KR20070113694A - A composition for promoting engraftment of hematopoietic stem cells - Google Patents

Abstract

A composition comprising mesenchymal stem cells derived from umbilical cord blood is provided to minimize or prevent side effects such as infection, bleeding, and anemia caused by delaying or failing engraftment of hematopoietic stem cells by promoting the engraftment of the hematopoietic stem cells using the mesenchymal stem cells, thereby decreasing medical costs and increasing the success rate of the hematopoietic stem cells' grafting. A composition for promoting engraftment of hematopoietic stem cells comprises mesenchymal stem cells derived from umbilical cord blood. The composition further comprises a cell culturing medium selected from the group consisting of RPMI 1640(Roswell Park Memorial Institute 1640), DMEM(Dulbecco's Modified Eagle's Medium), alpha-MEM (alpha-Minimum Essential Medium), McCoy's 5A medium, Eagle's basal medium, CMRL1066(Connaugh Medical Research Laboratories 1066), Glasgow minimum essential medium, Ham's nutrient mixtures medium, IMDM(Iscove's Modified Dulbecco's Medium), and Liebovitz' L-15 medium.

Description

A composition for promoting engraftment of hematopoietic stem cells

1 is a diagram showing the result of engraftment rate obtained by FACS analysis (mCD45 / hCD45) in mouse bone marrow after hematopoietic stem cell transplantation.

Figure 2 is a graph showing the average engraftment rate of each group obtained by FACS analysis (mCD45 / hCD45) in mouse bone marrow after hematopoietic stem cell transplantation.

Figure 3 is a diagram showing the results of engraftment measurement obtained by FACS analysis (mCD45 / hCD45) in mouse peripheral blood after hematopoietic stem cell transplantation.

Figure 4 is a graph showing the average engraftment rate of each group obtained by FACS analysis (mCD45 / hCD45) in mouse peripheral blood after hematopoietic stem cell transplantation.

Figure 5 is a diagram showing the result of engraftment rate obtained by FACS analysis (mCD45 / hCD41) in mouse peripheral blood after hematopoietic stem cell transplantation.

Figure 6 is a graph showing the average engraftment rate of each group obtained by FACS analysis (mCD45 / hCD41) in mouse peripheral blood after hematopoietic stem cell transplantation.

7 is a diagram showing colony production results obtained through CFU analysis (CFU-GEMM, CFU-GM, BFU-E, CFU-E) after hematopoietic stem cell transplantation.

Hematopoietic stem cells refer to cells having the ability to differentiate into blood cells such as red blood cells, white blood cells, and platelets, which constitute blood. Hematopoietic stem cells are produced in large quantities especially in the bone marrow and present in about 1% of normal bone marrow cells.

However, if hematopoietic stem cells are insufficient, such as aplastic anemia, hematopoietic stem cells, such as leukemia, myelodysplastic syndrome, etc., hematopoietic stem cells are damaged due to chemotherapy or irradiation. The parent cell cannot function properly and cannot produce blood cells.

In this case, if the hematopoietic stem cells are completely removed and the normal hematopoietic stem cells are administered to the patient, the hematopoietic stem cells migrate to the patient's bone marrow and regenerate normal blood cells to restore normal hematopoietic function. It is called hematopoietic stem cell transplantation. Source materials that can provide hematopoietic stem cells include bone marrow, umbilical cord blood, and peripheral blood. When hematopoietic stem cell transplantation is performed using each of them, each is referred to as bone marrow transplantation, cord blood transplantation, and peripheral blood transplantation. The type will depend on the condition of the donor and the availability of the donor. However, even after hematopoietic stem cell transplantation, hematopoietic stem cells return to the bone marrow and engraftment into the microenvironment of the bone marrow to allow for normal hematopoietic processes and generation of various blood cells. Delayed or unsuccessful side effects such as infection, bleeding, and anemia persist, which requires long-term treatment of antibiotics, red blood cells, white blood cells, and platelet transfusions. If unsuccessful, hematopoietic stem cell transplantation eventually fails, resulting in death of the patient.

