WO2000014203A1

WO2000014203A1 - Method for preparing cell fraction containing hematopoietic stem cells

Info

Publication number: WO2000014203A1
Application number: PCT/JP1999/004768
Authority: WO
Inventors: Osamu Natori; Masahiko Tamura
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1998-09-02
Filing date: 1999-09-02
Publication date: 2000-03-16
Also published as: AU5448099A

Abstract

Hematopoietic stem cells are successfully separated at a higher yield than those achieved in the prior art by separating cells having the Lin(-)/Sca-1(+)/c-kit(+)/CD48(-) phenotype from myeloid cells by using a cell sorter.

Description

Method for preparing cell fraction containing hematopoietic stem cells

The present invention relates to a method for preparing a cell fraction containing hematopoietic stem cells, a cell fraction produced by the method, and a use of the cell fraction. Background art

Hematopoietic stem cells have the ability to differentiate into all blood cells including granulocytic cells (myeloid), lymphoid cells (lymphoid), erythroid cells (erythroid), megakaryocytic cells, etc., and Its existence as a cell capable of self-replication has been suggested for a long time. Research on hematopoietic stem cells has been performed using mouse and human bone marrow cells. So far, CFU-S (Till JE et al., Radiat. Res. 14, 213, 1961), HPP-CFC (Bradley TR Blood 54, 1446, 1979) and LTC-IC (Sutherl and HJ et al., Blood 74, 1563, 1989) have been proposed as candidates for hematopoietic stem cells. However, these cells do not necessarily have the properties of hematopoietic stem cells, and are all defined based on the activity detected by a special bioassay, and it is not possible to isolate these cells themselves. could not.

On the other hand, the elucidation of cell surface antigens and the advance of cell separation technology represented by FACS have made it possible to separate hematopoietic stem cells as a somewhat uniform cell population using the expression of specific surface antigens as an index. For example, regarding mice, differentiation antigen (Lin antigen) negative (Lin (-)) in each lineage of lymphocytes, granulocytes, monocytes / macrophages, and erythrocytes, Thy-1.1 antigen negative (Thy-l.l ( -))) And a cell fraction (Lin (-) / Thy-) / Sca-1 (+)) that is positive for Sea-1 antigen (Sea-1 (+)), or using WGA or C-kit , Lin (-) / Sca-l (+) / WGA (+) or Lin (-) / Sca-l (+) / c-kit (+) phenotype Fractions have been reported as a cell population containing hematopoietic stem cells (Spangrude GJ et al., Science 241, 58, 1988: Jurecic R. et al., Blood 82, 2673, 1993: Okada S. et al., Blood 80, 3044, 1992. ). Regarding humans, CD34 (+) / DR (-) and CD34 (+) / CD38 (-) have been reported as cell fractions containing hematopoietic stem cells (Brandt J. et al., J. CI in. Invest. 82, 1017, 1988: Sauvaugeau G. et al., Proc. Natl. Acad. Sci. US A 91, 12223, 1995). Especially in mice, the properties of the cells belonging to these cell fractions have been studied relatively in detail, because they can be used to elucidate diseases at the level of hematopoietic stem cells and to conduct basic studies for the development of new therapeutic methods. However, there remains a problem that cells obtained by the above-mentioned separation methods using surface antigens as indicators have a low percentage of hematopoietic stem cells. For example, in a cell group having a surface antigen of Lin (-) / Sca-l (+) / c-kit (+), the percentage of hematopoietic stem cells was reported to be 1/30 (Osawa, M. Et al., J. Immunol., 156: 3207-3214 (1996)). More recently, hematopoietic stem cells have been isolated from mouse bone marrow cells using the cell fraction with the Lin (-) / Sca-l (+) / c-kit (+) / CD34 (-) phenotype. Have been developed (Osawa M. et al., Science 273, 242, 1996). In this method, the isolated cell fraction contains more hematopoietic stem cells than the conventional method, but the number of cells obtained from a single mouse is as small as 50 to 100 cells, and a large amount of hematopoietic stem cells is required. It was difficult to conduct experiments, especially screening experiments aimed at searching for hematopoietic stem cell growth factors and drugs. Disclosure of the invention

The present invention provides a method for efficiently preparing a substantially uniform cell fraction containing hematopoietic stem cells at a high frequency, a cell fraction prepared by the method, and uses of the cell fraction. The task is to

The present inventors have conducted intensive studies based on the above-mentioned knowledge on the method for separating hematopoietic stem cells with the aim of initiating a more efficient method for preparing hematopoietic stem cells. Surprisingly, surprisingly, CD48, known as a surface antigen expressed on lymphocytes, NK cells, and monocytes, was combined with a known index as a new index for the isolation of hematopoietic stem cells. It has been found that by using such a cell fraction, a cell fraction containing hematopoietic stem cells at a high frequency and containing a sufficient number of hematopoietic stem cells to be used for screening experiments and the like can be prepared. Further, from an experiment of transplantation into X-irradiated female C57B1 / 6N mice, it was found that the cell fraction contained hematopoietic stem cells having pluripotency and long-term hematopoietic ability. Furthermore, the present inventors have found that it is possible to carry out screening for hematopoietic stem cell growth factor or the like using the cell fraction thus prepared, and have completed the present invention.

