 Mac-1抗体、 PE標識抗 Gr-1抗体を加え、 氷中にて 3 0分間反応させた。 反応後、 染色バッファ一 （PBS (リン酸緩衝生理食塩水） 、 5% FCS (ゥシ胎児血清） 、 0.05% NaN3 ) で 2回洗い、 染色バッファーに懸濁した後、 セルソーター （FACS Vantage , Becton Di ckinson社） により単核球 （Mac-1陽性 ' Gr-1陰性細胞） 、 および頼粒球 （Mac-1陽性 · Gr-1陽性細胞） を分離した。 Anti-CD32 antibody was added to bone marrow cells removed from mouse femur bone marrow and placed on ice. After standing for 10 minutes, FITC-labeled anti-Mac-1 antibody and PE-labeled anti-Gr-1 antibody were added, and reacted on ice for 30 minutes. After the reaction, the cells were washed twice with a staining buffer (PBS (phosphate buffered saline), 5% FCS (CS fetal serum), 0.05% NaN 3 ), suspended in the staining buffer, and then suspended in a cell sorter (FACS Vantage, Mononuclear cells (Mac-1 positive 'Gr-1 negative cells) and granulocytes (Mac-1 positive and Gr-1 positive cells) were separated using Becton Dickinson.
( 2 ) T細胞の分離法 (2) T cell isolation method
胸腺細胞の懸濁液を、 細胞分離用高密度溶液 （Nycomed社、 Lymphoprep) に重層 し、 1500 rpm、 25°C、 30分間遠心し、 懸濁液と Lymphoprepとの界面に集まった細 胞を回収した。 細胞を染色バッファ一で 2回洗浄した後、 抗 CD32抗体、 FI TC標識 抗 CD4抗体、 PE標識抗 CD8抗体を加え、 氷中にて 30分間反応させた。 反応後、 染色 バヅファーで 2回洗い、 染色バッファーに懸濁した後、 セルソー夕一により未熟 T細胞 （CD4陰性 · CD8陰性細胞および CD4陽性 · CD8陽性細胞） 、 および成熟 T細 胞 （CD4陽性 · CD8陰性細胞および CD4陰性 · CD8陽性細胞） を分離した。 The thymocyte suspension is overlaid on a high-density cell separation solution (Nycomed, Lymphoprep), centrifuged at 1500 rpm for 30 minutes at 25 ° C, and cells collected at the interface between the suspension and Lymphoprep are removed. Collected. After the cells were washed twice with a staining buffer, an anti-CD32 antibody, a FITC-labeled anti-CD4 antibody, and a PE-labeled anti-CD8 antibody were added, and reacted on ice for 30 minutes. After the reaction, the cells were washed twice with a staining buffer and suspended in a staining buffer. Then, immature T cells (CD4 negative · CD8 negative cells and CD4 positive · CD8 positive cells) and mature T cells (CD4 positive · CD8-negative cells and CD4-negative and CD8-positive cells).
( 3 ) B細胞の分離法 (3) B cell isolation method
脾臓細胞の懸濁液を Lymphoprep (Nycomed社） に重層し、 1500 rpm、 25°C、 30分 間遠心し、 界面に集まった細胞を回収した。 細胞を染色バッファーで 2回洗浄し、 抗 CD32抗体を添加し氷中にて 1 0分放置した後、 F I TC標識抗 B220抗体、 PE標識抗 マウス I gM抗体を加え、 氷中にて 30分間反応させた。 反応後、 染色バッファーで 2 回洗い、 染色バッファーに懸濁した後、 セルソ一ターにより成熟 B細胞 （B220陽 性 - I gM陽性細胞） を分離した。 The suspension of spleen cells was overlaid on Lymphoprep (Nycomed) and centrifuged at 1500 rpm at 25 ° C for 30 minutes to collect the cells collected at the interface. Wash the cells twice with staining buffer, add anti-CD32 antibody, and leave on ice for 10 minutes, then add FITC-labeled anti-B220 antibody and PE-labeled anti-mouse IgM antibody, and on ice for 30 minutes Reacted. After the reaction, the cells were washed twice with the staining buffer, suspended in the staining buffer, and then mature B cells (B220-positive-IgM-positive cells) were separated using a cell sorter.