Bone marrow-derived mesenchymal stem cells, on the other hand, produce cytokines and extracellular matrix proteins that can differentiate into bone marrow stromal cells, which are the constituent cells of the bone marrow microenvironment, or help the proliferation of hematopoietic stem cells, return to bone marrow and engraftment. et al., Proc Natl Acad Sci USA 1992 89: 7350.7354., Clark et al., Ann NY Acad Sci 1995 770: 70.88., Majumdar et al., J Cell Physiol 1998 176: 57.66., Majumdar et al., J Hematother Stem Cell Res 2001 9: 841.848.), It is known that there is no significant toxicity in a single injection in humans (Koc et al., J Clin Oncol 2000 18: 307.316., Koc et al., Bone Marrow Transplant 2002) 30: 215.222.). Recently, in order to increase the success rate of hematopoietic stem cell transplantation using the characteristics of bone marrow-derived mesenchymal stem cells, a co-transplantation of bone marrow mesenchymal stem cells and hematopoietic stem cells isolated from different individuals has been attempted (Koc et al., Bone Marrow Transplant 2001). 27: 235.239), it is known to some extent that bone marrow-derived mesenchymal stem cells promote in vitro proliferation and early engraftment of hematopoietic stem cells and suppress immune responses.

However, most of the experiments on the promotion of engraftment of hematopoietic stem cells using bone marrow mesenchymal stem cells have been based on the heterologous transplantation of human bone marrow mesenchymal stem cells in animals with reduced immunity for transplantation of human hematopoietic stem cells. In the case of mesenchymal stem cell transplantation, the increase in hematopoietic stem cell engraftment rate in actual clinical trials is only the case of mesenchymal stem cells derived from related bone marrow with autologous bone marrow or histocompatibility antigens (HM Lazarus et al. , Biology of Blood and Marrow Transplantation 2005 May; 11 (5): 389-98.). According to the present report, when using bone marrow-derived mesenchymal stem cells, there is a disadvantage in that the donor's bone marrow matching histocompatibility antigens must be found to obtain mesenchymal stem cells for promoting hematopoietic stem cell engraftment. In addition, bone marrow-derived mesenchymal stem cells have a disadvantage in that it is difficult to culture cells from autologous bone marrow in cancer patients. Therefore, in order to make up for these drawbacks, studies on "universal mesenchymal stem cells" capable of transplantation even if the tissue-compatible antigens do not coincide are in progress, but no clinical cases have been reported.

In the present invention, it is found that umbilical cord blood-derived mesenchymal stem cells can promote the engraftment of hematopoietic stem cells after transplantation of hematopoietic stem cells, to solve the problems caused by delayed and failed engraftment of hematopoietic stem cells in conventional hematopoietic stem cell transplantation.

Accordingly, the present invention is to provide a composition for promoting hematopoietic stem cell engraftment including umbilical cord blood-derived mesenchymal stem cells.

In addition, the present invention is to provide a method for enhancing engraftment of hematopoietic stem cells by simultaneously or sequentially administering the hematopoietic stem cells and the present composition to a patient in need of transplantation.

The present invention provides a composition for promoting hematopoietic stem cell engraftment including umbilical cord blood-derived mesenchymal stem cells.

The composition for promoting hematopoietic stem cell engraftment of the present invention may be enhanced by engraftment of hematopoietic stem cells by simultaneously administering hematopoietic stem cells to a patient in need of hematopoietic stem cell transplantation or sequentially before or after administration of hematopoietic stem cells.

Hematopoietic stem cells that can be administered simultaneously or sequentially with the composition for promoting hematopoietic stem cell engraftment of the present invention refer to cells having the ability to differentiate into blood cells such as red blood cells, white blood cells and platelets constituting blood. Hematopoietic stem cells that can be used with the composition for promoting hematopoietic stem cell engraftment of the present invention means hematopoietic stem cells derived from bone marrow, umbilical cord blood, or peripheral blood. In particular, umbilical cord blood-derived hematopoietic stem cells generally do not cause an immune rejection reaction even if the histocompatibility antigens do not match completely, and are immunologically immature compared to bone marrow or peripheral blood, resulting in a slight degree of graft versus host reaction. The frequency is low, the necessary tests can be performed in advance, frozen storage, and can be supplied immediately when needed. The harvest is also noninvasive compared to the bone marrow, and successful transplantation with fewer hematopoietic stem cells than the bone marrow. Since there are many advantages, it can use preferably.