That is, the present invention relates to a method for efficiently preparing a cell fraction containing hematopoietic stem cells, a cell fraction prepared by the method, and a use of the cell fraction.

(1) Substantially containing mouse hematopoietic stem cells, characterized by separating cells having a Lin antigen-negative, Sea-1 antigen-positive, c-kit antigen-positive, and CD48 antigen-negative phenotype from mouse bone marrow cells Method for preparing a uniform cell fraction,

(2) The method according to (1), wherein cells further having a CD34 antigen-negative phenotype are isolated.

(3) a substantially uniform cell fraction containing mouse hematopoietic stem cells, prepared by the method according to (1) or (2);

(4) The cell fraction according to (3), which is an in vitro cultured cell,

(5) A cell composition comprising the cell fraction and the culture solution according to (3),

(6) a method for producing a chimeric mouse, which comprises transplanting the cell fraction according to (3),

(7) a chimeric mouse transplanted with the cell fraction according to (3),

(8) a chimeric mouse produced by transplanting the vesicle fraction described in (3) into a mouse with reduced or defective hematopoietic ability;

(9) Screen a compound that promotes the proliferation or differentiation of hematopoietic stem cells. The method

(a) administering the test compound to the chimeric mouse according to (8),

(b) detecting the proliferation or differentiation of hematopoietic stem cells transplanted into the chimeric mouse; and

(c) selecting a compound that promotes the proliferation or differentiation of hematopoietic stem cells as compared to the case where the test compound is not administered,

(10) A method for screening for a compound that promotes proliferation or differentiation of hematopoietic stem cells,

(a) contacting a test compound with the cell fraction according to (3),

(b) culturing the cell fraction contacted with the test compound,

(c) transplanting the cultured cell fraction into a mouse with reduced or defective hematopoietic ability,

(d) detecting the proliferation or differentiation of the hematopoietic stem cells transplanted into the mouse; and

(e) selecting a compound that promotes the proliferation or differentiation of hematopoietic stem cells as compared to the case where a cell fraction not contacted with the test compound is transplanted.

(11) A method for screening for a compound that promotes proliferation or differentiation of hematopoietic stem cells,

(a) contacting a test compound with the cell fraction according to (3),

(b) culturing a cell fraction contacted with the test compound to detect proliferation or differentiation of hematopoietic stem cells, and

(12) a compound which promotes proliferation or differentiation of hematopoietic stem cells, which can be isolated by the method according to any one of (9) to (11);

(13) The cell fraction according to (3), wherein the foreign gene has been introduced so as to be capable of expression. (14) The cell according to (13), wherein a foreign gene corresponding to the endogenous gene has been introduced into a non-human mammal having a specific expression system due to suppression of expression of the specific endogenous gene. Transplanting the fraction, and detecting whether or not the expression system is improved. A method for detecting a therapeutic effect on a disease caused by suppression of the expression of the specific endogenous gene,

(15) The cell fraction according to (13) for use in the detection method according to (14),

(16) A method for producing an antibody specific to hematopoietic stem cells, comprising a step of immunizing an animal with the cell fraction according to (3).

(17) An antibody specific to hematopoietic stem cells, which can be produced by the method according to (16),

(18) A method for isolating hematopoietic stem cells, comprising using the antibody according to (17),

(19) A method for quantifying hematopoietic stem cells, comprising using the antibody described in (17);

(20) A method for detecting a surface antigen specific to hematopoietic stem cells, comprising using the antibody according to (17), and

(21) A surface antigen specific to hematopoietic stem cells, which can be detected by the method described in (20).

The present invention firstly relates to a method for preparing a substantially uniform cell fraction containing hematopoietic stem cells at a high frequency. The method for preparing the cell fraction of the present invention includes Lin antigen negative, Sea-1 antigen positive, c-kit antigen positive, CD48 antigen negative (hereinafter referred to as Lin (-), Sea-1 (+), c- kit) and CD48 (-)) as an indicator. In the present invention, the “substantially uniform cell fraction” refers to a group of cells having the above phenotype. “High frequency” means that the proportion of hematopoietic stem cells in these cell groups is at least 1/24 or more, preferably 1/20 or more, more preferably 1/10 or more, and most preferably Preferably, it is 1/6 or more.

The ratio of hematopoietic stem cells contained in the cell group can be calculated, for example, from the ratio of the number of transplanted cells to the number of mice that have established kinulinism in the production of chimeric mice.