( 4 ) 造血前駆細胞、 および造血幹細胞の分離法 (4) Method for separating hematopoietic progenitor cells and hematopoietic stem cells
骨髄細胞の懸濁液を Lymphoprep (Nycomed社） に重層し、 1500 rpm、 25 C、 30分 間遠心し、 界面に集まった細胞を回収した。 細胞を染色バッファーで 2回洗浄し た後、 細胞を PBSで 2回洗浄し、 染色バッファーに懸濁した。 細胞懸濁液にビォチ ン化した分化抗原マーカーに対する抗体、 つまり抗 CD4抗体、 抗 CD8抗体、 抗 B220 抗体、 抗 Gr- 1抗体、 抗 Terl l9抗体を添加し、 氷中に 30分間放置した。 その後、 染 色バッファーで 2回洗浄の後、 アビジンをコートした磁性体ビーズ （アビジンマ
グネッ トビーズ、 Perseptive社） を添加し、 氷中で 30分放置した。 再度染色バッ ファーで 2回洗浄の後、 磁石を用いてアビジンマグネットビーズを集めて、 分化 抗原を提示している細胞群を除去し、 分化抗原陰性細胞（Lin- )を取得した。 分化 抗原陰性細胞溶液に、 F ITC標識抗 CD34抗体、 PE標識抗 Sea- 1抗体、 テキサスレッ ド 標識アビジン、 APC標識抗 C - Ki t抗体を添加し、 氷中 30分放置した。 2回染色バッ ファーで洗浄の後、 セルソ一夕一にて造血前駆細胞 （Sea- 1陰性 · C - kit陽性細胞 および CD34陽性 · Sca-1陽性 · C- ki t陽性細胞） および造血幹細胞 （CD34陰性〜弱 陽性 · Sea- 1陽性 · c-ki t陽性細胞） を選別した。 The bone marrow cell suspension was overlaid on Lymphoprep (Nycomed) and centrifuged at 1500 rpm at 25 C for 30 minutes to collect cells that had collected at the interface. After washing the cells twice with the staining buffer, the cells were washed twice with PBS and suspended in the staining buffer. An antibody against the biotinylated differentiation antigen marker, ie, an anti-CD4 antibody, an anti-CD8 antibody, an anti-B220 antibody, an anti-Gr-1 antibody, and an anti-Tell19 antibody, were added to the cell suspension, and the mixture was left on ice for 30 minutes. Then, after washing twice with the staining buffer, avidin-coated magnetic beads (Avidinma Gnet beads (Perseptive)) and left on ice for 30 minutes. After washing twice with a staining buffer again, avidin magnet beads were collected using a magnet, and the cells presenting the differentiation antigen were removed to obtain differentiated antigen-negative cells (Lin-). A FITC-labeled anti-CD34 antibody, a PE-labeled anti-Sea-1 antibody, a Texas red-labeled avidin, and an APC-labeled anti-C-Kit antibody were added to the differentiation antigen-negative cell solution, and left on ice for 30 minutes. After washing twice with a staining buffer, hematopoietic progenitor cells (Sea-1 negative, C-kit positive cells and CD34 positive, Sca-1 positive, C-kit positive cells) and hematopoietic stem cells (Selso overnight) CD34 negative to weak positive · Sea-1 positive · c-kit positive cells).
< 2 > cDNA合成と PCRによる発現の検出 <2> cDNA synthesis and expression detection by PCR
上記で得られた細胞を遠心し、 ペレッ トとした後、 RNAを抽出するため RNA抽出 用試薬（アイソジヱン、 日本ジ一ン社）を添加し、 試薬の使用説明書に従い RNAを取 得した。 これに対し、 5ユニッ トの DNase RNase free , GI BCO- BRL社） を加え 37 °Cで 30分間保温し、 混在するゲノム DNAを消化分解させ、 再びアイソジェン（日本 ジーン）を添加し、 純粋な RNAを取得した。 この MAから、 オリゴ dTをブライマ一 として cDNAを合成した。 つまり、 105 個の細胞にあたる RNAが 20マイクロリ ットル 反応液に相当するように反応液を調製した。 反応液の組成は、 逆転写酵素 （Supe rscript I K GIBCO- BRL社） の使用説明書に推奨されているものを使用した。 反応 は、 42°Cで 60分間実施し、 その後 72°C 10分の保温により逆転写酵素の活性を失活 させた。 After the cells obtained above were centrifuged to form pellets, a reagent for RNA extraction (Isodidin, Nippon Jin Co.) was added to extract RNA, and RNA was obtained according to the instructions for use of the reagent. To this, 5 units of DNase RNase free (GI BCO-BRL) were added, and the mixture was incubated at 37 ° C for 30 minutes to digest and degrade the mixed genomic DNA. RNA was obtained. From this MA, cDNA was synthesized using oligo dT as a primer. That, RNA corresponding to 10 5 cells to prepare a reaction liquid so as to correspond to 20 micro liters reaction. The composition of the reaction solution used was that recommended in the instruction manual for reverse transcriptase (Superscript IK GIBCO-BRL). The reaction was carried out at 42 ° C. for 60 minutes, and then the reverse transcriptase activity was inactivated by keeping the temperature at 72 ° C. for 10 minutes.
PCR反応に使用した各種ブライマ一の配列、 ァニーリング温度は以下の通りであ る。
The sequences and annealing temperatures of the various primers used in the PCR reaction are as follows.
