Composition comprising CCL5 for promoting activation of bone marrow-derived stem cells

Abstract

The present invention relates to a composition comprising C-C motif chemokine ligand 5 (CCL5) for promoting the activation of bone marrow-derived stem cells. It is identified that, when CCL5, which is a niche-enhancing factor, is injected together with growth related oncogene beta (GROβ), which is known to promote the mobilization of hematopoietic stem cells from the bone marrow into peripheral blood, and AMD3100, which is a CXCR4 antagonist, the migration of hematopoietic stem cells from bone marrow to peripheral blood increases, and the engraftment rate also increases when the hematopoietic stem cells are injected into recipients undergoing bone marrow transplantation. Therefore, the CCL5 protein, GROβ, and the CXCR4 antagonist AMD3100 are provided as a composition for promoting the activation of bone marrow-derived stem cells, and thus bone marrow-derived stem cells activated thereby is provided as a novel therapeutic means for bone marrow diseases requiring bone marrow transplantation.

Description

Composition for promoting the activation of bone marrow-derived stem cells containing CCL5

[0001] The present invention relates to a composition for promoting the activation of bone marrow-derived stem cells comprising CCL5. The present invention was carried out under Project No. KH140743 with the support of the Ministry of Health and Welfare of the Republic of Korea, the managing agency for the said project is the Korea Health Industry Development Institute, and the project title is "Development of Novel Erythrocyte Differentiation and Proliferation Technology through Elucidation of Differentiation Dynamics of High-Efficiency Mobilized Hematopoietic Stem Progenitor Cells Based on Nisch Enhancement Factor." Additionally, the present invention was carried out under Project No. 2022R1A5A2027161 with the support of the Ministry of Science and ICT of the Republic of Korea, the managing agency for the said project is the National Research Foundation of Korea, and the project title is "Molecular Control Research Center for Cancer Cell Diversity." In addition, the present invention was carried out under project number 00333287 with the support of the Ministry of Science and ICT of the Republic of Korea, the managing agency of the said project is the National Research Foundation of Korea, and the project title is "Research on the control of differentiation induction of immature leukemia / cancer stem cells based on novel differentiation-promoting factors in the bone marrow microenvironment and research on the treatment of blood cancer."

[0002] Hematopoietic stem cells are the original parent cells of blood cells, such as white blood cells, red blood cells, and platelets. Blood cells produced in the bone marrow migrate to the peripheral blood after maturation. Most patients with blood diseases have poor hematopoietic stem cells and are unable to produce healthy blood cells using their own stem cells. Therefore, most patients with blood diseases undergo treatment involving the transplantation of healthy hematopoietic stem cells, which is called hematopoietic stem cell transplantation.

[0003] Hematopoietic stem cell transplantation is a representative treatment for blood disorders such as various leukemias, aplastic anemia, lymphomas, blood cancers, and multiple myeloma, as well as immunodeficiency and autoimmune diseases. The procedure involves administering large doses of chemotherapy or irradiating the recipient with the entire body to suppress the function of problematic bone marrow cells, followed by the transplantation of bone marrow from a healthy donor. The transplantation requires the collection of hematopoietic stem cells from a donor, and the engraftment rate of the donor's stem cells upon infusion into the recipient determines the effectiveness of the treatment.

[0004] In most cases, hematopoietic stem cells are collected by inserting a needle into the bone behind the pelvis. However, recently, methods have been developed to collect hematopoietic stem cells from peripheral blood by injecting drugs that migrate them from the bone marrow to the peripheral blood. A representative method used is collecting hematopoietic stem cells using G-CSF, but this requires daily injections for four to five days, and to obtain a sufficient number of stem cells, 150 to 200 ml of blood per kilogram of the donor's body weight must be processed using a blood component apheresis device. Therefore, there are limitations in collecting a high volume of hematopoietic stem cells in a short period. Furthermore, it often takes a considerable amount of time for the stem cells to engraft after transplantation into the recipient, and the success of the treatment depends on how quickly bone marrow function recovers.

[0005] Therefore, in order to effectively treat diseases requiring bone marrow transplantation, methods are continuously being researched to not only easily collect hematopoietic stem cells from peripheral blood but also to improve the engraftment rate after transplantation.