Whether or not the cell fraction contains hematopoietic stem cells can be determined, for example, by determining whether or not the prepared cell group has long-term hematopoietic ability, as described in Example 2.

That is, after transplantation of the cell fraction containing hematopoietic stem cells, at least 60 days, preferably 140 days or more, more preferably 1 year or more, at least one year later, the peripheral blood of the recipient mouse is collected and subjected to hypotonic high temperature. If the presence of blood cells derived from Donna can be confirmed by analyzing genes obtained by disrupting the cells and detecting markers such as the Sry gene, etc. It can be determined. The cell fraction containing hematopoietic stem cells can be collected from bone marrow cells, spleen, and peripheral blood, but it is preferable to use bone marrow cells because of their high abundance.

Cells of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) can be used, for example, for Lin, Sea-1, c-kit, and CD48 as shown in Example 1. Using an antibody, separation can be performed using a cell saw. In order to further separate hematopoietic stem cells, a CD34 antigen-negative (hereinafter, referred to as CD34 (-)) indicator may be used in combination. Anti-CD34 antibody labeled with a fluorescent substance (eg, FITC, PE, RED613, APC, Texas Red, etc.) different from that used to label antibodies to Lin, Sea-1, c-kit, and CD48 after separation using the above indicators By separating the fractions by Celso overnight, the fraction of CD34 (-) can be further separated. Also, antibodies to CD34 may be combined together.

The cell fraction of the present invention can be cultured using a general culture solution, for example, DMEM, MEM, RPMI 1640, IMDM, or the like. This culture contains various additives used for normal cell culture, such as fetal bovine serum, insulin, IL-3, IL-6, etc. Bone marrow cells or bone marrow stromal cells whose proliferation ability has been deleted by X-ray treatment or the like can be used as a feeder or, in some cases, a feeder cell that supports the survival and growth of cells.

The cell fraction prepared in the present invention can be used, for example, for producing a chimeric mouse useful for screening for a factor that promotes proliferation or differentiation of hematopoietic stem cells. In the production of the chimeric mouse, hematopoietic ability is lost or reduced by subjecting the recipient mouse to a lethal or semi-lethal X-ray whole-body irradiation, and the separated cell fraction is transplanted from the tail vein. Lethal dose to recipient mice

When irradiated with X-rays, bone marrow cells collected by a common method from mice syngeneic with the recipient were transplanted simultaneously as rescue cells with approximately 2 x 10 ⁵ to 10 ⁶ cells, resulting in early death after transplantation. Can be prevented. It is preferable to perform a lethal dose of X-ray irradiation in order to keep the rate of hematopoietic deterioration constant. In addition, if different sexes, leukocyte surface antigens or isozymes are used as markers in the donor cell and the recipe, it is possible to confirm that kinulinism has been established by detecting these differences.

Screening for a factor that promotes the proliferation or differentiation of hematopoietic stem cells using the chimeric mouse thus produced comprises: (a) a step of administering a test compound to the chimeric mouse; Detecting the proliferation or differentiation of the hematopoietic stem cells transplanted into the chimeric mouse; and (c) selecting a compound that promotes the proliferation or differentiation of the hematopoietic stem cells as compared to the case where the test compound is not administered, It can be carried out by a method including: There is no particular limitation on the test compound used in the screening, and examples thereof include cell culture supernatants, purified proteins or peptides, synthetic compounds, and natural products derived from microorganisms and plants. The test compound can be administered to the mouse by oral, pulmonary, intravenous, intradermal, subcutaneous, intraperitoneal and / or intraventricular administration. The detection of the proliferation or differentiation of the hematopoietic stem cells transplanted into the chimeric mouse can be performed, for example, by detecting a certain period of time after the administration of the test compound. Peripheral blood or bone marrow cells are collected from chimeric mice, and the number of donor-derived hematopoietic stem cells or the number of various blood cells such as lymphocytes and granulocytes generated by differentiation can be measured.

The number of hematopoietic stem cells derived from the donor can be measured using the surface antigen as a marker. For example, when donor cells having Ly5.1 as a surface antigen are transplanted into a recipient having Ly5.2 as a surface antigen, a combination of Ly5.1 antigen positive (Ly5.1 (+)) and bone marrow cells By measuring the percentage of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) / Ly5.1 (+) cells contained in The number of hematopoietic stem cells from the included donor can be calculated. The number of hematopoietic stem cells contained in spleen and peripheral blood can be similarly measured. The number of various blood cells can be measured by further combining a surface antigen specific to various blood cells with the donor. That is, in the measurement of B cells, the ratio of Ly5.1 (+) / B220 (+) cells should be measured using FACS by combining B220 antigen positive (B220 (+)) specific to B cells. Can be used to calculate the number of donor-derived B cells. Similarly, for granulocytes, the number of donor-derived granulocytes can be calculated by combining Gr-1 antigen positive (Gr-1 (+)).

