Pharmaceutical composition for treating bone diseases

Abstract

Provided is a pharmaceutical composition that increases osteoblasts in a subject, the pharmaceutical composition containing high-purity mesenchymal stem cells. The pharmaceutical composition is used in combination with hematopoietic stem cell transplantation in the subject.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for treating bone diseases. [Background technology]

[0002] Hypophosphatasia (HPP) is an autosomal recessive genetic disease caused by mutations in the ALPL gene, which results in impaired mineralization due to a deficiency of alkaline phosphatase (ALP). The accumulation of pyrophosphate due to the decreased activity of ALP impairs mineralization, and the local phosphorus concentration decreases, causing hypomineralization of bones and rickets-like changes.

[0003] Serologically, elevated levels of ALP substrates, such as phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate, are observed. Bone-related clinical features include bone curvature, easy fracture, and tooth loss, while central nervous system symptoms include convulsions, hearing loss, and developmental delay. Severe cases can progress to a fatal clinical course, while even mild cases often limit daily life. In particular, patients with respiratory disorders due to hypoplasia of the ribs and other parts of the thorax have a poor prognosis. The mortality rate for patients who develop the disease during the most severe perinatal period is 50-100%. At present, there are an estimated 100-200 patients in Japan, and no effective treatment has been established (only symptomatic treatment is available).

[0004] The current treatment for HPP is ALP enzyme replacement therapy. The administration of this enzyme preparation improves bone mineralization, not only improving the prognosis of severe HPP patients, but also restoring motor function in mild cases. However, because it is a replacement therapy, it has problems such as the need for regular lifelong administration (subcutaneous injection three times a week), the fact that the effect is weakened by the production of antibodies against the enzyme, the fact that it does not pass through the blood-brain barrier and therefore does not improve central nervous system symptoms, and the high medical costs (even for patients receiving the lowest dosage, the annual medical costs are over 20 million yen).

[0005] Mesenchymal stem cells (MSCs) can be relatively easily isolated from various tissues, such as bone marrow, adipose tissue, placenta, dental pulp, and amnion. Focusing on their tissue repair and immunosuppressive properties, hundreds of clinical trials are underway for a variety of diseases. However, only a few trials have reached the late clinical stage, and only a limited number of drugs have been approved by regulatory authorities in various countries, such as Temcel (JCR-Pharma) for severe GvHD in Japan and Alfisel (Tigenix), which was recommended for approval in Europe in early 2019 for anal fissures caused by Crohn's disease.

[0006] A variety of factors have been pointed out as reasons for the difficulty in putting MSCs to practical use, including the difficulty of quality control when producing large quantities of uniform cells for administration, the difficulty of delivering them to the affected area after administration, and the lack of non-invasive cell tracking technology (making it difficult to understand their dynamics in the body).

[0007] Previously, the present inventors demonstrated that human MSCs can be selected extremely efficiently by using two antibodies, LNGFR (CD271) and Thy1 (CD90), and developed a technique to directly isolate human MSCs from bone marrow, peripheral blood, placental chorion, and dental pulp using a cell sorter. The cell population obtained by this technique (Rapidly Expanding Clone: ​​REC) has been confirmed to exhibit excellent proliferation, differentiation, and migration abilities, and to be high-quality, high-purity human MSCs (Patent Document 1, Non-Patent Document 2).

[0008] In addition, the present inventors performed bone marrow transplantation combined with allogeneic bone marrow-derived MSC transplantation therapy on severe HPP patients based on the "Guidelines for clinical research using human stem cells" (Non-Patent Document 1). This clinical study suggested that by replacing donor-derived immune cells (bone marrow cells) by bone marrow transplantation and then transplanting MSCs, some of the transplanted MSCs would engraft in the bone marrow without being rejected, resulting in the formation of normal bones. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 6363950 [Patent Document 2] International Publication No. 2016 / 017795 Brochure [Non-patent literature]