In the composition for promoting hematopoietic stem cell engraftment of the present invention, umbilical cord blood, which is a tissue of mesenchymal stem cells, is defined as blood collected from the umbilical vein of the umbilical cord (umbilical cord) connecting the placenta and the fetus.

Among the cell components of the composition for promoting hematopoietic stem cell engraftment of the present invention, mesenchymal stem cells isolated from umbilical cord blood have multipotent unlike typical bone marrow stromal cells, and thus, bone, cartilage under appropriate differentiation conditions. It can be differentiated into mesenchymal tissues such as adipose tissue, muscle, tendons, and the like. In addition, since cord blood-derived mesenchymal stem cells have the ability to self-renewal, they are cells that can proliferate in undifferentiated state under appropriate conditions. Umbilical cord blood-derived mesenchymal stem cells are obtained from immature individuals in developmental stages compared to mesenchymal stem cells isolated from normal mesenchymal tissues such as bone marrow, fat, muscle, and skin, which are mature adult tissues. .

Cord blood-derived cell components included in the composition for promoting hematopoietic stem cell engraftment of the present invention require multiple operations in that the cord blood, which is a by-product naturally occurring in childbirth, is used in the collection and acquisition of the tissue of origin. It is much easier to collect than normal mesenchymal tissue such as bone marrow. Moreover, cord blood is easier to obtain donors through the infrastructure already established and activated than the bone marrow transplant.

Umbilical cord blood-derived cell components included in the composition for promoting hematopoietic stem cell engraftment of the present invention are cells that do not express histocompatibility antigen HLA-DR (class II), which is the most important cause of rejection in tissue or organ transplantation, Implantation can be minimized by minimizing or minimizing immune responses, such as rejection, which can be a problem during transplantation.

Composition for promoting hematopoietic stem cell engraftment of the present invention can be used for the treatment of patients in need of transplantation of hematopoietic stem cells, specifically, acute leukemia, chronic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, germ cell tumor Solid tumors, such as breast cancer, ovarian cancer, small cell lung cancer, neuroblastoma tumors, aplastic anemia, erythremia, Gaucher's disease, Hunter's syndrome, ADA enzyme deficiency, Wiskot-Aldrich syndrome, immune diseases such as various metabolic diseases and rheumatoid arthritis, It can also be implanted in patients with autoimmune diseases such as systemic lupus erythematosus, multiple sclerosis and the like. It can also be transplanted into patients with hematopoietic stem cells damaged by chemotherapy or radiation. That is, it can be used to promote hematopoietic stem cell engraftment for any disease requiring hematopoietic stem cell transplantation due to damage, deficiency, disease, etc. of hematopoietic stem cells.

The composition for promoting hematopoietic stem cell engraftment of the present invention includes a medium for suspending water and cellular components. For example, RPMI 1640 (Roswell Park Memorial Institute 1640), DMEM (Dulbecco's Modified Eagle's Medium), α-MEM (alpha-Minimum Essential Medium), McCoy's 5A medium, Eagle's basal medium, CMRL 1066 (Connaugh Medical Research Laboratories 1066) Commonly used cell culture media such as Glasgow Minimum Essential Medium, Ham's nutrient mixtures medium, Iscove's Modified Dulbecco's Medium (IMDM), and Liebovitz 'L-15 medium are all possible.

One or more accessory ingredients may be added to the composition for promoting hematopoietic stem cell engraftment according to the present invention, including serum of fetal calf, horse or human, serum or plasma components such as albumin (albumin), and dextran. 40 (dextran 40), injection solutions such as saline, penicillin G to prevent microbial contamination, antibiotics such as streptomycin sulfate and gentamycin, and amphotericin B ) And one or more selected antifungal agents such as nystatin.

Method for collecting cord blood, which is a raw material of cellular components used in the composition for promoting hematopoietic stem cell engraftment of the present invention, and a method for separating mesenchymal stem cells from cord blood is as follows.