In another aspect of the present invention, a screening of a factor that promotes proliferation or differentiation of hematopoietic stem cells is performed by transplanting the cell fraction of the present invention into a mouse after contacting a test compound with a test compound, and detecting proliferation or differentiation of hematopoietic stem cells. About the method. This screening method comprises: (a) a step of bringing a test compound into contact with the cell fraction of the present invention; (b) a step of culturing the cell fraction contacted with the test compound; (c) a cultivated cell fraction (D) a step of detecting the proliferation or differentiation of hematopoietic stem cells transplanted into the mouse, and (e) cells not contacting the test compound. Selecting a compound that promotes the proliferation or differentiation of hematopoietic stem cells as compared to the case where the fraction is transplanted. The test compound to be brought into contact with the cell fraction of the present invention in the screening includes, for example, the above-mentioned cell culture supernatant, purified tan Examples include proteins or peptides, synthetic compounds, natural products derived from microorganisms and plants, and supporting cells that are expected to produce factors that promote the growth or differentiation of the cell fraction of the present invention. And a compound produced by the feeder cells can be used as a test compound.

As a specific example of this screening, first, a test compound is added to the cell fraction of the present invention, or the cell fraction is transformed into some kind of supporting cells (for example, bone marrow-derived stromal cells, fibroblasts or And incubate it for a certain period of time. Then cultured cells were harvested and transplanted into 2 x l0 ^⁵ ~5 x l0 ⁵ cells rescue cells are both recipient mice were irradiated lethal dose X-ray. When using semi-lethal X-irradiated recipient mice, it is not necessary to transplant rescue cells. From the viewpoint of eliminating variations among individuals, it is preferable to perform a lethal dose of X-ray irradiation. In parallel with this, the same number of cell fractions as used for culture are transplanted into the same recipient mice. After a certain period of time after transplantation, it is possible to screen for factors that promote proliferation or differentiation of hematopoietic stem cells by comparing the percentage of donor-derived blood cells in the peripheral blood of the recipient transplanted with each cell. it can.

Another embodiment of the screening for a factor that promotes the proliferation or differentiation of hematopoietic stem cells of the present invention relates to in vitro screening. This screening comprises: (a) a step of bringing a test compound into contact with the cell fraction of the present invention; and (b) culturing the cell fraction contacted with the test compound, thereby expanding or differentiating hematopoietic stem cells. And (c) selecting a compound that promotes the proliferation or differentiation of hematopoietic stem cells as compared to the case where the test compound is not administered. The test compounds used in the screening include, as in the above-mentioned screening, cell culture supernatants, purified proteins or peptides, synthetic compounds, natural products derived from microorganisms or plants, and factors promoting proliferation or differentiation of hematopoietic stem cells. Compounds released by supporting cells that are expected to be produced are included. Of hematopoietic stem cells Proliferation can be measured by calculating the percentage of hematopoietic stem cells contained in the cells before and after the treatment. The proportion of hematopoietic stem cells contained in the cells can be measured by changing the number of cells to be transplanted during the production of the chimeric mouse and measuring the number of chimerism-established individuals. The differentiation of hematopoietic stem cells can be measured by FACS or a fluorescent antibody method using an antibody against an antigen specific to the differentiated cells. For example, B220 can be used as a specific antigen for B cells, Gr_l as a specific antigen for granulocytes, and TER119 as a specific antigen for erythroid cells. In addition, it can be measured by a colony assay such as CFU-GM, CFU-Mix, and CFU-S.

As a result of the above screening, if significant proliferation or differentiation of hematopoietic stem cells is detected, the test compound used in the screening is determined to be a factor that promotes proliferation or differentiation of hematopoietic stem cells. Such factors are particularly useful in developing therapeutic drugs for hematological diseases.

The cell fraction of the present invention can be used for judging the effect of gene therapy by introducing a foreign gene and transplanting it into a model experimental animal.

Specifically, a cell fraction into which a foreign gene has been introduced is transplanted into an animal in which the production of the gene product is not observed or reduced, and the production of the gene product is observed or reduced. The efficacy of treatment can be assessed based on whether the underlying phenotype improves.

For example, a C-kit gene can be introduced into a cell fraction containing hematopoietic stem cells derived from a W mouse, and this can be transplanted into a W mouse to evaluate the improvement of anemia. W mice are mice with abnormalities in the W locus on chromosome 5, and have the characteristic symptoms of macrocytic anemia, mast cell deficiency, infertility, and albinism (Russell, ES, Adv. Genet. 20, 357-459, 1979). c-kit is a receptor type 1 tyrosine kinase present at the W locus (Chabot, B., Nature, 335, 88-89, 1988), and may be expressed in immature hematopoietic cells of bone marrow cells. (0gawa, M., J. Exp. Med. 174, 63-71, 1991). Therefore, immature hematopoietic cells with mutations in c-kit It is thought to be anemic without functioning.