[0010] [Non-Patent Document 1] T.Taketani et al., “Ex vivo expanded allogeneic mesenchymal stem cells with bone marrow transplantation improved osteogenesis in fan ts with severe hypophosphatasia,”Cell Transplant.,vol.24,no.10,pp.19 31-1943,2015 [Non-Patent Document 2] Mabuchi Y et al., “LNGFR+ Thy-1+ Vcam-1hi+ cells reveal fun ctionally distinct subpopulations in mesenchymal stem cells”.Stem Cell Re ports 1(2):152-165,2013 Summary of the Invention [Problem to be solved by the invention]

[0011] The clinically significant effects of allogeneic bone marrow-derived MSC transplantation in combination with bone marrow transplantation include (i) improved bone mineralization and life prognosis, and (ii) improved central nervous system symptoms and mental development. However, to form normal bone structure in patients after MSC transplantation and obtain further efficacy, it is necessary to use highly purified MSCs with higher migration and osteogenic differentiation ability. [Means for solving the problem]

[0012] As a result of intensive research into solving the above problems, the inventors have succeeded in solving the above problems by combining the administration of highly purified mesenchymal stem cells with hematopoietic stem cell transplantation, thereby completing the present invention. That is, the present invention is as follows. [1] A pharmaceutical composition for increasing osteoblasts in a subject, comprising highly purified mesenchymal stem cells, the pharmaceutical composition being used in combination with hematopoietic stem cell transplantation into the subject. [2] The pharmaceutical composition described in [1], wherein the hematopoietic stem cells are derived from umbilical cord blood. [3] The pharmaceutical composition described in [1] or [2], wherein the hematopoietic stem cells are derived from the bone marrow of a donor other than the subject. [4] The pharmaceutical composition according to any one of [1] to [3], wherein the subject is a patient with a congenital bone disease. [5] The pharmaceutical composition described in [4], wherein the congenital bone disease is hypophosphatasia. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the highly purified mesenchymal stem cells are fast-proliferating mesenchymal stem cells derived from human bone marrow. [7] The pharmaceutical composition described in [6], wherein the fast-proliferating mesenchymal stem cells are a cell population of stem cell clones that are co-positive for LNGFR (CD271) and Thy-1 (CD90) and satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [7-1] The composition described in [6], wherein the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and which satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [7-2] The composition described in [6], wherein the highly purified mesenchymal stem cells derived from human bone marrow are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less [8] Cells were administered at a dose of 1x10 per kg of subject body weight. 7 The pharmaceutical composition according to any one of [1] to [7-2], wherein the cells are administered once a week, and this administration is repeated four times. [9] At least 1x10 cells 6 The pharmaceutical composition according to [8], used at a concentration of cells / ml.

[10] The composition described in any one of [1] to [9], wherein the HLA of the donor of the highly purified mesenchymal stem cells does not match the HLA of the subject.

[11] The composition described in any one of [1] to [9], in which the HLA of the donor of the highly purified mesenchymal stem cells matches at least 4 of the 6 antigens at 3 loci of the HLA of the subject.

[12] The composition according to any one of [1] to [9], wherein the HLA of the donor of the highly purified mesenchymal stem cells matches at least three of the six antigens at three loci of the HLA of the subject. Effect of the Invention

[0013] According to the present invention, it is possible to increase osteoblasts in a subject, and as a result, the pharmaceutical composition of the present invention can be used for congenital bone system diseases such as hypophosphatasia. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows a transplantation test method for verifying the efficacy of highly purified mesenchymal stem cells REC-01 for hypophosphatasia pathological model mice. [Diagram 2]FIG. 1 shows the survival curve of Alpl- / - mice treated with asfotase alfa (Strensic (registered trademark)) (verification results of the life-prolonging effect). [Diagram 3] This is a diagram showing the change in body weight of each mouse after cell transplantation. The figure shows the change in body weight over time in Alpl- / - mice (left) and Alpl+ / - mice (normal group: right) after cell transplantation. Alpl+ / - mice used for comparison were Alpl- / - littermate mice (littermates). Vertical axis: Body weight on the day of transplantation is set as 1, and the change in body weight after transplantation is shown as a ratio. Horizontal axis: Number of days after transplantation. [Figure 4] This is a diagram showing the results of investigating ALP activity in bone marrow two months after REC-01 transplantation. The figure shows ALP staining images of frozen sections of mouse femurs. Alpl- / - mice (left) administered only mouse bone marrow cells, Alpl- / - mice (middle left and middle right) administered mouse bone marrow cells + REC01, and Alpl+ / - mice (right), which are the positive control group for ALP activity. The areas indicated by the arrows (blue) show positive ALP staining (blue-purple). [Diagram 5] This figure shows long-term engraftment and intrabone marrow ALP activity after REC-01 transplantation. The figure shows ALP staining images of frozen sections of mouse femur and skull 3 months after transplantation. The arrows (blue) indicate the ALP-positive areas (blue-purple). [Figure 6] FIG. 1 shows the survival curves of Alpl− / − mice with or without REC-01 transplantation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] 1. Overview Generally, the mechanisms of therapeutic effects of cell therapy can be broadly divided into two categories: cellular replacement, in which transplanted cells are incorporated into the recipient's tissues and organs and replace the recipient's cells with the transplanted cells, thereby restoring function; and trophic action, in which the transplanted cells produce trophic factors, cytokines, and extracellular matrices that have a protective effect on the host's cells or enhance the tissue's repair ability, thereby restoring function.