表 1 table 1
PCR反応は、 Taqポリメラ一ゼとして ExTaq (宝酒造（株）） を使用し、 铸型 cDNAに は上記にて作製した各種血球 cDNAを 2マイクロリッ トルずつ使用し、 緩衝液、 核 酸の組成は添付の使用説明書に推薦される条件で全量 25マイク口リッ トルで実施 した。 反応は、 94°Cで 2分インキュベートした後、 94°C 30秒；表 1に記載の各 ブライマー毎に設定されたアニーリング温度、 30秒； 72°C 1分のサイクルを 35 回行い、 さらに 72°Cで 7分インキュベートすることにより行った。 PCR反応産物 1 0マイクロリ ツ トルを、 1 .2%ァガロースゲル電気泳動に供しェチジゥムブ口マイ ド 染色により増幅バンドを確認した。 The PCR reaction used ExTaq (Takara Shuzo Co., Ltd.) as the Taq polymerase, the various blood cell cDNAs prepared above were used for type II cDNA in 2 microliter increments, and the buffer and nucleic acid compositions were as follows. A total of 25 mic mouth liters were performed under the conditions recommended in the attached instruction manual. After incubating at 94 ° C for 2 minutes, the reaction was performed at 94 ° C for 30 seconds; annealing temperature set for each primer in Table 1 for 30 seconds; 72 ° C for 1 minute 35 times. This was performed by incubating at 72 ° C for 7 minutes. Ten microliters of the PCR reaction product was subjected to 1.2% agarose gel electrophoresis, and an amplified band was confirmed by ethidium mouth opening staining.
< 3 >結果 <3> Results
上記の増幅バンドの濃さにより 発現量を評価した結果を表 2に示す。 表中の 記号は、 次のとおりである。 Table 2 shows the results of evaluating the expression level based on the intensity of the amplified band. The symbols in the table are as follows.
一 発現が認められない No expression is observed
+ ： 発現が認められる +: Expression is observed
+ + ： 強く発現している + +: Strongly expressed
土 ： 発現が認められないか 弱く発現している
表 2 Sat: no expression or weak expression Table 2
表 2に示すように、 Notch-し Notch-2は調べた造血細胞の全てで発現していた。 Notch- 3は、 未熟 T細胞、 成熟 T細胞、 及び B細胞に細胞種特異的な発現が確認さ れる。 Notch-4は造血系の細胞には発現が認められなかった。 一方、 Notch/Del ta のシグナル伝達系の下流に存在している HES-1 HES-3は、 調べた全ての細胞で発 現が認められた。 HES-5は、 造血幹細胞、 造血前駆細胞以外の細胞で細胞種特異的 に発現が認められた。 As shown in Table 2, Notch- and Notch-2 were expressed in all of the examined hematopoietic cells. Notch-3 is confirmed to be cell type-specific expression in immature T cells, mature T cells, and B cells. Notch-4 was not expressed in hematopoietic cells. On the other hand, HES-1 HES-3, which is present downstream of the Notch / Delta signaling system, was expressed in all the cells examined. HES-5 was expressed in a cell type-specific manner in cells other than hematopoietic stem cells and hematopoietic progenitor cells.
以上の結果は、 Notchのシグナル伝達系がこれら造血系の細胞中で実際に機能し ていることを示している。 前述のように Not ch/D e 1 taのシグナル伝達系は神経系や 筋肉形成の分化制御に重要な役割を果たしていることが分かっている。 これらの ことより、 Notch/De ltaのシグナル伝達系は血液細胞の分化調節についても密接 に関与していると推測できる。 実施例 2 造血幹細胞での HES- 1の強制発現 The above results indicate that the signaling system of Notch actually functions in these hematopoietic cells. As described above, it is known that the signaling system of Notch / De1ta plays an important role in controlling the differentiation of the nervous system and muscle formation. From these facts, it can be inferred that the signaling system of Notch / Delete is closely related to the regulation of blood cell differentiation. Example 2 Forced expression of HES-1 in hematopoietic stem cells
< 1 > EGFP-HES-1発現ベクターの構築 <1> Construction of EGFP-HES-1 expression vector
( 1 ) レトロウイルスで遺伝子移入した細胞内で、 HES- 1の発現及び局在をモニタ
一することを可能にするため、 HES-1と蛍光発光タンパクである EGFP (Enhanced green fluorescent fluorescence protein、 Clontech社) が融合タンノ クとして 発現するようにベクターを構築した （図 1〜4参照） 。 (1) Monitor expression and localization of HES-1 in cells transfected with retrovirus To make it possible to construct a vector, a vector was constructed so that HES-1 and a fluorescent protein, EGFP (Enhanced green fluorescent fluorescence protein, Clontech) were expressed as a fusion protein (see FIGS. 1 to 4).