The introduction of the foreign gene into the cell fraction of the present invention can be performed by the calcium phosphate method (Viology (1973) 52, 456-467) or the electroporation method (EMBO J. (1982) 1, 841-845). It can be performed by a method known to a trader.

Further, the present invention relates to an antibody specific to hematopoietic stem cells. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and various special antibodies (for example, Fab, F (ab) ₂ , single-chain antibodies, humanized antibodies, human antibodies, and the like). The antibody of the present invention can be prepared by a method known to those skilled in the art. In the case of a polyclonal antibody, for example, the cell fraction of the present invention can be obtained by immunizing a foreign animal such as rat, mouse, and egret by a conventional method, and purifying the antibody fraction from serum. it can. Monoclonal antibodies can also be prepared by preparing hybridomas from spleen cells of immunized animals by a conventional method. Cell fusion can be performed basically according to a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

To determine whether the obtained antibody is specific to hematopoietic stem cells, the ability of the obtained antibody to bind to hematopoietic stem cells using FACS was determined using a fluorescently labeled antibody that recognizes immunoglobulin in immunized animals. This can be done by analysis. Commercially available fluorescently labeled antibodies can be used.

If an antibody specific to hematopoietic stem cells, which is found by the above method, is used, hematopoietic stem cells can be easily isolated. For example, the cells to which the antibody binds can be collected by Celso overnight using the fluorescently labeled antibody. In addition, hematopoietic stem cells can be quantified using the antibody. For example, the number of cells to which the fluorescently labeled antibody has bound can be measured by FACS. Furthermore, if a surface antigen of hematopoietic stem cells to which the antibody binds is identified, it is possible to find a surface antigen specific to hematopoietic stem cells. For example, using cells in which the surface antigen has been confirmed to be expressed, the membrane fraction is separated from the cells, and after solubilization, the antibody is isolated. Purification methods such as affinity chromatography, gel filtration chromatography, ion-exchange chromatography, and HPLC can be used to purify the antibody with the ability to bind to an antibody as an index. The purified protein can be analyzed for amino acid to identify a partial sequence of the amino acid, and further, a probe having a corresponding nucleotide sequence can be used to identify a gene from the cDNA library. In addition, a cDNA library of cells in which the surface antigen has been confirmed to be expressed can be prepared, and the gene can be isolated using the expression-cloning method using the binding ability to the antibody as an index.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the gate settings used for cell separation by Yuichi Celsoo.

Bone marrow cells having a specific gravity of 1.063 to 1.077 were gated for normal cells (R1 in the figure) by 88 (side scatter) and FS (forward scatter). Next, the gate of Lin (-) / c-kit (+) cells (R2 in the figure) was set with Lin (Red613) and c-kit (Cy5), and finally, Sca-l (PE) and CD48 (FITC ), Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) (R3 in the figure) and Lin (-) / Sca-l (+) / The gate of c-kit (+) / CD48 (+) (R4 in the figure) was set. The number of cells obtained by these gate settings and the calculated percentage of bone marrow cells are also shown.

Figure 2 shows Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells (denoted as CD48 (-) in the figure) and Lin (-) / Sca-l (+) / c-kit (+) / CD48 (+) cells (denoted as CD48 (+) in the figure) were transplanted at 4 (6) and 40 (5) cells, respectively, on day 147 after transplantation. The results of PCR using mouse peripheral blood are shown.

In both figures, the left end shows the molecular weight. ΒΜ (ιη) is a bone marrow cell of a male mouse, and BM (f) is a result of PCR using a bone marrow cell of a female mouse, which shows a positive control and a negative control, respectively. The results at 147 days after transplantation in Table 1 are also shown below the figure. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Separation of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells

The femur was excised from the male C57B1 / 6N mouse, and both ends were cut. Then, bone marrow cells were collected using 2 ml of IMDM medium (GIBC0) containing 10% FBS. The obtained bone marrow cell-containing solution was once passed through a mesh to remove bone fragments, centrifuged at 1,000 rpm (200 xg) for 5 minutes (Hitachi, 05RP22), and the supernatant was removed to obtain a pellet of bone marrow cells. . The pellet of the obtained bone marrow cells was suspended in 2 ml of Nicodenz (specific gravity: 1.063) (Nycomed). This was gently layered on 2 ml of Nicodenz (specific gravity 1.077), and then centrifuged at 2300 rpm (1000 X g) for 30 minutes to collect cells that had stopped at the boundary region between the two Nicodenz layers with different specific gravities. . The obtained cells were washed twice with one FACS buffer (PBS containing 2% FCS), and finally suspended in 50-1 FACS buffer. To this cell suspension, a solution of a biotin-labeled antibody against the following molecules was added. Mouse CD3 £, mouse CD4 5R (B220), mouse Ly-6G (Gr-1), mouse CDllb (Mac-1), mouse TER119 (all purchased from Pha rmingen). The concentration of each of these antibody solutions was 0.5 mg / ml, and lg (21) was added per 1 × 10 ⁶ cells. The cell suspension to which the antibody was added was allowed to stand on ice for 30 minutes. One milliliter of FACS buffer was added, and the mixture was centrifuged at 5,000 rpm (2000 × g) for 1 minute (KUBOTA, KM-15200). The obtained cells were suspended again in FACS buffer 50-1. An avidin-coated magnetic bead suspension (Immunotech) was added to the cells in an amount of ^{8 to 1} per lxlO ⁶ cells, allowed to stand on ice for 30 minutes, and then resuspended in 1 ml of FACS buffer. The cells were collected by centrifugation (KUB0TA, KM-15200) at 5,000 rpm (2000 xg) for 1 minute. 50〃 back Nigoshi suspended in 1 FACS buffer one, the following fluorescent-labeled antibody solution was added L〃g per cell IX 10 ^6. Strepta vidin-Red 613 (0.25 mg / ml) (GIBC0 BRL), APC-labeled anti-mouse c-kit antibody (0.1 mg / ml) (Pharmingen), PE-labeled anti-Sca-1 antibody (0.2 mg / ml) ( Pharmingen), FITC-labeled anti-mouse CD48 antibody (0.5 mg / ml) (Pharmingen). The cell suspension to which these fluorescent-labeled antibodies had been added was allowed to stand under ice cooling for 30 minutes, centrifuged at 5000 rpm (2000 × g) for 1 minute, and then suspended in 1 ml of FACS buffer. The obtained cell suspension was separated by Celso Ichiichi (Cole Yuichisha, EPICS elite ESP), and a cell fraction having a desired phenotype was collected. Fig. 1 shows cell separation by Celso overnight. Finally, the frequency of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells in femur bone marrow cells was about 0.0006%, Finally, 400 to 500 cells were obtained from two femurs of male C57B1 / 6N mice.

[Example 2] Evaluation of long-term hematopoietic ability

After subjecting female C57B1 / 6N mice to 9 Gy whole body X-ray irradiation, a certain number of hematopoietic stem cell fraction cells derived from male mice and 1 × 10 ⁶ bone marrow cells derived from female mice as a recipient X The whole body was irradiated from the tail vein of a mouse. Peripheral blood of these mice is collected periodically after transplantation, and the Sry gene specifically expressed in OS cells is detected by PCR (Kunieda T. et al., Biol. Reproduction 46, 692, 1992). ) Thus, the presence or absence of hematopoiesis by hematopoietic stem cells derived from male mice was evaluated. That is, peripheral blood obtained by orbital blood collection from recipient mice was diluted 10-fold with MilliQ water (Millipore), boiled in a boiling water bath for 15 minutes, cooled with water, and cooled at 14,000 rpm for 5 minutes. Centrifuged supernatant (T0MY, MRX-150) 20-1 was used as type II PCR. The following synthetic oligo DNA (requested by Cymedia for synthesis) was used as a primer. Sryl (SEQ ID NO: 1 / 5'-GTGAGAGGCACAAGTTGGC-3 '), Sry2 (SEQ ID NO: 2/5, -TCTT AAACTCTGAAGAAGAGAC-3'), Sry3 (SEQ ID NO: 3/5, -CTCTGTGTAGGATCTTCAATC-3,) ヽ Sry4 (SEQ ID NO: 4/5, -GTCTTGCCTGTATGTGATGG-3,). Buffer for PCR reaction (Yukara Co., Ltd.) for type II of 20〃1, 10〃1, 2.5 mM dNTP solution (Yukara Co., Ltd.) 8〃1, 20 pM Sry2 Primer 2、1, 20 pM Sry4 Primer 2 〃1 ぉ ^^ 111 (3 water 57 .5 / 1 was added to give a reaction solution. After 3 cycles of reaction at 94 ° C for 8 minutes and 60 ° C for 2 minutes, 0.51 of Taq polymerase (Yukara) was added. C 1 minute, 60 ° C 2 minutes 30 seconds, 72 ° C 2 minutes 30 seconds reaction was performed 30 cycles. Then, add 10 反応 1, a buffer for PCR reaction 10 PCR1, 2.5 mM dNTP solution 8〃1, 20 pM Sryl primer 2〃1, 20 pM Sry3 primer ΙμΛ MilliQ water 67.5〃1 and Taq polymer 0.5〃1 of Ize was added, and 30 cycles of reaction at 94 ° C for 1 minute, 60 ° C for 2 minutes and 30 seconds, and 72 ° C for 2 minutes and 30 seconds were performed. The solution 7.5-5 after the PCR reaction was subjected to electrophoresis on a 4 ° agarose gel (NuSieve GTG agarose, Yukara), and a 147 bp Sryl-3 product band was detected by a transilluminator. The measurement was performed using male bone marrow cells and female bone marrow cells as positive and negative controls, respectively. FIG. 2 shows the results of PCR using peripheral blood of mice 147 days after transplantation.