Umbilical cord blood collection may be obtained from the umbilical vein, which has been left out of the uterus after birth, in the case of normal vaginal delivery, or from the umbilical vein, with the placenta also delivered out of the uterus in the case of cesarean section. Is implemented.

In the present invention, when the cord blood is collected from the umbilical vein extracted from the uterus after delivery, the umbilical vein is collected from the umbilical vein connecting the placenta and the fetus after birth. The umbilical cord blood may be collected from the uterus after birth or before umbilical cord detachment occurs. In the case of cesarean section, placenta umbilical cord blood is collected after placental detachment.

After securing the umbilical vein, the cord blood is collected in a cord blood collection bag (bag) containing anticoagulant using a sampling needle.

By removing the plasma components by centrifuging the cord blood collected as described above, and obtaining a mononuclear cell layer, the isolated mononuclear cells are isolated from CD34 antibodies, which are common markers for immature hematopoietic cells, including hematopoietic stem cells. In response, CD34 positive cells can be isolated and used as a component of hematopoietic stem cells.

Separation and culture of mesenchymal stem cells from umbilical cord blood can be used, including the methods of Korean Patent No. 0489248 (Pittinger MF, Mackay AM, et al. Science 1999; 284: 143-7. , Lazarus HM, Haynesworth SE, et al. Bone Marrow Transplant 1995; 16: 557-64.), For example:

The collected cord blood is centrifuged to separate mononuclear cells, and then washed several times to remove foreign substances. After washing, the mononuclear cells are planted in a culture vessel at an appropriate density and cultured to form a monolayer. Among them, the colony of long-shaped spindle cells is homogeneous and homogeneous as observed by a phase contrast microscope. The cells that proliferate in) form mesenchymal stem cells. Subsequently, when cells grow to the appropriate level, medium is cultured and grown until the required number of cells is reached.

The mesenchymal stem cells isolated and cultured by the above method may be used for transplantation immediately, and may be frozen or stored and thawed at a necessary time, and then used again or expanded.

The present invention also provides a method for enhancing engraftment of hematopoietic stem cells by simultaneously or sequentially administering the hematopoietic stem cells and the composition to a patient in need thereof.

Here, "patients requiring hematopoietic stem cell transplantation" include, but are not limited to, acute leukemia, chronic myeloid leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, germ cell tumors, breast cancer, ovarian cancer, small cell lung cancer, neuroblastoma tumors, and the like. Immune diseases such as solid tumors, aplastic anemia, erythrocytosis, Gaucher's disease, Hunter syndrome, ADA enzyme deficiency, Wiskot-Aldrich syndrome, various metabolic diseases and autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis Any patient who needs transplantation of hematopoietic stem cells, such as a patient suffering from, or a patient whose hematopoietic stem cells have been damaged due to chemotherapy or irradiation.

According to the present invention, the composition comprising umbilical cord blood-derived mesenchymal stem cells may be administered to a patient at the same time as hematopoietic stem cells or before or after transplantation of hematopoietic cells to promote engraftment of hematopoietic stem cells.

In the present invention, hematopoietic stem cells and mesenchymal stem cells may be administered in vivo according to a conventional method for administering cell therapy. Preferably it can be administered intravenously or directly to the bone marrow.

It will be apparent to those skilled in the art that the dosage of the composition will vary depending on the desired effect. The optimal dosage to be administered can be readily determined by one skilled in the art and includes a variety of conditions, including the severity, severity, age, weight, health status, sex, diet and time of administration, route of administration, duration of treatment, and concurrent drug use. It can be adjusted according to the factor.

Preferably, hematopoietic stem cells are administered at a dose of 0.1 × 10 ⁶ to 2.0 × 10 ⁷ cells / kg and cord blood-derived mesenchymal stem cells at a dose of 0.02 × 10 ⁶ to 2.0 × 10 ⁸ cells / kg. .