[0016] Cell replacement is essential for the treatment of hypophosphatasia (HPP), and the most important factors are the delivery of cells to the affected area and their long-term survival and differentiation. In addition, this disease is a bone formation imperfect disease that shows systemic symptoms, so intravenous systemic administration is required. However, most normal mesenchymal stem cells (MSCs) have lost their migration ability, and there is a risk of side effects such as pulmonary embolism and thrombosis when a large amount of cells are administered intravenously, so the number of cells that can be transplanted at one time is limited. In addition to HPP, there are more than 450 types of genetic bone formation disorders for which treatment has not been established. Congenital skeletal diseases (CSDs) have no established treatment, and are either fatal or significantly impair daily life. In CSDs, the ossification pathway of MSCs, the origin of osteoblasts that form bone, is impaired, so bone regenerative medicine using allogeneic MSCs with normal ossification ability is considered a promising treatment.

[0017] The present invention was completed by focusing on mesenchymal stem cells that have excellent differentiation and migration capabilities and rapid proliferation capabilities, i.e., highly purified mesenchymal stem cells, and is characterized by its use in increasing osteoblasts and, ultimately, as a medicine for bone diseases. That is, the present invention provides a pharmaceutical composition for increasing osteoblasts in a subject, comprising highly purified mesenchymal stem cells, and the pharmaceutical composition of the present invention is used in combination with hematopoietic stem cell transplantation into a subject.

[0018] 2.High-purity mesenchymal stem cells The mesenchymal stem cells used in the present invention are somatic stem cells derived from mesodermal tissue (mesenchyme), and are expected to be applied to regenerative medicine, such as the reconstruction of bones, blood vessels, and cardiac muscle. Mesenchymal stem cells can be obtained from various tissues such as bone marrow, adipose tissue, placental tissue, dental pulp, or umbilical cord tissue. The purification process is, for example, as follows.

[0019] A small amount of fat fragments collected from humans or non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, horses, goats, etc.) are enzymatically treated to obtain a mixed population of cell types. A floating fat cell population is separated by centrifugation, and fibroblast-like cells that sink and grow on the bottom surface when left in contact with the ceiling surface of a culture vessel filled with culture medium are then proliferated by subculture. In the present invention, it is also possible to use mesenchymal stem cells derived from iPS cells or commercially available mesenchymal stem cells.

[0020] In a previous study, the present inventors succeeded in isolating a rapidly expanding cell clone (REC) from LNGFR (CD271)-positive mesenchymal stem cells (CD271+ cells) or LNGFR (CD271) and Thy-1 (CD90)-copositive mesenchymal stem cells (CD271+CD90+ cells). This REC clone is a type of "high-purity mesenchymal stem cell" in the present invention.

[0021] RECs are cells that can reach confluence in two weeks when seeded one cell at a time onto a 96-well plate and cultured, and have over 1000 times the proliferation, differentiation, and migration capabilities of mesenchymal stem cells obtained by conventional methods. In particular, because they retain their migration capabilities, they can be administered intravenously and are applicable to severe systemic diseases such as osteochondrogenesis imperfecta. REC, a type of highly purified mesenchymal stem cell, is produced by isolating LNGFR (CD271) positive or LNGFR (CD271) and Thy1 (CD90) co-positive cells from the mononuclear cell fraction of bone marrow using a cell sorter, and then seeding the cells individually onto a plate to select only the rapidly proliferating MSCs, which are an extremely homogenous cell population.