A similar experiment was performed to detect male-derived cells 42 days and 72 days after transplantation and to evaluate long-term hematopoietic ability. Table 1 shows the results. In the table, "CD48 (-)" indicates Lin (-) / Sea-l (+) / c-kit (+) / CD48 (-) cells, and "CD48 (+)" indicates Lin (-) / Sca -l (+) / c-kit (+) / CD48 (+) cells.

table 1

Long-term hematopoietic activity of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells Transplanted cells Transplanted cells Number of recipients with donor-derived hematopoiesis 42 days after transplantation Transplant 72 Days after transplant 147 days after transplant

CD48 (-) 4 pieces 8/10 5/8 1/6 CD48 (-) sure 5/5 5/5 4/5 CD48 (+) 4 pieces 0/10 0/7 0/6 CD48 (+) 40 pieces 5/8 4/6 1/5

In mice transplanted with 4 or 40 Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells, 1/6 (17) Hematopoiesis derived from Lin (-) / Sea-l (+) / c-kit (+) / CD48 (-) cells was observed at a rate of 4/5 (80). On the other hand, when Lin (-) / Sca-l (+) / c-kit (+) / CD48 (+) cells, which are counterparts of these cells, were transplanted, 147 days after transplantation In the case of mice transplanted with 4 cells, only 0 (6%) of mice transplanted with 4 cells, and only 1 out of 5 mice (20%) transplanted with 40 cells had Lin (-) / Sca-l ( +) / c-kit (+) / CD48 (+) cells only showed hematopoiesis. Therefore, it was shown that most of the hematopoietic stem cells having long-term hematopoietic ability were contained in the fraction of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-).

[Example 3] Evaluation of pluripotency to lymphocytes, monocytes / macrophage cells, female C57B1 / 6N (Ly5.2) mouse (Charles River Japan, Inc.) Bone marrow cells were subjected to density gradient centrifugation to obtain specific gravity of 1.063. Cells floating in the 1.077 border region were collected. These cells were used for mouse CD3e, mouse CD45R (B220), mouse Ly-6G (Gr-1), mouse CDllb (Mac -1), a biotin-labeled antibody solution to mouse TER119 (0.5 mg / ml) (all Pharmingen) was added at l〃g per 1 × 10 ⁶ cells, suspended, and allowed to stand under ice-cooling for 30 minutes. After washing with 1 ml of FACS buffer, add a magnetic beads suspension (available from Avidin Co., Ltd., I-Unotech) 8 細胞¹ per ⁶ cells of lxlO, allow to stand under ice-cooling for 30 minutes, and add 1 ml of FACS buffer. Resuspended. The cells were collected by magnetic separation at Perseptive Diagnostics (Solo-Sep), and the fraction not adsorbed to the magnet was collected and centrifuged at 5000 rpm (2000 xg) for 1 minute (KUB0TA, KM-15200). Lin (-) fraction Cells were used.

A female C57B1 / 6N (Ly5.2) mouse (Nippon Charles River Co., Ltd.) subjected to 9 Gy X-ray whole-body irradiation was used as a recipient mouse, and 100 male C57B1 / 6-Ly5.1 mice (0 sawa, M. et al., J. Immunology, Vol. 156, 3207 (1996)) from Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) fraction cells and ⁴ lxlO cells. The Lin (-) fraction cells were transplanted from the tail vein. On the 71st day after transplantation, peripheral blood of recipient mice was collected, and the presence or absence of hematopoiesis by hematopoietic stem cells derived from Ly5.1 mice was evaluated. That is, an equal amount of a 0.7% citrate-containing MEM medium (GIBC0) containing 0.5 μl of Fc block (0.5 mg / ml) (Pharmingen) was added to 20 μl of peripheral blood obtained by orbital blood collection from a recipient mouse. After mixing at room temperature for 10 minutes, 0.5 μl of TRC-labeled anti-mouse CDllb (Mac-1) antibody (O.lmg / ml) (Caltag), 0.5 μl of PE-labeled anti-Thyl.2 antibody (Caltag) 0.2 mg / ml) (Pharmingen), 1.5 μl of PE-labeled anti-CD45R (B220) antibody (O.lmg / ml) (Caltag), 0.5 μl of FITC-labeled anti-CD45.2 (Ly5.1) antibody ( 0.5 μg / ml) (Pharmingen) and 10 μ 10 of a MEM medium supplemented with 0.7% citrate were added, followed by standing at room temperature for 30 minutes. To this, 1 ml of IxFACS Lysing solution (Becton Dickinson) was added, mixed, allowed to stand at room temperature for 10 minutes, washed twice with 1 ml of FACS buffer, and resuspended in 250 μ 懸濁 of FACS buffer. Ly5.1 (+) / (B220 (+) + Thyl.2 (+)) cells and Ly5.1 (+) / Mac-1 () in this cell suspension using FACScan (Beet on Dickinson) +) The percentage of cells was measured, confirming lymphoid hematopoiesis and monocyte / macrophage hematopoiesis from donors, respectively. Table 2