When the hematopoietic stem cells and the umbilical cord blood-derived mesenchymal stem cells are administered at the same time or sequentially with the composition for promoting hematopoietic stem cell engraftment of the present invention, the ratio of 1: 0.2 to 1:10 is preferred, and 1: 0.5 to 1: 5. Is more preferable. The ratio of hematopoietic stem cells and umbilical cord blood-derived mesenchymal stem cells that can promote the engraftment of hematopoietic stem cells may vary depending on the patient, and may be appropriately controlled by a doctor who has rich experience in transplantation of hematopoietic stem cells.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the present embodiments are intended to complete the disclosure of the present invention, and those skilled in the art to which the present invention pertains. It is provided to fully inform the scope of the invention, and the invention is defined only by the scope of the claims.

Example

In the following examples, it was intended to evaluate the effectiveness of umbilical cord blood-derived mesenchymal stem cells to promote engraftment of hematopoietic stem cells through animal model experiment. As an animal model, 8-week-old NOD / SCID mice, which are genetically deficient in a part of the immune system and used as a main model for engraftment and immunity-related studies, were purchased from Jackson Laboratory (Marine, USA). Hematopoietic stem cells and umbilical cord blood-derived mesenchymal stem cells were prepared by MediPost. Bone marrow-derived mesenchymal stem cells were purchased from Cambrex, USA (Walkersville, MD, USA).

All test results were expressed using mean and standard deviation, and statistical comparison between each group was ANOVA, and the significance was verified at 5% significance level compared to the control group.

Isolation of Hematopoietic Stem Cells

Umbilical cord blood was transferred to a 50 ml conical tube and centrifuged at 3000 rpm for 30 minutes. Plasma is removed, the buffy coat layer is transferred to a new tube, and then suspended in an equal amount of PBS (2% FBS with 2 mM EDTA), and the suspension is carefully placed on a 15 ml histopaque, 400 × Centrifuged in g for 40 minutes. Plasma components were removed, the monocyte layer was transferred to a new tube, and washed with PBS (200 × g, 10 minutes). After erythrocyte lysis buffer was added to the cell pellet for 15 minutes, the cells were centrifuged to remove the buffer and washed with PBS again (200 × g, 10 minutes). 1 × 10 ⁸ isolated mononuclear cells were suspended in 300 μl PBS and 100 μl of Fc blocking antibody was added. 100 µl of a CD34 antibody with magnetic beads was added and allowed to react at 4 ° C for 30 minutes. Washed with PBS (1200 rpm, 5 min), suspended in 5 ml PBS, and isolated CD34 positive cells using autoMACS. The number of isolated CD34 positive cells was counted and used for the hematopoietic stem cell transplantation step.

Preparation of Mesenchymal Stem Cells

Bone marrow-derived or umbilical cord blood-derived mesenchymal stem cells (four passages) stored at −196 ° C. were thawed and cultured by adding α-MEM medium containing 10% fetal bovine serum in a 175 cm ² flask. Once the cells grew under 37 ° C., 5% CO ₂ conditions, the cells were separated from the culture flasks by treatment with 0.25% trypsin-EDTA and used for the hematopoietic stem cell transplantation step after counting.

Hematopoietic Stem Cell Transplantation

Four to six hours before transplantation of hematopoietic stem cells into NOD / SCID mice, three mice per urine were injected and irradiated at 300 cGy, a sublethal irradiation level, to autologous hematopoietic stem cells of NOD / SCID mice. Damaged. After 4 hours of irradiation, the animals were treated with G1: RPMI medium (phenol red-free) only (control, 2), G2: group with hematopoietic stem cells (4), as shown in Table 1. G3: Hematopoietic stem cells and bone marrow-derived mesenchymal stem cells (4 mice) and G4: hematopoietic stem cells and cord blood-derived mesenchymal stem cells (4 mice) were divided into four groups, and the test substance was administered through the microvenous vein. .

