JP7166514B2 - cord blood hematopoietic stem cell support - Google Patents

Description

The present invention relates to novel uses of adipose tissue-derived mesenchymal stem cells. Specifically, it relates to the application of adipose tissue-derived stem cells to hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation is becoming widely used as a treatment for blood diseases (eg, various leukemias and aplastic anemia). There are currently three sources of hematopoietic stem cells: bone marrow, peripheral blood, and cord blood. Usually, various conditions and restrictions are comprehensively considered to determine the optimal transplantation technique. Cord blood transplantation is less successful than the other two types of transplantation, but compared to bone marrow transplantation, the burden on the donor is less, it can be transplanted immediately because it is cryopreserved, and there is less graft-versus-host disease (GVHD). The number of enforcement cases is increasing due to the advantages of However, cord blood transplantation also has drawbacks. Although the cause is unknown, there are many cases of poor engraftment and delayed recovery of hematopoiesis, and patients require frequent blood transfusions, and in some cases, hemorrhage and infection are life-threatening. Therefore, cord blood with a cell number below a certain standard is not used, and a valuable cell source is wasted.

On the other hand, adipose tissue-derived mesenchymal stem cells (ADSCs) have the ability to differentiate into various cells such as osteocytes, chondrocytes, and cardiomyocytes, and are being investigated for tissue reconstruction and treatment of various diseases. there is The research group of the present inventors established mouse ADSCs and examined their properties in order to find new uses for ADSCs. In an analysis using a mouse model for bone marrow transplantation, it was found that concomitant use of ADSCs resulted in faster engraftment and engraftment with a smaller number of hematopoietic stem cells (Non-Patent Document 1). On the other hand, it has also been found that human ADSCs exhibit supporting ability for peripheral blood-derived hematopoietic stem cells (Non-Patent Document 2).

Nakao N. et al., Am J Pathol. 2010 Aug;177(2):547-54. Nishiwaki S. et al., Int J Hematol. 2012 Sep;96(3):295-300

As noted above, although cord blood transplantation has its advantages, it suffers from several challenges. In particular, poor engraftment and delayed hematopoietic recovery are major obstacles to the expansion of the use of umbilical cord blood transplantation. Accordingly, an object of the present invention is to provide effective means for improving engraftment failure and hematopoietic recovery delay, and to improve the success rate or treatment results of cord blood transplantation.

Hematopoiesis in adult humans takes place in the bone marrow, but the presence of hematopoietic stem cells alone is not sufficient and a supporting hematopoietic microenvironment is required. The microenvironment is constructed by mesenchymal stem cells (MSCs) in the bone marrow and osteoblasts differentiated from them. MSCs generally refer to bone marrow-derived ones (BMSCs), but it is not practical to establish MSCs from the patient's bone marrow tissue exhausted by chemotherapy, etc., and the possibility of blood malignant cell contamination cannot be denied.

As described above, the research group of the present inventors conducted research focusing on the ability of adipose tissue-derived mesenchymal stem cells (ADSCs) to support hematopoietic stem cells, and reported certain results (Non-Patent Documents 1 and 2). Based on the findings obtained so far and focusing on the high clinical need for constructing a hematopoietic microenvironment in cord blood transplantation, the present inventors decided to investigate the usefulness of ADSCs in cord blood transplantation. . Detailed studies revealed that ADSCs have a supportive effect on umbilical cord hematopoietic stem cells, and the effect is higher than that of bone marrow-derived MSCs. That is, according to the studies of the present inventors, it is surprising that ADSCs not only replace BMSCs in constructing the microenvironment necessary for hematopoiesis by umbilical cord hematopoietic stem cells, but also exhibit superior hematopoietic support ability. insight has been provided. The following inventions are mainly based on this finding.
[1] A cord blood transplant cell preparation containing adipose tissue-derived mesenchymal stem cells , which is intramedullary administered together with a cord blood transplant material .
[2] The cell preparation for cord blood transplantation of [1], wherein the adipose tissue-derived mesenchymal stem cells are human cells.
[3] The cell preparation for cord blood transplantation according to [1] or [2], which is administered into the bone marrow after being mixed with cord blood or hematopoietic stem cells in the cord blood.
[4] The cell preparation for cord blood transplantation of [1] or [2], which is administered in a therapeutically effective amount together with cord blood or hematopoietic stem cells in the cord blood to a patient in need of cord blood transplantation.
[5] Use of adipose tissue-derived mesenchymal stem cells for producing a cell preparation for cord blood transplantation that is intramedullary administered together with a cord blood transplant material .