Pluripotency of Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells Individual number Donna-derived lymphocytes Donna-derived monocytes / macrophage

Percentage of lineage cells (%) Percentage of lineage cells (%)

1 22.27 21.04

2 23.43 5.52

3 0.46 9.75

Mice transplanted with Lin (-) / Sca-l (+) / c-kit (+) / CD48 (-) cells showed that Lin (-) / Sca-L Hematopoiesis derived from -l (+) / c-kit (+) / CD48 (-) cells was observed in both lymphocyte and monocyte / macrophage lineages. Therefore, it was shown that the Lin (-) / Sea-l (+) / c-kit (+) / CD48 (-) fraction contains pluripotent and long-term hematopoietic stem cells. Was. Industrial applicability

According to the present invention, a method for preparing a cell fraction containing hematopoietic stem cells at a high frequency was provided. The cell fraction of the present invention can exhibit high long-term hematopoietic activity in chimeric mice. The cell fraction of the present invention can be used in a wide range of applications, such as screening for factors that induce proliferation and differentiation of hematopoietic stem cells, judging the effects of gene therapy, and screening for antibodies or antigens specific to hematopoietic stem cells.

Claims

The scope of the claims

1. Substantially containing mouse hematopoietic stem cells, characterized by separating cells with phenotype of Lin antigen negative, Sea-1 antigen positive, c-kit antigen positive and CD48 antigen negative from mouse bone marrow cells Method for preparing a homogeneous cell fraction.

2. The method according to claim 1, wherein cells further having a CD34 antigen-negative phenotype are isolated.

3. A substantially uniform cell fraction containing mouse hematopoietic stem cells, prepared by the method according to claim 1 or 2.

4. The cell fraction according to claim 3, which is an in vitro cultured cell.

5. A cell composition comprising the cell fraction according to claim 3 and a culture solution.

6. A method for producing a chimeric mouse, comprising transplanting the cell fraction according to claim 3.

7. A chimeric mouse into which the cell fraction according to claim 3 has been transplanted.

8. A chimeric mouse produced by transplanting the cell fraction according to claim 3 into a mouse having reduced or defective hematopoietic ability.

9. A method for screening for a compound that promotes proliferation or differentiation of hematopoietic stem cells,

(a) administering the test compound to the chimeric mouse according to claim 8,

(c) a step of selecting a compound that promotes the proliferation or differentiation of hematopoietic stem cells as compared to the case where the test compound is not administered.

10. A method for screening for a compound that promotes proliferation or differentiation of hematopoietic stem cells,

(a) contacting a test compound with the cell fraction according to claim 3, (b) culturing the cell fraction contacted with the test compound,

1 1. A method for screening for a compound that promotes proliferation or differentiation of hematopoietic stem cells,

(a) contacting a test compound with the cell fraction according to claim 3,

(c) selecting a compound that promotes the proliferation or differentiation of hematopoietic stem cells as compared to the case where the test compound is not administered.

12. A compound which promotes proliferation or differentiation of hematopoietic stem cells, which can be isolated by the method according to any one of claims 9 to 11.

13. The cell fraction according to claim 3, wherein the exogenous gene has been introduced so that it can be expressed.

14. The cell fraction according to claim 13, wherein a foreign gene corresponding to the endogenous gene has been introduced into a non-human mammal having a specific expression system by suppressing the expression of the specific endogenous gene. A method for detecting a therapeutic effect on a disease caused by suppression of expression of the specific endogenous gene, wherein the method comprises detecting whether or not the expression system is improved.

1 5. The cell fraction according to claim 13 for use in the detection method according to claim 14.

1 6. A method for producing an antibody specific to hematopoietic stem cells, comprising a step of immunizing an animal with the cell fraction according to claim 3.

17. An antibody specific to hematopoietic stem cells, which can be produced by the method according to claim 16.

18. A method for isolating hematopoietic stem cells, comprising using the antibody according to claim 17.

19. A method for quantifying hematopoietic stem cells, comprising using the antibody according to claim 17.

20. A method for detecting a surface antigen specific to hematopoietic stem cells, comprising using the antibody according to claim 17.

21. A surface antigen specific to hematopoietic stem cells, which can be detected by the method according to claim 20.