[0006] The objective of the present invention is to provide a new means for improving the collection and therapeutic effects of bone marrow-derived stem cells that can be used to treat diseases requiring bone marrow transplantation, by confirming that the niche reinforcing factor CCL5 (CC motif chemokine ligand 5) not only increases the migration of bone marrow-derived hematopoietic stem cells into peripheral blood but also increases the engraftment rate of the hematopoietic stem cells migrated into peripheral blood within the recipient.

[0007] The present invention provides a composition for promoting the activation of bone marrow-derived stem cells, comprising CCL5 (CC motif chemokine ligand 5) protein as an active ingredient.

[0008] In addition, the present invention provides a pharmaceutical composition for treating bone marrow disease comprising, as an active ingredient, bone marrow-derived stem cells isolated from a patient who was administered CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist.

[0009] In addition, the present invention provides a method for treating a bone marrow disease comprising the step of administering to a subject a composition containing, as an active ingredient, bone marrow-derived stem cells isolated from a patient who received CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist.

[0010] According to the present invention, when CCL5 (CC motif chemokine ligand 5), a niche enhancing factor, is injected together with GROβ (growth related oncogene beta) and AMD3100, a CXCR4 antagonist known to promote the migration of hematopoietic stem cells from bone marrow to peripheral blood, not only is the migration of hematopoietic stem cells from bone marrow to peripheral blood increased, but the engraftment rate is also increased when the hematopoietic stem cells are injected into a recipient receiving a bone marrow transplant. By confirming this, CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist, are provided as a composition for promoting the activity of bone marrow-derived stem cells, thereby providing activated bone marrow-derived stem cells as a new therapeutic means for bone marrow diseases requiring bone marrow transplantation.

[0011] Figures 1A, 1B, and 1C show the results of evaluating the effects of niche factors FGF21 (fibroblast growth factor 21) and CCL5 (CC motif chemokine ligand 5) on hematopoietic stem cell proliferation in bone marrow cells.

[0012] Figure 2 shows the results of evaluating the effects of GROβ and AMD3100 on the number of hematopoietic stem cells and progenitor cells in bone marrow cells.

[0013] Figures 3A and 3B show the results of evaluating the effects of GROβ and AMD3100 on the migration of hematopoietic stem cells in mice.

[0014] Figures 4A and 4B show the results of evaluating the effect of co-administration of GROβ, AMD3100, and Nisch enhancing factor on the migration of hematopoietic stem cells in mice.

[0015] Figure 5 shows the results of evaluating whether the engraftment ability of activated hematopoietic stem cells in recipient mice changes as a result of co-administering GROβ, AMD3100, and CCL5 to donor mice.

[0016] Figures 6A and 6B show the results of analyzing the effect of CCL5 deficiency on the migration of hematopoietic stem cells increased by GROβ and AMD3100.

[0017] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0018] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0019] The present invention will be described in more detail below.

[0020] The present invention provides a composition for promoting the activation of bone marrow-derived stem cells, comprising CCL5 (CC motif chemokine ligand 5) protein as an active ingredient.

[0021] The above composition further includes AMD3100, a GROβ (growth related oncogene beta) and CXCR4 antagonist.

[0022] The above bone marrow-derived stem cells are hematopoietic stem cells and progenitor cells, and specifically, the above bone marrow-derived stem cells are LK (Lin - cKit + ) cells, LKS(Lin - cKit + Sca1 + ) cells, HPC (hematopoietic progenitor cells) cells (Lin - cKit + Sca1 + CD48 + ), and HSC (hematopoietic stem cells) cells (Lin - cKit + Sca1 + CD48 - CD150 + It is one or more selected cells among ).

[0023] The activation of the bone marrow-derived stem cells means that the migration of hematopoietic stem cells present in the bone marrow to the peripheral blood increases upon injecting the above composition; furthermore, this means that not only is the collection of hematopoietic stem cells advantageous due to the migration to the peripheral blood, but the engraftment rate is also significantly superior in recipients requiring bone marrow transplantation because the collected hematopoietic stem cells are activated.