Results of colony assay (left) and co-culture assay (right). Matrigel assay results. Results of safety tests using pigs. After infusion of ADSCs into the iliac bone marrow, hemodynamics were examined. Results of safety tests using pigs. One month after administration of ADSC, the animals were sacrificed and each organ was pathologically examined. Results of safety tests using pigs. One month after ADSC administration, the animals were sacrificed, and each organ was pathologically examined (histological staining). Blood biochemistry test results. wbc: white blood cell count, lym: lymphocyte count, mon: monocyte count, granulocyte: granulocyte count, rbc: red blood cell count, hb: hemoglobin, hct: hematocrit, plt: platelet count, alb: albumin level, alp: alkaline phosphatase , alt: Alt value, amy: amylase, t-bil: total bilirubin, bun: urea nitrogen, ca: calcium, phos: inorganic phosphorus, cre: creatinine, glu: blood sugar, na: sodium, k: potassium, tp: total protein, glb: globulin

The present invention relates to cell preparations for cord blood transplantation. Umbilical cord blood used for cord blood transplantation is fetal blood, and it contains hematopoietic stem cells (umbilical cord hematopoietic stem cells) that are immature and highly proliferative. The process of cord blood transplantation is generally divided into pretreatment (administration of anticancer drugs, irradiation, administration of immunosuppressants, etc.), transplantation of hematopoietic stem cells (transfusion of cord blood), and recovery (engraftment, hematopoiesis). be. The cell preparation of the present invention is used in the second step, “transplantation of hematopoietic stem cells”. Cord blood can be obtained, for example, from cord blood banks. In Japan, the cords of the Japanese Red Cross Hokkaido Cord Blood Bank, the Japanese Red Cross Kanto Koshinetsu Cord Blood Bank, the Chubu Cord Blood Bank, the Japanese Red Cross Kinki Cord Blood Bank, the Hyogo Cord Blood Bank, the Japanese Red Cross Kyushu Cord Blood Bank, etc. A blood bank has been established. In addition, the cord blood may be provided without going through the cord blood bank.

As used herein, the term "cell preparation" means an agent containing cells as an active ingredient. In the cell preparation of the present invention, adipose tissue-derived mesenchymal stem cells (sometimes abbreviated as “ADSC” in this specification) are used as active ingredients. That is, the cell preparation of the present invention is most characterized by containing adipose tissue-derived mesenchymal stem cells. In the present invention, "adipose tissue-derived mesenchymal stem cells (ADSC)" refer to somatic stem cells contained in adipose tissue, but as long as pluripotency is maintained, the culture of the somatic stem cells ( cells obtained by subculturing) also fall under "adipose tissue-derived mesenchymal stem cells (ADSC)". Usually, ADSCs are prepared in an "isolated state" as cells constituting a cell population (including cells other than ADSCs derived from adipose tissue) using adipose tissue isolated from a living body as a starting material. The “isolated state” here means a state in which it has been removed from its original environment (that is, a state in which a part of the living body is formed), that is, it exists in a state different from its original state due to artificial manipulation. means that there is In addition, adipose tissue-derived mesenchymal stem cells are ADSC (Adipose-derived mesenchymal stem cells / Adipose tissue derived stem cells), ASC (adipose-derived stem / stromal cells), ADRC (Adipose-derived cells regeneration), AT-MSC ( Adipose-derived mesenchymal stem cells), AD-MSC (Adipose-derived mesenchymal stem cells), etc. The following terms are used interchangeably herein: adipose tissue-derived mesenchymal stem cells, ADSCs, ASCs, ADRCs, AT-MSCs, AD-MSCs.