詳しくは、 PSV2CMVHES- 1 (京都大学、 影山龍一郎博士より分与、 Sasai, Y.， G enes Dev., 6:2620, 1992 参照） を EcoRI消化し、 pFLAG-CMV2ベクター (Kodak社) の EcoRIサイ 卜に、 FLAGの転写方向と同一方向になるようにサブクロ一ニングした PFLAG-CMV2-HES-1.1 (図 1 ) を作成した。 一方で、 HES-1の翻訳開始コドンを変更 し、 EGFPと HES-1が融合タンパクとして発現するように HES- 1の翻訳開始コ ドンを 改変し、 Bglllサイ トに変更した。 つまり、 翻訳開始点 ATGを Bglllに変更するよう な配列を有する合成オリゴヌクレオチド （配列番号 1 5) と、 HES-1遺伝子の中に 存在する Pstlサイ 卜より下流に位置する部位のアンチセンスオリゴヌクレオチド (配列番号 16) をブライマーに用いて、 PSV2CMVHES- 1を铸型として、 PCRを行つ た。 この PCR産物を、 Bglllと Pstlで消化して得られる HES-1遺伝子を含む断片を、 上記に記載の pFLAG- CMV2- HES- 1.1の Bglllと Pst- 1サイ 卜にクロ一ニングした。 こ のブラスミ ドを PFLAG-CMV2- HES-1.2 (図 2) とした。 For details, PSV2CMVHES-1 (Kyoto University, distributed by Dr. Ryuichiro Kageyama, see Sasai, Y., Genes Dev., 6: 2620, 1992) is digested with EcoRI and EcoRI digested with pFLAG-CMV2 vector (Kodak) A subcloned PFLAG-CMV2-HES-1.1 (Fig. 1) was prepared in the same direction as the FLAG transfer direction. On the other hand, the translation initiation codon of HES-1 was changed, and the translation initiation codon of HES-1 was modified so that EGFP and HES-1 were expressed as a fusion protein, and changed to the Bglll site. That is, a synthetic oligonucleotide (SEQ ID NO: 15) having a sequence that changes the translation start point ATG to Bglll, and an antisense oligonucleotide at a site located downstream from the Pstl site in the HES-1 gene. (SEQ ID NO: 16) was used as a primer, and PCR was performed using PSV2CMVHES-1 as type II. A fragment containing the HES-1 gene obtained by digesting this PCR product with Bglll and Pstl was cloned into Bglll and Pst-1 sites of pFLAG-CMV2-HES-1.1 described above. This brassmid was named PFLAG-CMV2-HES-1.2 (Fig. 2).
PFLAG-CMV2-HES-1.2を、 Bgll Iと EcoRIで消化して得られる HES- 1を含む断片を、 pEGFP-CI (Clontech社） の Bglll/EcoRIサイ トにクローニングし、 pEGFP- CI-HES - 1と名づけた （図 3) 。 これにより、 EGFPと HES-1がーつの転写単位に乗った遺伝 子が構築できた。 さらに、 pEGFP- CI- HES- 1を Eco47IIIと Sailで消化して得られる HES-1を含む断片を、 Hpalと Xholで消化した PMSCV2.1ベクタ一 （トロント大学、 R. Hawley博士より入手、 Hawley, R.G., Gene Ther.， 1:136, 199 参照） にクロー ン化した。 このべクタ一を pMSCV-EH (図 4) とした。 以後、 このブラスミ ドを HE S- 1感染用のレトロウィルス産生用に用いた。 A fragment containing HES-1 obtained by digesting PFLAG-CMV2-HES-1.2 with Bgll I and EcoRI was cloned into the Bglll / EcoRI site of pEGFP-CI (Clontech), and pEGFP-CI-HES- 1 (Figure 3). As a result, a gene was constructed in which EGFP and HES-1 were on one transcription unit. In addition, a fragment containing HES-1 obtained by digesting pEGFP-CI-HES-1 with Eco47III and Sail was extracted from Hpal and Xhol using a PMSCV2.1 vector (obtained from Dr. R. Hawley, University of Toronto, Hawley , RG, Gene Ther., 1: 136, 199). This vector was named pMSCV-EH (Fig. 4). Thereafter, this plasmid was used for producing a retrovirus for HES-1 infection.
( 2 ) ネガティブコントロールとして、 EGFP遺伝子のみを移入するレトロウィル スベクターを構築した。 pEGFP- CIを Eco47IIIと Sailで消化して得られる EGFPを含 む断片を、 Hpalと Xholで消化した PMSCV2.1ベクタ一に挿入した。 このべクタ一を pMSCV-E した。 (2) As a negative control, a retrovirus vector into which only the EGFP gene was transferred was constructed. A fragment containing EGFP obtained by digesting pEGFP-CI with Eco47III and Sail was inserted into a PMSCV2.1 vector digested with Hpal and Xhol. This vector was pMSCV-E.