TABLE 1

group Route of administration Dosage (cells / head) Marisu G1 excipient control Vein Vein 0 2 G2 test group Vein Vein Umbilical Cord Blood Stem Cells 5x10 ⁴ cells 4 G3 test group Vein Vein Umbilical cord blood hematopoietic stem cells 5x10 ⁴ cells + bone marrow-derived mesenchymal stem cells 2.5x10 ⁵ cells 4 G4 test group Vein Vein Umbilical cord blood stem cells 5x10 ⁴ cells + Umbilical cord blood-derived mesenchymal stem cells 2.5x10 ⁵ cells 4

Outcome Analysis of Hematopoietic Stem Cell Transplantation

The transplantation result of hematopoietic stem cells was observed until 10 weeks after administration of the test substance. General symptoms such as changes in general status, motility, appearance, and autonomic nerves were observed at least five times a week during the observation period, and the presence or absence of dead animals was measured. It was.

In order to determine the outcome of hematopoietic stem cell transplantation, the engraftment rate (%) was confirmed by analyzing human-derived blood cell antigens expressed in all cell groups through FACS analysis.CFU (colony forming unit) was obtained by collecting bone marrow after cell transplantation. ) Analysis was performed.

FACS analysis

Bone marrow was collected from the tibia and femur and peripheral blood from the inferior vena cava from mice maintained aseptically for 10 weeks in a SPF (specific pathogen free) facility.

The collected bone marrow was washed once with PBS (2% FBS-PBS) containing 2% fetal bovine serum, and then erythrocytes were destroyed at room temperature for 15 minutes by adding erythrocyte lysis buffer and washed once with 2% FBS-PBS.

In order to immunostain using FACS, 5 × 10 ⁵ cells per tube were suspended in 100 μl, and the antibody to be analyzed was added in the indicated amount and reacted at 4 ° C. for 30 minutes (FITC-mCD45 / PE-hCD45, hCD41). ).

For analysis of the collected peripheral blood, 75 μl of blood was reacted with the same amount of antibody added to the bone marrow sample, and then erythrocytes were destroyed for 15 minutes at room temperature with 2 ml of erythrocyte lysis buffer. Was the same as

2 ml of 2% FBS-PBS was added and washed, followed by centrifugation (1500 rpm, 5 minutes) to remove the supernatant. 250 μl of 4% PFA (paraformaldehyde) was added, mixed lightly, and analyzed using FACS.

① Result of engraftment rate in mouse bone marrow (mCD45 / hCD45)

As a result of measuring the engraftment rate using the monoclonal antibody against hCD45 (human CD45) by separating the cells from the bone marrow of each mouse, as shown in FIG. 1, 1.77% in the test group administered only hematopoietic stem cells, hematopoietic stem cells And bone marrow-derived mesenchymal stem cells treated with 9.46%, and hematopoietic stem cells and cord blood-derived mesenchymal stem cells treated with 13.06% (mean values). When bone marrow or umbilical cord blood-derived mesenchymal stem cells were transplanted with hematopoietic stem cells, engraftment rate was increased in mouse bone marrow than group that only transplanted hematopoietic stem cells alone, and umbilical cord blood-derived mesenchymal stem cells showed higher engraftment rate than bone marrow-derived mesenchymal stem cell transplant group. The tendency to further increase was confirmed. Mean engraftment rate analyzed in each group is shown in the graph of FIG.

② Result of measurement of engraftment rate in peripheral blood of mouse (mCD45 / hCD45)

As a result of measuring the engraftment rate using a monoclonal antibody against hCD45 (human CD45) by separating the cells from the blood of each mouse, as shown in FIG. 3, 0.19% in the excipient control group, and only the hematopoietic stem cells were administered. 0.24% in the test group, 0.40% in the test group administered with hematopoietic stem cells and bone marrow-derived mesenchymal stem cells, and 0.29% in the test group administered with hematopoietic stem cells and cord blood-derived mesenchymal stem cells (mean values). The engraftment rate was less than 1% in both hematopoietic stem cell transplantation alone or bone marrow or cord blood-derived mesenchymal stem cells and hematopoietic stem cells. Mean engraftment rate in peripheral blood analyzed in each group is shown in the graph of FIG.

③ Analysis of platelet production ability in peripheral blood of mouse (mCD45 / hCD41)

As a result, the cells were isolated from blood of each mouse, and platelet production capacity was measured using a monoclonal antibody against hCD41 (human CD41). As shown in FIG. 5, 0.05% of the excipient control group was administered only with hematopoietic stem cells. 0.18% in the group, 0.98% in the test group administered with hematopoietic stem cells and bone marrow-derived mesenchymal stem cells, and 0.44% in the test group administered with hematopoietic stem cells and cord blood-derived mesenchymal stem cells (mean values).