As used herein, the term “for cord blood transplantation” refers to cord blood transplantation, that is, cord blood or hematopoietic stem cells contained therein (hereinafter, for convenience of explanation, these are collectively referred to as “cord blood transplantation material”). It means to be used together during administration (transplantation, transfusion). Therefore, the cell preparation of the present invention is typically administered together with the cord blood transplant material during cord blood transplantation. The timing of administration of the cord blood transplant material and the cell preparation is not particularly limited, and both are administered to the recipient (patient) at the same time or with a predetermined time interval. Preferably, both are administered simultaneously. "Simultaneously" here does not require strict simultaneity. Therefore, it is possible to administer both of them without a time lag, such as administering them to the recipient after mixing them together, and of course, administering both of them, such as administering one immediately after administering the other. The concept of "simultaneous" here also includes the case of performing under conditions with substantially no time difference, in which case it is preferable to set the time difference as short as possible. For example, one is administered within 15 minutes, preferably within 10 minutes, more preferably within 5 minutes after administration of the other. On the other hand, when one is administered with a predetermined time difference after the other is administered, for example, the other is administered within 6 hours (preferably 2 to 5 hours, more preferably 3 to 5 hours) after administration of one.

The umbilical cord blood transplant material is not particularly limited in its form, preparation method, etc., as long as it contains umbilical cord hematopoietic stem cells. For example, cord blood, cord blood concentrates, cord blood purified products (which can be purified, such as by magnetic bead separation, etc.) are used as cord blood transplant materials.

In one aspect, the cell preparation of the present invention contains umbilical cord blood or hematopoietic stem cells contained therein in addition to the active ingredient ADSCs. That is, the cell preparation of the present invention is provided as a formulation in which ADSCs and a cord blood transplant material are mixed. For example, after separately preparing ADSCs and a cord blood transplant material, the two are mixed to form the cell preparation of this embodiment. After hematopoietic stem cells in cord blood are co-cultured with ADSCs, the proliferated cells may be collected and used as the active ingredient of this embodiment.

Embodiments of cell preparations containing ADSCs and cord blood transplant materials are not limited to the above examples (ie, formulations). For example, the cell preparation of the present invention can be provided in the form of a kit comprising a first component containing ADSCs and a second component containing cord blood transplant material. In this case, both components will be administered to the recipient at the same time or with a predetermined time interval.

Preparation of ADSC should just follow a conventional method. ADSC is widely used for various purposes, and can be easily prepared by those skilled in the art by referring to literatures and books. Cells distributed from public cell banks or commercially available cells may be used. Examples of methods for preparing ADSCs are described below.

<Method for preparing ADSC>
ADSCs are prepared through processes such as separation of stem cells from adipose matrix, washing, concentration, and culturing. The ADSC preparation method is not particularly limited. For example, a known method (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr;24(4):150-4. Epub 2006 Feb 20. Review.; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec;13(12):4279-95.; Zuk PA et al. (2001), Multilineage cells from human ADSCs can be prepared according to adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr;7(2):211-28. Devices for preparing ADSCs from adipose tissue (for example, Celution (registered trademark) device (Cytori Therapeutics, Inc., San Diego, USA)) are also commercially available, and ADSCs are prepared using the device. You can decide. Using this device, a cell population containing ADSCs can be separated from adipose tissue (K. Lin. et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). Specific examples of methods for preparing ADSC are shown below.

(1) Preparation of cell population from adipose tissue Adipose tissue is collected from an animal by means of excision, aspiration, or the like. The term "animal" as used herein includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc.). In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (autologous adipose tissue) from the same individual as the subject (patient) to whom the cell preparation of the present invention is applied. However, this does not preclude the use of adipose tissue from animals of the same species (allogeneic) or adipose tissue from different species.

Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat can be collected very easily under local anesthesia, and thus it can be said to be a preferable cell source because it places less burden on the donor during collection. One kind of adipose tissue is usually used, but two or more kinds of adipose tissue can be used in combination. In addition, adipose tissue (which does not have to be of the same type) collected in multiple batches may be mixed and used for subsequent operations. The amount of adipose tissue to be collected can be determined in consideration of the type of donor, the type of tissue, or the required amount of ADSCs, and is, for example, about 0.5 to 500 g. When a human donor is used, it is preferable that the amount collected at one time is about 10 to 20 g or less in consideration of the burden on the donor. The collected adipose tissue is optionally subjected to the enzymatic treatment described below after removal of blood components adhering thereto and fragmentation. Blood components can be removed by washing the adipose tissue in an appropriate buffer or culture medium.

Enzymatic treatment is carried out by digesting adipose tissue with enzymes such as collagenase, trypsin and dispase. Such enzymatic treatment may be performed by techniques and conditions known to those skilled in the art (see, for example, R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). . Cell populations obtained by the above enzymatic treatments include pluripotent stem cells, endothelial cells, stromal cells, hematopoietic cells, and/or their progenitor cells. The types and proportions of the cells that make up the cell population depend on the origin and type of adipose tissue used.

(2) Acquisition of sedimentation cell population (SVF fraction: stromal vascular fractions) The cell population is then subjected to centrifugation. The sediment by centrifugation is collected as a sedimented cell population (herein also referred to as "SVF fraction"). The centrifugation conditions vary depending on the type and amount of cells, but are, for example, 800 to 1500 rpm for 1 to 10 minutes. Prior to centrifugation, it is preferable to subject the enzyme-treated cell population to filtration or the like to remove enzyme-undigested tissues and the like contained therein.

The "SVF fraction" obtained here contains ADSCs. The type and ratio of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the enzyme treatment conditions, and the like. In addition, WO2006/006692A1 pamphlet shows the characteristics of the SVF fraction.

(3) Selective culture of adherent cells (ADSC) and collection of cells
The SVF fraction contains ADSCs as well as other cell components (endothelial cells, stromal cells, hematopoietic cells, progenitor cells thereof, etc.). Therefore, in one aspect of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. Then, the cells obtained as a result are used in the present invention as ADSCs.

First, the SVF fraction is suspended in an appropriate medium, seeded on a culture dish, and cultured overnight. Floating cells (non-adherent cells) are removed by medium exchange. After that, the culture is continued while appropriately replacing the medium (for example, once every 2 to 4 days). Perform subculture as necessary. The number of passages is not particularly limited, but from the viewpoint of maintaining pluripotency and proliferation ability, it is not preferable to repeat passages excessively (preferably up to about 5 passages). As the culture medium, a normal animal cell culture medium can be used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., etc.), α-MEM (Dainippon Pharmaceutical Co., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Research Institute), and the like can be used. Media supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or serum replacement (Knockout serum replacement (KSR), etc.) may be used. The amount of serum or serum substitute to be added can be set, for example, within the range of 5% (v/v) to 30% (v/v).

Adherent cells are selectively survived and proliferated by the above operation. The proliferated cells are then harvested. The recovery operation may follow a conventional method, and for example, the cells can be easily recovered by detaching the cells after enzyme treatment (trypsin or dispase treatment) with a cell scraper, pipette, or the like. In addition, when sheet culture is performed using a commercially available temperature-sensitive culture dish or the like, it is possible to collect the cells in the form of a sheet as they are without enzymatic treatment. By using the cells (ADSCs) collected in this manner, a cell population containing highly purified ADSCs can be prepared.

(4) Low-serum culture (selective culture in low-serum medium) and cell recovery In one aspect of the present invention, instead of the operation (3) or after the operation (3), the following low-serum culture I do. Then, the cells obtained as a result are used in the present invention as ADSCs.