< 2 >GP+E-86細胞への遺伝子導入
上記方法により調製した pMSCV-EHならびに pMSCV-Eを制限酵素 Sealで消化し、 線 状 D N Aとした。 線状 pMSCV- EHをリポフエクシヨン法 （TransIT LT- 宝酒造 (株）） を用いて GP+E- 86細胞 （レトロウイルス産生細胞株 （ATCC CRL-9642) 、 筑波大学 中内啓光教授より入手、 Markowi ts, D.， J. Virol . , 62 : 1120, 1994 参照） に トランスフエクトした。 TransITによる導入は、 添付の使用説明書に従い行なった c 8時間のトランスフエクシヨンの後、 10% FCSを添加した MEM-ひ培地を添加し、 そ のまま 2日間培養を継続した。 その後、 GP+E- 86細胞をトリブシンにて剥離し、 G418 ( lmg/ml ) を含む 10% FCSを添加した MEM-ひ培地中にて再度培養し増殖してく る、 G418耐性 GP+E- 86細胞を選別した。 これを再度トリブシンにより培養皿より剥 離し、 PBSに懸濁し、 フローサイ トメ一夕一 （FACS Vantage , Becton Dickinson) に供した。 EGFPの発現状況を検出し、 EGFPの発現の高い株を選別し、 分取した。 このようにして得られた EGFP高発現細胞を 10% FCSを添加した MEM-ひ培地中にて 培養して得た培養上清を、 感染ウィルス溶液として使用した。 <2> Gene transfer into GP + E-86 cells PMSCV-EH and pMSCV-E prepared by the above method were digested with the restriction enzyme Seal to obtain linear DNA. Linearized pMSCV-EH was obtained from GP + E-86 cells (retrovirus-producing cell line (ATCC CRL-9642) using the Lipofextion method (TransIT LT-Takara Shuzo Co., Ltd.), Prof. Keimitsu Nakauchi, University of Tsukuba, Markowi ts , D., J. Virol., 62: 1120, 1994). Introduction by TransIT after the transflector Ekushi Sauvignon c 8 hours was conducted in accordance with the instructions attached, it was added MEM- non medium supplemented with 10% FCS, was continued 2 days of culture remains fully. Thereafter, the GP + E-86 cells are detached with trypsin, and cultured and grown again in MEM-h medium supplemented with 10% FCS containing G418 (lmg / ml). 86 cells were sorted. This was again detached from the culture dish with trypsin, suspended in PBS, and supplied to a flow cytometer overnight (FACS Vantage, Becton Dickinson). The expression status of EGFP was detected, and strains with high EGFP expression were selected and sorted. The culture supernatant obtained by culturing the EGFP-high-expressing cells thus obtained in a MEM-hi medium supplemented with 10% FCS was used as an infectious virus solution.
< 3 >造血幹細胞への HES— 1遺伝子移入 <3> HES-1 gene transfer into hematopoietic stem cells
( 1 ) マウス胎児肝臓由来造血幹細胞の選別 (1) Selection of hematopoietic stem cells from mouse fetal liver
妊娠 C57B1/6マウスは、 日本チヤ一ルスリバ一株式会社より購入した。 妊娠 14日 目のマウスを開腹し、 胎児を無菌的に摘出した。 胎児肝臓を他の組織が混入しな いように慎重に分離した後、 21ゲージの注射針をつけたシリンジで細胞を分散さ せ、 Lymphoprep ( Nycomed社） に重層した。 これを 1 5 0 0 rpm、 25°C、 30分遠心 し界面に集まった細胞を回収した。 細胞を PBSで 2回洗浄した後、 染色バッファー (PBS、 5% FCSヽ 0.05% NaN3 ) に懸濁した。 Pregnant C57B1 / 6 mice were purchased from Nippon Charls River Inc. On the 14th day of pregnancy, the mouse was laparotomized and the fetus was aseptically removed. After carefully separating the fetal liver from contaminating other tissues, the cells were dispersed with a syringe fitted with a 21-gauge injection needle and layered on Lymphoprep (Nycomed). This was centrifuged at 1500 rpm at 25 ° C. for 30 minutes to collect cells collected at the interface. After washing the cells twice with PBS, the cells were suspended in a staining buffer (PBS, 5% FCS ヽ 0.05% NaN 3 ).
以後の細胞分画法の基本的な手技は、 おおむね、 Herzenberg, L.A. 「Weir' s H andbook of Experimental Immunology, 5th editionj , Blackwel l Science Inc . The basic procedures of the subsequent cell fractionation method are generally described in Herzenberg, L.A., `` Weir's H andbook of Experimental Immunology, 5th editionj, Blackwell Science Inc.