[0024] In addition, the present invention provides a pharmaceutical composition for treating bone marrow disease comprising, as an active ingredient, bone marrow-derived stem cells isolated from a patient who was administered CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist.

[0025] The above-mentioned bone marrow-derived stem cells have significantly superior engraftment ability within the recipient.

[0026] The above bone marrow disease is one or more diseases selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, aplastic anemia, multiple myeloma, malignant lymphoma, myeloproliferative neoplasm, myelodysplastic syndrome, paroxysmal nocturnal hemoglobinuria, immunodeficiency, hemoglobin dysplasia, Fanconi anemia, systemic lupus erythematosus, and induced secondary bone marrow failure.

[0027] The pharmaceutical composition of the present invention may be manufactured in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0028] The above-mentioned pharmaceutically acceptable carriers are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0029] In the present invention, the content of the additive included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional formulations.

[0030] The above pharmaceutical composition may be formulated into one or more external forms selected from the group consisting of injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, and cataplasms.

[0031] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and a diluent for formulation. The pharmaceutically acceptable carrier and diluent include, but are not limited to, excipients such as starch, sugar, and mannitol; fillers and extenders such as calcium phosphate; cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose; binders such as gelatin, alginates, and polyvinylpyrrolidone; lubricants such as talc, calcium stearate, hydrogenated castor oil, and polyethylene glycol; disintegrants such as povidone and crospovidone; and surfactants such as polysorbate, cetyl alcohol, and glycerol. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically affinity to the target. Examples of diluents include, but are not limited to, saline solution, aqueous buffer solution, solvent, and / or dispersion media.

[0032] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. For oral administration, it may be formulated into tablets, troches, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. For parenteral administration, it may be formulated into injectable solutions, suppositories, powders for respiratory inhalation, aerosols for sprays, ointments, powders for topical application, oils, creams, etc.

[0033] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and weight, age, gender, health status, dietary constitutional specificity, properties of the formulation, degree of disease, time of administration of the composition, method of administration, duration or interval of administration, excretion rate, and form of the drug, and may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg / kg, but is not limited thereto, and may be administered once or several times a day.

[0034] The above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method. The pharmaceutical effective amount and effective dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, method of administration, time of administration and / or route of administration, etc., and a person skilled in the art can easily determine and prescribe a dosage effective for the intended treatment. The pharmaceutical composition of the present invention may be administered once a day or divided into several doses.

[0035] In addition, the present invention provides a method for treating a bone marrow disease comprising the step of administering to a subject a composition containing, as an active ingredient, bone marrow-derived stem cells isolated from a patient who received CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist.

[0036] Since the present method utilizes the composition described above, redundant details are omitted to avoid excessive complexity in this specification.

[0037]

[0038] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0039]

[0040] Example 1. Effects of Nisch-enhancing factors FGF21 and CCL5 on hematopoietic stem cell proliferation

[0041] The effects of FGF21 (fibroblast growth factor 21) and CCL5 (CC motif chemokine ligand 5), factors of the bone marrow hematopoietic stem cell niche, on hematopoietic stem cell proliferation were evaluated in mouse bone marrow cells. The FGF21 and CCL5 used in this example were purchased from PeproTech. After separating the femur and tibia of mice, both ends of the bones were cut, and bone marrow was extracted by passing flow cytometry buffer into the bone using a syringe. Red blood cells were removed from the bone marrow using ACK lysis buffer, and lymphocytes were obtained. These lymphocytes were then treated with FGF21 or CCL5 at a concentration of 200 ng / mL and cultured for 48 hours. After 48 hours of culture, lymphocytes were collected, treated with fluorescent antibodies, and then subjected to flow cytometry to identify undifferentiated hematopoietic stem cells, specifically LKS cells (Lin - cKit + Sca1 + ) and HPC cells, which are hematopoietic progenitor cells (Lin - cKit + Sca1 + CD48 + The ratio of ) was analyzed.