In low serum culture, the SVF fraction (using the cells collected in (3) when performing this step after (3)) is cultured under low serum conditions, and the desired pluripotent stem cells (i.e. ADSC ) are selectively propagated. Since the low-serum culture method requires only a small amount of serum, it is possible to use the subject's (patient's) own serum to which the cell preparation of the present invention is administered. That is, culture using autologous serum becomes possible. By using autologous serum, xenogenic animal materials are excluded from the production process, and a highly safe cell preparation that can be expected to have a high therapeutic effect is provided. As used herein, the term "low serum conditions" refers to conditions in which the medium contains 5% or less serum. The cells are preferably cultured in a medium containing 2% (V/V) or less serum. More preferably, the cells are cultured in a medium containing 2% (V/V) or less serum and 1 to 100 ng/ml of fibroblast growth factor-2 (bFGF).

Serum is not limited to fetal bovine serum, and human serum, sheep serum and the like can be used. Human serum is preferably used, and more preferably serum of the subject to which the cell preparation of the present invention is applied (ie, autologous serum).

As the medium, an ordinary medium for animal cell culture can be used, provided that it contains a low amount of serum when used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., etc.), α-MEM (Dainippon Pharmaceutical Co., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Research Institute), and the like can be used.

By culturing by the above method, pluripotent stem cells (ADSCs) can be selectively proliferated. In addition, since pluripotent stem cells (ADSC) proliferate under the above culture conditions have high proliferative activity, the number of cells required for the present invention can be easily prepared by subculturing. Incidentally, WO2006/006692A1 pamphlet shows the characteristics of cells that selectively proliferate by culturing the SVF fraction with low serum.

Cells selectively proliferated by the low serum culture described above are then collected. The recovery operation may be performed in the same manner as in the case of (3) above. By using the collected cells (ADSCs), a cell preparation containing ADSCs in high purity can be obtained.

(5) Formulation
The cells of the SVF fraction, the cells obtained as a result of the above selective culture (3), or the cells obtained as a result of the above low serum culture (4) are A cell preparation can be obtained by suspending in a medium such as A single dose may contain, for example, 1×10 ⁶ to 1×10 ⁹ cells so that a therapeutically effective amount of cells is administered. The content of cells can be appropriately adjusted in consideration of the purpose of use, target disease, sex, age, body weight, condition of the affected area, condition of cells, etc. of the target (recipient).

Dimethyl sulfoxide (DMSO), serum albumin, etc. for the purpose of protecting cells, antibiotics for the purpose of preventing bacterial contamination, and various ingredients for the purpose of activating, proliferating or inducing differentiation of cells. (vitamins, cytokines, growth factors, steroids, etc.) may be contained in the cell preparation of the present invention. Examples of cytokines are interleukin (IL), interferon (IFN), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO), activin, oncostatin M (OSM). CSF, G-CSF, EPO, etc. are also growth factors. On the other hand, examples of growth factors are hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF, FGF2), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) , transforming growth factor (TGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). In addition, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) It may be contained in the cell preparation of the present invention.

In the above method, cell preparations are constructed using cells proliferated by culturing the SVF fraction with low serum. Cells proliferated by low-serum culture may be used as ADSCs to prepare cell preparations. That is, in one aspect of the present invention, ADSCs are cells proliferated when a cell population obtained from adipose tissue is cultured with low serum. In addition, the SVF fraction (containing adipose tissue-derived mesenchymal stem cells), instead of the pluripotent stem cells obtained by selective culture (above (3) and (4)), is directly used to form a cell preparation. can be The cell preparation of this embodiment includes (a) a sedimented cell population (SVF fraction) collected as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate, or (b) fat After the tissue is treated with protease, it contains a sedimented cell population (SVF fraction) recovered as a sediment by centrifugation without filtration. Here, "use as it is" means using as an active ingredient of a cell preparation without undergoing selective culture.