1997 にしたがって行なった。 抗 CD32抗体を添加し氷中に 10分放置した後、 細胞 懸濁液にピオチン化した分化抗原マ一力一、 つまり抗 CD8抗体、 抗 B220抗体、 抗 G Γ-l抗体、 抗 Terll9抗体を添加し、 氷中に 30分間放置した。 その後、 染色バッファ —で 2回洗浄の後アビジンマグネッ トビーズを添加し、 氷中 30分放置した。 再度 2 回染色バッファ一で洗浄の後、 磁石を用いて分化抗原を提示している細胞群を除
去し、 分化抗原陰性細胞（Lin—)を取得した。 分化抗原陰性細胞溶液に FITC標識抗 CD34抗体、 PE標識抗 Sca-1抗体、 テキサスレッ ド標識アビジン、 APC標識抗 c-kit抗 体を添加し、 氷中 30分放置した。 2回染色バッファ一で洗浄の後、 セルソー夕 —にて Lin— Sea- 1 +c-kit+細胞を選別した。 1997. After adding the anti-CD32 antibody and leaving it on ice for 10 minutes, the cell suspension was incubated with the differentiation antigen, which was biotinylated, that is, anti-CD8 antibody, anti-B220 antibody, anti-GII-l antibody, and anti-Terll9 antibody. And left on ice for 30 minutes. Then, after washing twice with a staining buffer, avidin magnet beads were added, and the mixture was left on ice for 30 minutes. After washing twice with the staining buffer again, remove the cells presenting the differentiation antigen using a magnet. Then, differentiated antigen-negative cells (Lin-) were obtained. FITC-labeled anti-CD34 antibody, PE-labeled anti-Sca-1 antibody, Texas Red-labeled avidin, and APC-labeled anti-c-kit antibody were added to the differentiation antigen-negative cell solution, and left on ice for 30 minutes. After washing twice with the staining buffer, Lin-Sea-1 + c-kit + cells were selected on a cell saw.
( 2) HES- 1遺伝子発現レトロウィルスの造血幹細胞への感染 (2) Infection of hematopoietic stem cells with retrovirus expressing HES-1 gene
レトロネクチン（宝酒造（株）） をコートした 24穴プレートに、 pMSCV-EHをトラン スフェク トした 0.5mlの GP+E-86上清、 さらに SCF(10ng/ml)、 Iい 6(10ng/ml)を含 む MEM -ひ、 10¾ FCSを 0.5ml添加した。 ここへ、 Lin— c-KIT+Sca- Γ 造血幹細胞を添 加し、 1週間培養した。 その後、 細胞を回収し、 コロニー形成能について検討した ( コロニー形成能は、 MEM-ひ培地に 0.9%メチルセルロース、 30% FCS、 0. 1%ゥシ血 清アルブミン、 及び 10ng/mlずつの SCF、 Iい 3、 IL- 6、 EP0、 TP0、 さらに、 0. 5mg /mlG418の存在下で実施した。 14日間の培養の後、 出現してくるコロニーの形態、 ならびに数を検出した。 尚、 上記で用いた各種造血因子は、 いずれもリコンビナ ント体であり、 純粋なものである。 On a 24-well plate coated with RetroNectin (Takara Shuzo Co., Ltd.), 0.5 ml of GP + E-86 supernatant transfected with pMSCV-EH, SCF (10 ng / ml), and I6 (10 ng / ml) And 0.5 ml of 10-FCS was added. To this, Lin-c-KIT + Sca-II hematopoietic stem cells were added and cultured for 1 week. Thereafter, the cells were collected and examined for colony forming ability (the colony forming ability was determined by adding 0.9% methylcellulose, 30% FCS, 0.1% serum albumin, and 10 ng / ml SCF The culture was performed in the presence of I-3, IL-6, EP0, TP0, and 0.5 mg / ml G418.After 14 days of culture, the morphology and number of emerging colonies were detected. The various hematopoietic factors used in the above are all recombinant and pure.
< 4〉結果 <4> Results
上記条件における実施結果を表 3に示す。 表中の数字は、 感染させた造血幹細 胞 2,000個あたりのコロニー数である。 表 3 感染レト Πウイ コロニーの形態 （ /2，000造血幹細胞） Table 3 shows the results under the above conditions. The numbers in the table are the number of colonies per 2,000 infected hematopoietic stem cells. Table 3 Morphology of infected lettuce wiwi colonies (/ 2,000 hematopoietic stem cells)
Large E-mix CFU-GM CFU-G CFU-M CFU-E 計 Large E-mix CFU-GM CFU-G CFU-M CFU-E Total
EGFP 0 25.5 8 89.5 41 164 EGFP 0 25.5 8 89.5 41 164
EGFP-HES-1 6.5 4 5.5 4 20.5 40.5EGFP-HES-1 6.5 4 5.5 4 20.5 40.5
1
表に示したコロニー数は G418に抵抗性のコロニーであり、 EGFP遺伝子のみ、 あ るいは EGFP- HES-1の融合遺伝子が導入された細胞のみを示す。 さらに、 これらの コロニーにおいて目的の遺伝子の発現は、 蛍光顕微鏡下で EGFPの発現を観察する ことでも確認した。 EGFP遺伝子のみを移入した細胞では、 細胞質全体が蛍光を発 しており核内での局在は確認されなかった。 一方、 EGFP-HES- 1の融合遺伝子が導 入された株では、 核が特異的蛍光を発していることが蛍光顕微鏡による観察から 確かめられ、 この HES-1が正常に発現され核内で機能するものと推測された。 1 The number of colonies shown in the table is a colony resistant to G418, and indicates only the EGFP gene alone or only the cell into which the EGFP-HES-1 fusion gene has been introduced. Furthermore, the expression of the target gene in these colonies was also confirmed by observing the expression of EGFP under a fluorescence microscope. In cells transfected with the EGFP gene alone, the entire cytoplasm fluoresced, and no localization in the nucleus was confirmed. On the other hand, in the strain into which the EGFP-HES-1 fusion gene was introduced, it was confirmed by fluorescence microscopy that the nucleus emitted specific fluorescence, and this HES-1 was normally expressed and functioned in the nucleus. Was speculated to be.