[0042] As shown in Figure 1A, treatment of mouse bone marrow with the Nisch enhancing factor FGF21 resulted in an increase in the number of hematopoietic stem cells and progenitor cells. As shown in Figure 1B, treatment of the bone marrow of UT2-deficient (UT2 KO) mice (Mx1 Cre) with FGF21 resulted in a greater increase in the number of hematopoietic stem cells and progenitor cells compared to wild-type (WT). As shown in Figure 1C, treatment of mouse bone marrow with the Nisch enhancing factor CCL5 resulted in a decrease in the number of hematopoietic stem cells and progenitor cells, and the number of hematopoietic stem cells and progenitor cells decreased further in the bone marrow of UT2-deficient (UT2 KO) mice.

[0043] The above results confirmed that the Nisch enhancing factor FGF21 increases the number of hematopoietic stem cells and progenitor cells, while CCL5 decreases the number of hematopoietic stem cells and progenitor cells. These results indicate that FGF21 and CCL5 have different effects on the proliferative capacity of hematopoietic stem cells and progenitor cells, and that a deficiency of UT2 in hematopoietic stem cells and progenitor cells enhances the effects of FGF21 and CCL5.

[0044]

[0045] Example 2. Effects of GROβ and AMD3100 on hematopoietic stem cells and progenitor cells

[0046] GROβ (growth-related oncogene beta), a CXCR2 agonist, and AMD3100, a CXCR4 antagonist, promote the migration of hematopoietic stem cells from the bone marrow to peripheral blood. The effects of GROβ and AMD3100 on the number of hematopoietic stem cells and progenitor cells in the bone marrow were evaluated. The GROβ and AMD3100 used in this example were purchased from PeproTech. After separating the femur and tibia of mice, both ends of the bones were cut, and bone marrow was extracted by passing flow cytometry buffer into the bones using a syringe. Red blood cells were removed from the bone marrow using ACK lysis buffer, and lymphocytes were obtained; subsequently, the lymphocytes were treated with 5 μL / mL of AMD3100 and / or 2.5 μg / mL of GROβ and cultured for 48 hours. Lymphocytes were collected after 48 hours of culture, treated with fluorescent antibodies, and then analyzed via flow cytometry to identify undifferentiated hematopoietic stem cells, specifically Lineage-negative cells (Lin-) and LK cells (Lin - cKit + ), LKS cells (Lin - cKit + Sca1 + ), hematopoietic progenitor cells, specifically HPC cells (Lin - cKit + Sca1 + CD48 + ), hematopoietic stem cells known as HSC cells (Lin - cKit + Sca1 + CD48 - CD150 + The ratio of ) was analyzed.

[0047] As shown in Figure 2, when AMD3100 was treated alone in mouse bone marrow, the number of LKS cells and HPC cells decreased, and when GROβ was treated, the number of LK cells, LKS cells, HPC cells, and HSC cells all decreased.

[0048] The above results confirmed that GROβ reduces the number of hematopoietic stem cells and progenitor cells more significantly than AMD3100. By demonstrating the effects of GROβ and AMD3100 on undifferentiated hematopoietic stem cells, these results indicate that GROβ has a superior effect on undifferentiated hematopoietic stem cells compared to AMD3100.

[0049]

[0050] Example 3. Effects of GROβ and AMD3100 on the migration of hematopoietic stem cells in mice

[0051] To evaluate the effects of GROβ and AMD3100 on hematopoietic stem cell migration, UT2 (Upstream of mTORC2) mice (UT2 fl / fl ) and mice lacking UT2 in hematopoietic cells (Mx1Cre UT2 fl / fl It was evaluated in ). CXCL2 (Groβ) at a concentration of 2.5 mg / kg and AMD3100 (Plefixafor) at a concentration of 5 mg / kg were injected subcutaneously into mice. After 15 minutes, blood was collected from the mice, placed in 10 mM EDTA to prevent coagulation, red blood cells were removed using ACK lysis buffer, and lymphocytes were extracted. After treating the lymphocytes with fluorescent antibodies, undifferentiated hematopoietic stem cells, specifically Lineage-negative cells (Lin-) and LKS cells (Lin - cKit + Sca1 + The ratio of ) was analyzed.