[0022] The RECs isolated and purified in this way can be expanded to more than 1012 cells from a single cell while retaining their differentiation, proliferation, and migration abilities. In particular, because they retain their migration ability, they can be administered intravenously, and are expected to be applied to severe systemic diseases such as osteochondrodysplasia. In the present invention, among the above-mentioned REC clones, cell clones with less variation in differentiation potential and proliferation potential can be used.

[0023] In the present invention, mesenchymal stem cells positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90) can be obtained according to the method described in WO2009 / 31678. The outline of the method is as follows.

[0024] First, mesenchymal stem cells are highly enriched by selecting a cell fraction positive for LNGFR (CD271) (CD271+) or co-positive for CD271 and CD90 (CD271+CD90+) from a cell population containing human mesenchymal stem cells. If the cell population containing human mesenchymal stem cells contains blood cells, a step of selecting cells co-negative for CD45 and CD235a (CD45-CD235a-) may be added to select non-blood cells.

[0025] Cell populations containing mesenchymal stem cells can be prepared by flow cytometry or affinity chromatography. The material for obtaining this cell population is not particularly limited, but examples thereof include bone marrow, adipose tissue, umbilical cord blood, peripheral blood (including peripheral blood after administration of G-CSF), etc. In addition, bone marrow from the spine, sternum, ilium, etc. may be used. In addition, examples of cells include ES cells and iPS cells.

[0026] During cell preparation, if the material is in the form of a cell mass involving mesenchymal stem cells, the material can be subjected to physical treatment such as pipetting, or enzyme treatment with trypsin, collagenase, etc. If the material is contaminated with red blood cells, it is preferable to hemolyze the red blood cells in advance. The cell population prepared as described above is used to select CD271+ cells or CD271+CD90+ cells.

[0027] Methods for selecting CD271+ cells or CD271+CD90+ cells include, for example, methods using antibodies. The antibodies are anti-CD271 antibodies and / or anti-CD90 antibodies capable of selecting CD271+ cells or CD271+CD90+ cells. When flow cytometry is used for selection, live cells can be selected in a short time by using anti-CD271 antibodies labeled with different fluorescent dyes such as FITC, PE, and APC, or an appropriate combination of anti-CD271 antibodies and anti-CD90 antibodies. In addition to flow cytometry, CD271+CD90+ cells can be selected by methods using magnetic beads or affinity chromatography.

[0028] Before using these methods, dead cells may be removed by reacting a cell population with a fluorescent dye (e.g., PI) that stains dead cells and removing the fluorescently stained cells. Next, the selected LNGFR-positive cells or LNGFR and Thy1 co-positive cells are cultured as single cells (clones), and a lot showing rapid proliferation is selected to obtain RECs with excellent proliferation, differentiation and migration capabilities.

[0029] Here, "fast proliferation" and "rapid proliferation" mean that when one cell is seeded into each well of a 96-well culture plate and cultured, the cell has a proliferation rate such that the culture plate becomes confluent or semi-confluent 2 weeks or earlier after the start of culture (doubling time is 26±1 hours).

[0030] Confluence is a state in which 90% or more of the culture vessel surface (culture surface) is covered with cultured cells. Semi-confluence is a state in which 70-90% of the culture vessel surface (culture surface) is covered with cultured cells. The size and type of culture equipment used can be changed appropriately depending on the cell growth rate. Moderately / Slowly Expanding Cells, that is, cells that do not become semi-confluent or confluent even after 2 weeks of single cell culture, are discarded. The RECs collected from each well selected as RECs are transferred to a culture flask for each well and further cultured until they become confluent (expansion culture). The expanded cells are then collected separately. RECs from one well are counted as one lot.

[0031] The RECs used in the present invention are obtained by clonal sorting in which one cell is seeded per well, and therefore the genetic traits of the expanded cells are all identical. Therefore, in the present invention, the entire cell population may be referred to as a "clone," or each individual cell constituting the cell population may be referred to as a "clone."