EGFP遺伝子のみを遺伝子移入したものでは、 未分化な造血細胞である large E- mixコロニーの形成は見られない。 一方、 HES- 1を遺伝子移入した細胞群では larg e E- mixコロニーの出現が観察されている。 これらのコロニーは非常に巨大なコロ ニーを形成しており、 造血幹細胞の分化が抑制されていたことを物語っている。 さらに EGFP遺伝子と EGFP- HES-1の融合遺伝子の遺伝子移入用のレトロウィルスの 感染効率について評価していないので、 全コロニー数についての比較はできない が、 EGFP遺伝子のみを移入した細胞群の方が、 全コロニー数が多いにも関わらず large E-mixコロニーは全く出現していない。 これらの結果をまとめると、 HES-1 を強制発現していない培養系では、 造血幹細胞は分化し、 造血前駆細胞である GM - CFC、 G-CFC、 M-CFCのみが出現してくるが、 HES- 1を高発現させた培養系では未分 化な造血細胞が維持されており、 造血幹細胞の分化を HES-1の遺伝子移入により調 節することに成功した。 産業上の利用性 本発明により、 造血幹細胞及び/または造血前駆細胞を、 分化を抑制した状態 で生存させ、 増殖させることができる。
When only the EGFP gene was transfected, the formation of large E-mix colonies, which are undifferentiated hematopoietic cells, was not observed. On the other hand, in the cell group into which HES-1 was transfected, the appearance of larg e-mix colonies was observed. These colonies form a very large colony, indicating that hematopoietic stem cell differentiation was suppressed. Furthermore, since the infection efficiency of the retrovirus for gene transfer of the EGFP gene and the EGFP-HES-1 fusion gene has not been evaluated, it is not possible to compare the total number of colonies, but the cell group to which only the EGFP gene was transferred was Despite the large number of total colonies, no large E-mix colonies appeared. To summarize these results, in a culture system that does not express HES-1, hematopoietic stem cells differentiate and only hematopoietic progenitor cells GM-CFC, G-CFC, and M-CFC appear. In the culture system in which HES-1 was highly expressed, undifferent hematopoietic cells were maintained, and the differentiation of hematopoietic stem cells was successfully regulated by HES-1 gene transfer. INDUSTRIAL APPLICABILITY According to the present invention, hematopoietic stem cells and / or hematopoietic progenitor cells can survive and proliferate in a state where differentiation is suppressed.
Claims
1. 哺乳動物の造血幹細胞中で分化抑制遺伝子の発現を強化すると共に、 血 液細胞刺激因子を作用させることにより、 前記造血幹細胞及び/または前記造血 幹細胞から分化した造血前駆細胞の分化および増殖を調節する方法。 1. The differentiation and proliferation of the hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells can be enhanced by enhancing the expression of the differentiation inhibitory gene in mammalian hematopoietic stem cells and by acting a blood cell stimulating factor. How to adjust.
2. 哺乳動物がヒトであり、 分化抑制遺伝子及び血液細胞刺激因子がヒト由 来またはその類縁体である請求項 1記載の方法。 2. The method according to claim 1, wherein the mammal is a human, and the differentiation inhibitory gene and the blood cell stimulating factor are derived from a human or an analog thereof.
3. 造血幹細胞が、 臍帯血、 胎児肝臓、 骨髄、 胎児骨髄、 または末梢血から 得られるものである請求項 1記載の方法。 3. The method according to claim 1, wherein the hematopoietic stem cells are obtained from cord blood, fetal liver, bone marrow, fetal bone marrow, or peripheral blood.
4. 分化抑制遺伝子が HE S— 1遺伝子、 HE S— 3遺伝子、 HE S— 5遺 伝子から選ばれるものである請求項 1記載の方法。 4. The method according to claim 1, wherein the differentiation-suppressing gene is selected from the HES-1 gene, the HES-3 gene, and the HES-5 gene.