[0052] As shown in Figure 3, the number of undifferentiated bone marrow cells in the peripheral blood of mice administered GROβ and AMD3100 was found to increase compared to the untreated control group. In UT2-deficient mice, the migration of LKS cells also increased in mice administered GROβ and AMD3100, but at a lower level compared to UT2 mice.

[0053] Through the above results, it was confirmed that GROβ and AMD3100 have the effect of inducing the migration of hematopoietic stem cells in the bone marrow to the peripheral blood, and it was confirmed that in the case of UT2 deficiency in hematopoietic stem cells and progenitor cells, the effect of GROβ and AMD3100 on the migration of hematopoietic stem cells is reduced. The above results demonstrated the stem cell migration ability of GROβ and AMD3100 and indicate that UT2 is involved in the stem cell migration ability of GROβ and AMD3100.

[0054]

[0055] Example 4. Effects of Co-administration of GROβ, AMD3100, and Nisch Enhancement Factor on Blood Stem Cell Migration in Mice

[0056] To evaluate the effect of co-administration of GROβ, AMD3100, and Nisch enhancing factor on hematopoietic stem cell migration, UT2 mice (UT2 fl / fl It was evaluated in ). 2.5 mg / kg of CXCL2 (Groβ), 5 mg / kg of AMD3100 (Plefixafor), 1 mg / kg of CCL5, and 1 mg / kg of FGF21 were injected subcutaneously into mice. After 15 minutes, blood was collected from the mice, placed in 10 mM EDTA to prevent coagulation, erythrocytes were removed using ACK lysis buffer, and lymphocytes were extracted. After treating the lymphocytes with fluorescent antibodies, LKS cells (Lin), an undifferentiated hematopoietic stem cell population, were identified through flow cytometry. - cKit + Sca1 + The ratio of ) was analyzed.

[0057] As shown in Figure 4, the number of undifferentiated bone marrow cells in the peripheral blood of mice administered GROβ and AMD3100 increased compared to the untreated control group. When CCL5 was administered along with GROβ and AMD3100, the number of LKS cells in the peripheral blood increased by more than twofold. Through these results, it was confirmed that the combined administration of GROβ, AMD3100, and CCL5 has the effect of activating hematopoietic stem cells and significantly increasing their migration.

[0058]

[0059] Example 5. Evaluation of the effect of combined administration of GROβ, AMD3100, and CCL5 on the engraftment ability of donor hematopoietic stem cells

[0060] To evaluate the engraftment ability of hematopoietic stem cells migrated into peripheral blood by the concomitant administration of GROβ, AMD3100, and CCL5, hematopoietic stem cells were collected from the peripheral blood of donor mice after concomitant administration of GROβ, AMD3100, and CCL5, and then administered to recipient mice to analyze the engraftment of donor-derived hematopoietic stem cells. Boy / J CD45.1 mice were used as donor mice, and CXCL2 (Groβ), AMD3100 (Plefixafor) at a concentration of 2.5 mg / kg, and CCL5 at a concentration of 5 mg / kg, were injected subcutaneously into the donor mice. After 15 minutes, blood was collected from the mice, placed in 10 mM EDTA to prevent blood coagulation, red blood cells were removed using ACK lysis buffer, and lymphocytes were extracted. To evaluate the engraftment ability of donor-derived hematopoietic stem cells, the extracted lymphocytes were mixed in a 2:1 ratio with lymphocytes extracted from the bone marrow of C57BL / 6 CD45.2 mice of similar genotype for competitive transplantation. C57BL / 6 CD45.2 mice, which are similar genotypes to the donor mice, were used as recipient mice. The mixed lymphocytes were injected into the tail vein of recipient mice that had been injected with a busulfan to remove bone marrow cells. One month after transplantation, blood was collected from the recipient mice, placed in 10 mM EDTA to prevent blood coagulation, red blood cells were removed using ACK lysis buffer, and lymphocytes were extracted. After treating the lymphocytes with fluorescent antibodies, the engraftment rate of the cells derived from the donor mice was confirmed by flow cytometry.