[0032] In the present invention, the REC to be used for selection can be evaluated in advance using a REC marker (anti-Ror2). For example, after the expansion culture, the attached and proliferated cells are collected from all the lots, and a portion of each lot (1 to 3 × 10 5 A small number of cells (approximately 1000 cells) are selected and single-stained with an anti-Ror2 monoclonal antibody. A method for single-staining with an anti-Ror2 monoclonal antibody is known (WO2016 / 17795). In summary, the ratio of REC marker-positive cells in the collected cells is determined by flow cytometry analysis using the REC marker. The ratio may be determined by quantifying the mRNA expression of Ror2 using quantitative PCR, or the ratio may be determined manually using a microscope. A lot (cell population) with the above-mentioned positive ratio of a certain value (e.g., 65%) or more is considered to be acceptable, and this can also be used for selection.

[0033] The cell population containing the cell clone of the present invention is a population of mesenchymal stem cell clones that are co-positive for LNGFR (CD271) and Thy-1 (CD90) and rapidly proliferate, and satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less

[0034] In one embodiment of the present invention, the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and the cell population satisfies at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less

[0035] In one embodiment of the present invention, the highly purified human bone marrow-derived mesenchymal stem cells are a cell population of rapidly proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and satisfy at least one of the following characteristics (a) and (b): (a) The coefficient of variation of forward scatter in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less

[0036] In the present invention, by examining the cell proliferation ability, fat differentiation ability, expression level of REC-specific markers, and uniformity of cell size of each lot of REC clones and analyzing the correlations between them, it has become possible to select RECs that are highly pure, uniform, and have higher cell performance.

[0037] In the present invention, the coefficient of variation (CV value) of forward scattered light and the average size of cells are used as indices for selection. Forward scatter is light that is scattered at a small angle in the forward direction relative to the axis of the laser light. Forward scatter is made up of scattered light, diffracted light, and refracted light of the laser light generated on the cell surface, and provides information about the size of the sample.

[0038] The coefficient of variation (CV) is the standard deviation divided by the mean, and is used to relatively evaluate the variation in data with different units, or the relationship between the data and the variation in relation to the mean. In the present invention, a cell population having a CV value of 40% or less is selected. A cell population having a CV value of 40% or less is a cell population composed of cells of uniform size. Preferably, the CV value is 35% or less, 30% or less, 25% or less, or 20% or less. Furthermore, the average size of cells in the cell population selected according to the present invention is 20 μm or less, preferably 18 μm or less, and is in the range of 14 μm to 18 μm.

[0039] 3. Composition of the Present Invention (1) Target The compositions of the present invention are applied to humans or non-human organisms, such as birds and non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses), and are used to proliferate osteoblasts and promote bone regeneration.

[0040] The diseases to which this treatment applies are skeletal diseases that require an increase in osteoblasts, regardless of whether they are congenital or acquired. Skeletal diseases are a general term for diseases that cause skeletal abnormalities due to disorders in the tissues that form the skeleton, such as bones and cartilage. Congenital skeletal diseases are broadly divided into two categories: dysostosis and osteochondrodysplasia.

[0041] Examples of congenital bone system diseases include thoracic insufficiency syndrome, hypophosphatasia, achondroplasia, hypochondroplasia, osteogenesis imperfecta, osteopetrosis, multiple cartilaginous exostoses, enchondromatosis, type II collagen dysplasia-related disease, punctate chondrodysplasia, pseudoachondroplasia, Larsen syndrome, fibrodysplasia ossificans progressiva, TRPV4 dysplasia, osteosclerotic disease, Beals syndrome, etc. In particular, hypophosphatasia, which is a congenital bone system disease, is preferred in the present invention.

[0042] (2) Cell concentration and dosage Cells should be at least 1x10 6 Used in cell populations with a concentration of 1x10 cells / ml. 6 cell / ml, 5x10 6 cell / ml, 1x10 7 The amount is, for example, cell / ml, and can be set appropriately depending on the purpose of use. Cells were administered at a concentration of 1x10 per kg of subject body weight. 7 The cell population of cells is administered once a week, and this is repeated 1 to 4 times (one course of administration is 1 to 4 weeks), preferably 4 times.