<Applicable diseases>
The cell preparation of the present invention is used for cord blood transplantation. Therefore, the cell preparation of the present invention can be applied to various diseases for which cord blood transplantation is effective. Examples of applicable diseases include acute lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, juvenile myelomonocytic leukemia, myelodysplastic syndrome, malignant lymphoma, multiple myeloma, aplastic anemia, Hemoglycemia, congenital immunodeficiency, congenital metabolic disorder.

<Dose/number of cells>
The dose of the cell preparation of the present invention can be set in consideration of the route of administration (transplantation site), the degree of symptoms of the recipient (patient), and the like. For example, we will administer 1.0×10 ⁷ to 1.0×10 ⁹ ADSCs in a single cord blood transplant (based on a recipient weighing 50 kg). The amount of ADSCs in the cell preparation may be set so as to achieve the desired dosage.

<Administration route>
The cells of the present invention as long as an environment in which ADSCs, which are active ingredients, coexist with hematopoietic stem cells in transplanted cord blood in the recipient's body (in other words, a state in which ADSCs can act on hematopoietic stem cells) is formed. The administration route of the formulation is not particularly limited. However, typically, the cell preparations of the present invention are administered into the bone marrow such as the iliac bone. The cord blood transplant material is administered into the bone marrow in a state of being mixed with the cell preparation or separately from the cell preparation.

2. Cord blood transplantation (treatment of various diseases)
As a second aspect, the present invention provides a cord blood transplantation method (method for treating various diseases by cord blood transplantation). In the transplantation method of the present invention, ADSCs and a cord blood transplant material are administered to a recipient (patient) in need of cord blood transplantation. Typically, ADSCs and cord blood transplant materials are administered using the cell preparation of the present invention, but it is also possible to perform the transplantation method of the present invention using ADSCs that have not been prepared in the form of preparations. is. The amount of ADSC used conforms to the above description. That is, the number of ADSCs used in one treatment (based on a recipient weighing 50 kg) can be, for example, 1.0×10 ⁷ to 1.0×10 ⁹ . The routes of administration are as described above.

Aiming to improve the success rate of cord blood transplantation and treatment results, we compared the support and differentiation ability of human adipose tissue-derived MSCs (ADSCs) against human cord blood CD34-positive hematopoietic stem cells by colony assay and co-culture assay. Moreover, the effects of transplantation into the living body were confirmed by Matrigel assay.

1. Materials and Methods (1) Establishment of Mesenchymal Stem Cells Bone marrow-derived MSCs (BMSCs) were established from two individuals, and adipose tissue-derived MSCs (ADSCs) were established from three individuals. After the fat mass was minced with a scalpel and scissors, the fat piece was melted using collagenase I, and the liberated cells were collected. After that, culture was started in a medium containing 20% human serum in DMEM. When the cells became confluent, they were subcultured, and after 3 to 4 weeks, the cells were harvested and used for experiments. As a result of analyzing cell surface markers, 90% or more of the cells were CD45-negative, CD73-positive, CD90-positive, and CD105-positive, confirming that they were MSCs.

(2) Colony assay A methylcellulose medium was prepared, and 500 human umbilical cord CD34-positive hematopoietic stem cells alone, 500 human umbilical cord CD34-positive hematopoietic stem cells and 5000 human BMSCs, or 500 human umbilical cord CD34-positive hematopoietic stem cells were added to 0.5 ml of the medium. and 5000 human ADSCs. It was plated on a 12-well plate and cultured for 8 days at 37°C, 5% CO ₂ in a humidified incubator. Colony types and numbers were then evaluated using an inverted microscope.

(3) Co-culture Assay Human BMSCs or human ADSCs were added to wells of a 24-well plate precoated with gelatin (5000 per well). When the cells became 80-90% confluent, the culture supernatant was aspirated, and 5000 human umbilical cord CD34-positive hematopoietic stem cells suspended in αMEM supplemented with 12.5% bovine serum and 12.5% horse serum were added per well. 0.5 mL of fresh medium was added every week, culture was continued for 3 weeks, and the number of differentiated leukocytes was counted.