[0061] As shown in Figure 5, the proportion of cells derived from CD45.1 donor mice in the blood of recipient mice 4 weeks after bone marrow transplantation was determined. Compared to the case where only GROβ and AMD3100 were administered, lymphocytes extracted from donors administered GROβ, AMD3100, and CCL5 in combination appeared in a higher proportion in the blood of recipient mice. These results demonstrate that bone marrow collected by administering GROβ, AMD3100, and CCL5 in combination to bone marrow donor mice exhibits a higher engraftment ability in recipient mice.

[0062]

[0063] Example 6. Effect of CCL5 Deficiency on Hematopoietic Stem Cell Migration Increased by GROβ and AMD3100

[0064] WT mice and CCL5 KO mice lacking CCL5 were injected subcutaneously with 2.5 mg / kg of CXCL2 (Groβ) and 5 mg / kg of AMD3100 (Plefixafor). After 15 minutes, blood was collected from the mice, placed in 10 mM EDTA to prevent coagulation, erythrocytes were removed using ACK lysis buffer, and lymphocytes were extracted. After treating the lymphocytes with fluorescent antibodies, flow cytometry was performed to identify LKS cells (Lin - cKit + Sca1 + ) and HPC cells, which are hematopoietic progenitor cells (Lin - cKit + Sca1 + CD48 + The ratio of ) was analyzed.

[0065] As shown in Figure 6, it was confirmed that the number of LKS cells and HPC cells in the peripheral blood of WT mice administered GROβ and AMD3100 increased compared to control WT mice that received no treatment, indicating an increased migration of LKS cells and HPC cells into the peripheral blood. However, in CCL5-deficient mice, it was confirmed that the administration of GROβ and AMD3100 reduced the increase in the migration of LKS cells and HPC cells into the peripheral blood. These results demonstrate that CCL5 deficiency reduces the migration of hematopoietic stem cells into the peripheral blood by GROβ and AMD3100.

[0066] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

[0067] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

Claims

1. A composition for promoting the activation of bone marrow-derived stem cells, comprising CCL5 (CC motif chemokine ligand 5) protein as an active ingredient.

2. A composition for promoting the activation of bone marrow-derived stem cells, wherein, in claim 1, the composition further comprises AMD3100, which is a GROβ (growth-related oncogene beta) and CXCR4 antagonist.

3. A composition for promoting the activation of bone marrow-derived stem cells, characterized in that, in claim 1, the bone marrow-derived stem cells are hematopoietic stem cells and progenitor cells.

4. In paragraph 1, the bone marrow-derived stem cells are LK (Lin - cKit + ) cells, LKS(Lin - cKit + Sca1 + ) cells, HPC (hematopoietic progenitor cells) cells (Lin - cKit + Sca1 + CD48 + ), and HSC (hematopoietic stem cells) cells (Lin - cKit + Sca1 + CD48 - CD150 + A composition for promoting the activation of bone marrow-derived stem cells, characterized by being one or more cells selected from ).

5. A composition for promoting the activation of bone marrow-derived stem cells according to claim 1, wherein the activation of the bone marrow-derived stem cells is characterized by an increased migration of stem cells from the bone marrow to peripheral blood.

6. A pharmaceutical composition for treating bone marrow disease comprising, as an active ingredient, bone marrow-derived stem cells isolated from a patient administered CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist.

7. A pharmaceutical composition for treating bone marrow disease according to claim 6, wherein the bone marrow-derived stem cells have excellent engraftment ability within the recipient.

8. A pharmaceutical composition for treating a bone marrow disease according to claim 6, wherein the bone marrow disease is one or more diseases selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, aplastic anemia, multiple myeloma, malignant lymphoma, myeloproliferative neoplasm, myelodysplastic syndrome, paroxysmal nocturnal hemoglobinuria, immunodeficiency, hemoglobin dystrophy, Fanconi anemia, systemic lupus erythematosus, and induced secondary bone marrow failure.

9. A method for treating a bone marrow disease comprising the step of administering to a subject a composition containing, as an active ingredient, bone marrow-derived stem cells isolated from a patient who received CCL5 (CC motif chemokine ligand 5) protein, GROβ (growth related oncogene beta), and AMD3100, a CXCR4 antagonist.

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