5. 血液細胞刺激因子がサイ ト力イン、 インタ一ロイキン、 増殖刺激因子、 インタ一フエロン、 ケモカインから選ばれる請求項 1記載の方法。 5. The method according to claim 1, wherein the blood cell stimulating factor is selected from the group consisting of cytokin, interleukin, growth stimulating factor, interferon, and chemokine.
6. 血液細胞刺激因子が S C F、 I L— 3、 I L— 6、 GM— C S F、 TP 0、 Wnt、 D l l— 1、 D l l— 3、 J ag ge d— 1、 J agge d— 2、 D LKから選ばれる請求項 5記載の方法。 6. Blood cell stimulating factors are SCF, IL-3, IL-6, GM-CSF, TP0, Wnt, Dll-1, Dll-3, Jagge d-1, Jagge d-2, D 6. The method according to claim 5, wherein the method is selected from LK.
7. 分化抑制遺伝子の発現を、 ウィルスベクターに組込まれた分化抑制遺伝 子を造血幹細胞に導入することにより強化する請求項 1記載の方法。 7. The method according to claim 1, wherein the expression of the differentiation-suppressing gene is enhanced by introducing the differentiation-suppressing gene incorporated into the viral vector into hematopoietic stem cells.
8. ウィルスベクターが、 レトロウイルスベクター、 アデノウイルスベクタ ―、 ヘルぺスウィルスベクター、 H I Vベクターから選ばれる請求項 7記載の方 法。 8. The method according to claim 7, wherein the virus vector is selected from a retrovirus vector, an adenovirus vector, a herpes virus vector, and an HIV vector.
9. 分化抑制遺伝子が、 発現調節を受けるものである請求項 1記載の方法。 9. The method according to claim 1, wherein the differentiation-suppressing gene is one whose expression is regulated.
10. 分化抑制遺伝子の発現調節が、 テトラサイクリンによる調節、 ェクダイ ソンによる調節、 I P T Gによる調節から選ばれる請求項 9記載の方法。 10. The method according to claim 9, wherein the regulation of the expression of the differentiation-suppressing gene is selected from regulation by tetracycline, regulation by ecdysone, and regulation by IPTG.
1 1. 分化抑制遺伝子がウィルスベクタ一に組込まれた、 遺伝子導入べクタ一 ( 1 1. differentiation suppressor gene has been incorporated into the virus vector of all, gene transfer base Kuta one (
12. 分化抑制遺伝子が HE S― 1遺伝子、 HE S— 3遺伝子、 HE S— 5遺 伝子から選ばれる請求項 1 1記載の遺伝子導入ベクター。 12. The gene transfer vector according to claim 11, wherein the differentiation-suppressing gene is selected from HES-1 gene, HES-3 gene, and HES-5 gene.
13. ウィルスベクタ一がレトロウイルスベクタ一、 アデノウイルスベクター、 ヘルぺスウィルスベクタ一、 H I Vベクターから選ばれる請求項 1 1記載の遺伝
子導入べクタ一。 13. The gene of claim 11, wherein the virus vector is selected from a retrovirus vector, an adenovirus vector, a herpes virus vector, and an HIV vector. Child introduction vector.
1 4 . 分化抑制遺伝子の発現が強化された哺乳類の造血幹細胞。 1 4. Mammalian hematopoietic stem cells with enhanced expression of differentiation-suppressing genes.
1 5 . 分化抑制遺伝子の発現が強化された哺乳類の造血幹細胞又はこの造血幹 細胞から分化した造血前駆細胞を、 血液細胞刺激因子を作用させつつ培養するこ とを特徴とする造血幹細胞及び/または造血前駆細胞の生産方法。
15. A hematopoietic stem cell and / or a hematopoietic stem cell characterized in that a mammalian hematopoietic stem cell in which the expression of a differentiation inhibitory gene is enhanced or a hematopoietic progenitor cell differentiated from the hematopoietic stem cell is cultured with a blood cell stimulating factor. A method for producing hematopoietic progenitor cells.
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| WO2004072264A2 (en) * | 2003-02-12 | 2004-08-26 | Johns Hopkins University School Of Medicine | Fate determination by hes 1 in hematopoietic stem-progenitor cells and uses thereof |
| WO2004090121A2 (en) * | 2003-04-08 | 2004-10-21 | Yeda Research And Development Co. Ltd | Stem cells having increased sensitivity to a chemoattractant and methods of generating and using same |
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| WO1992018615A1 (en) * | 1991-04-09 | 1992-10-29 | Indiana University Foundation | System and process for supporting hematopoietic cells |
| WO1993018137A1 (en) * | 1992-03-04 | 1993-09-16 | Systemix, Inc. | Culturing of hematopoietic stem cells and their genetic engineering |
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| WO1992018615A1 (en) * | 1991-04-09 | 1992-10-29 | Indiana University Foundation | System and process for supporting hematopoietic cells |
| WO1993018137A1 (en) * | 1992-03-04 | 1993-09-16 | Systemix, Inc. | Culturing of hematopoietic stem cells and their genetic engineering |
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