(4) Matrigel assay
Cytell red-labeled human umbilical cord blood and human BMSCs or human ADSCs were mixed with Matrigel as follows.
Control group: 2×10 ⁶ human umbilical cord blood/400 μL Matrigel
BMSC-added group: 2×10 ⁶ human cord blood and 2×10 ⁶ human BMSCs/400 μL Matrigel
ADSC-added group: 2×10 ⁶ human umbilical cord blood and 2×10 ⁶ human ADSC/400 μL Matrigel

Each mixture was administered subcutaneously into the flank of NOD/SCID immunodeficient mice (3 per group). After 48 hours the mice were sacrificed and the solidified Matrigel was removed and enzymatically lysed. Cytell red positive viable cells in the lysate were counted using FACS and trypan blue method.

(5) Safety test using pigs Miniature pigs (female, weight 20.8 kg) were used. First, cryopreserved syngeneic adipose tissue-derived mesenchymal stem cells (ADSCs) were thawed in a constant temperature bath at 37°C and suspended in RPMI. A minipig was implanted with an SG catheter (for pulmonary artery pressure measurement) under general anesthesia. ADSCs were infused into the iliac bone marrow while monitoring hemodynamics. A dose of 1×10 ⁶ cells/kg was administered. A total of 3 administrations were performed while changing the puncture site. Monitoring was performed for about 30 minutes after the end of administration. On the other hand, before and after ADSC infusion and about 1 month after, blood was collected and biochemical examination was also performed.

2. result
BMSC and ADSC were established and used from 2 and 3 persons, respectively. Colony assays and co-culture assays confirmed that human ADSCs also have supportive capacity for umbilical cord CD34-positive hematopoietic stem cells (Fig. 1). ADSCs were much higher than BMSCs in their supportive action on progenitor cells (Fig. 1, left) and in their differentiation-promoting action into leukocytes (Fig. 1, right). On the other hand, experiments (Matrigel assay) using living bodies (NOD/SCID immunodeficient mice) also confirmed the high supporting capacity of ADSCs (Fig. 2).

Next, safety was confirmed using pigs. Pre-established syngeneic ADSCs were transfused into the iliac bone under hemodynamic monitoring, but no acute injury such as embolism was observed (FIGS. 3 and 6, left). One month after the transfusion, the animals were sacrificed and pathologically examined, but no organ damage or ADSC-induced tumor formation was observed (Figs. 4 and 5). Also, no significant change was observed in each component in the blood (Fig. 6).

3. DISCUSSION It was found that human ADSCs exhibited extremely high supporting capacity against human umbilical cord CD34-positive hematopoietic stem cell blood. It was also confirmed that ADSC is highly safe. Therefore, ADSCs are highly expected to be clinically applied as supporting cells for cord blood transplantation.

The cell preparation of the present invention can improve the success rate of cord blood transplantation or treatment results. The cell preparation of the present invention can be applied to all indications for cord blood transplantation.

The present invention is by no means limited to the description of the above embodiments and examples of the invention. Various modifications are also included in the present invention within the scope of those skilled in the art without departing from the description of the claims. The contents of articles, published patent publications, patent publications, etc., identified herein are incorporated by reference in their entirety.

Claims (5)

A cell preparation for cord blood transplantation, which contains adipose tissue-derived mesenchymal stem cells and is administered into the bone marrow together with a cord blood transplant material . The cell preparation for cord blood transplantation according to claim 1, wherein the adipose tissue-derived mesenchymal stem cells are human cells. 3. The cell preparation for cord blood transplantation according to claim 1 or 2, which is administered into the bone marrow after being mixed with cord blood or hematopoietic stem cells in the cord blood. 3. The cell preparation for cord blood transplantation according to claim 1 or 2, which is administered to a patient in need of cord blood transplantation in a therapeutically effective amount together with cord blood or hematopoietic stem cells in the cord blood. Use of adipose tissue-derived mesenchymal stem cells for producing a cell preparation for cord blood transplantation , which is administered into the bone marrow together with a material for cord blood transplantation.

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