The engraftment rate was less than 1% in both hematopoietic stem cell transplantation alone or bone marrow or cord blood-derived mesenchymal stem cells and hematopoietic stem cells. Average engraftment rate in peripheral blood analyzed in each group is as shown in the graph of FIG.

CFU (colony forming unit) analysis (CFU-GEMM (CFU-granulocyte, erythrocyte, macrophage, megakaryocyte), CFU-GM (CFU-granulocyte, macrophage), BFU-E (burst forming unit-erythrocyte), CFU-E (CFU-E) -erythrocyte))

Ten weeks after administration of the test substance, bone marrow was collected from each subject and colony forming unit assay was performed. A part of the collected sample (5 × 10 ⁵ cells) was suspended in 300 μl IMDM, aliquoted into methyl cellulose medium, and then cultured for 14 days. After 14 days, the formed colonies were counted by observing at 40 times magnification under an optical microscope.

As a result of using the whole 5 × 10 ⁵ cells in the CFU analysis, as shown in FIG. 7, 20 colonies formed after 14 days were observed, and more colonies were found in the group transplanted with bone marrow or cord blood mesenchymal stem cells. It showed a tendency to be observed a lot.

Evaluation of FACS and CFU Assays

After 10 weeks of administration of the test group, differences in engraftment rates were observed among test animal subjects. However, the engraftment rate of hematopoietic stem cells was increased in the group in which bone marrow or umbilical cord blood-derived mesenchymal stem cells were administered together with the hematopoietic stem cells, compared with the group administered with hematopoietic stem cells alone. The engraftment rate was highest in the group transplanted.

The composition for promoting hematopoietic stem cell engraftment including umbilical cord blood-derived mesenchymal stem cells according to the present invention promotes engraftment of hematopoietic stem cells using umbilical cord blood-derived mesenchymal stem cells, thereby delaying or failing engraftment of hematopoietic stem cells during hematopoietic stem cell transplantation. It is possible to minimize or prevent side effects such as infection, bleeding, and anemia that occur, to reduce related medical expenses, and to increase the success rate of hematopoietic stem cell transplantation.

Claims (6)

Composition for promoting hematopoietic stem cell engraftment including umbilical cord blood-derived mesenchymal stem cells. The composition for promoting hematopoietic stem cell engraftment according to claim 1, wherein the cord blood is autologous cord blood. The composition for promoting hematopoietic stem cell engraftment according to claim 1, wherein the cord blood is taga-derived cord blood. The solid tumor according to any one of claims 1 to 3, including acute leukemia, chronic myeloid leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, germ cell tumor, breast cancer, ovarian cancer, small cell lung cancer, neuroblastoma Suffering from autoimmune diseases, including aplastic anemia, erythrocytosis, Gaucher's disease, Hunter syndrome, ADA enzyme deficiency, immune diseases including Wiskot-Aldrich syndrome, metabolic diseases, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis A composition for promoting hematopoietic stem cell engraftment for transplantation into a patient or a patient whose hematopoietic stem cell is damaged by radiation or the like. The method according to claim 1, wherein the RPMI 1640 (Roswell Park Memorial Institute 1640), DMEM (Dulbecco's Modified Eagle's Medium), α-MEM (alpha-Minimum Essential Medium), McCoy's 5A medium, Eagle's basal medium, CMRL 1066 (CMRL 1066) Connaugh Medical Research Laboratories 1066), Glasgow minimal essential medium, Ham's nutrient mixtures medium, IMDM (Iscove's Modified Dulbecco's Medium), or a culture medium for promoting hematopoietic stem cell adhesion medium selected from Liebovitz 'L-15 medium. The method of claim 5, wherein the cell culture medium is at least one auxiliary component selected from serum of fetal calf, horse or human, albumin (albumin), dextran 40 (physiological saline), physiological saline, injection, antibiotics or antifungal agents Hematopoietic stem cell engraftment promotion composition further comprising.

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