[0043] (3) Composition In the present invention, the mesenchymal stem cells are administered in the form of a pharmaceutical composition. In one form, such a composition comprises a pharma- ceutically acceptable carrier and / or excipient. The administration form is injection, such as intravenous injection or intravenous drip. "Carrier" and "excipient" refer to compositions commonly used in the art to facilitate the storage, administration, and / or biological activity of cells.

[0044] Carriers used in the compositions of the present invention include, for example, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, etc. Excipients include starch, cellulose, glucose, lactose, etc. The composition containing the mesenchymal stem cells of the present invention is prepared as a suitable liquid suspension, for example in a buffer solution or culture medium. Suspensions for use in injection may contain sodium carboxymethylcellulose, sorbitol, dextran, and the like.

[0045] 4. Combination with hematopoietic stem cell transplantation Generally, only a small number of mesenchymal stem cells (MSCs) will engraft in the bone marrow when administered intravenously. Therefore, in the present invention, hematopoietic stem cell transplantation is used in combination with administration of mesenchymal stem cells.

[0046] First, MSCs derived from a subject (patient) with impaired osteogenic ability are replaced with MSCs with normal osteogenic ability. To achieve this, patient-derived MSCs are removed using anticancer drugs, radiation therapy, or the like. At this time, patient-derived hematopoietic stem cells are also removed, so when administering the high-purity mesenchymal stem cells of the present invention, hematopoietic stem cell transplantation is performed in combination to maintain blood cells. "Combined use" means that administration of high-purity mesenchymal stem cells and hematopoietic stem cell transplantation are used in one course of treatment, and the timing of hematopoietic stem cell transplantation may be before, after, or simultaneously with administration of high-purity mesenchymal stem cells. "Simultaneous use" includes preparing a suspension in which high-purity mesenchymal stem cells and hematopoietic stem cells are mixed in the above-mentioned carrier and then injected. However, in the present invention, it is preferable to administer high-purity mesenchymal stem cells after hematopoietic stem cell transplantation.

[0047] Because the transplanted hematopoietic stem cells recognize the simultaneously administered MSCs as self and differentiate into blood cells, there is very little chance that the transplanted MSCs will be rejected by the recipient's immune cells.

[0048] In the present invention, the donor that is the source of the high-purity mesenchymal stem cells and the donor that is the source of the hematopoietic stem cells in hematopoietic stem cell transplantation are both derived from a person different from the target patient (e.g., a healthy person). In addition, the donor of the high-purity mesenchymal stem cells and the donor of the hematopoietic stem cells may be the same or different. In hematopoietic stem cell transplantation, it is considered important that there is a matching rate of four loci (eight antigens), namely, the A locus, the B locus, the C locus, and the DR locus, of the HLA type. However, the present invention can be applied even if the HLA of the donor of the high-purity mesenchymal stem cells is not a perfect match or a mismatch with the HLA of the target.

[0049] In other words, the HLA of the donor of highly purified mesenchymal stem cells can be used which matches at least three of the six antigens at three loci (HLA-A, B, DR-B) of the subject's HLA, or at least four of the six antigens.

[0050] The present invention will be described in more detail below with reference to examples, although the scope of the present invention is not limited to these examples. [Production Example] The highly purified mesenchymal stem cells used in the present invention were obtained by separating mononuclear cells from bone marrow fluid collected from healthy donors by density gradient centrifugation, selectively isolating only cells that are co-positive with CD90 and CD271 antibodies, converting them into single cells, and further expanding the culture up to P4, and selecting cell clones with a CV value of 40% or less. The resulting cell clone was named "REC-01."

[0051] [Test example] Verification of the survival benefit of Strensiq administration in Alpl- / - mice We generated and bred 179 hypophosphatasia model mice (alkaline phosphatase-deficient mice: ALP(- / -) mice) and administered Strensiq (asfotase alpha) subcutaneously every other day.

[0052] As a result, 41 (22.9%) of the 179 Alpl- / - mice died on the day of birth (including stillbirths). The results of subcutaneously administering an enzyme (Strensic) every other day to mice that survived until genetic determination are shown in Figure 2. 88 mice (49.2%) died within 2 weeks of birth, 98 mice (54.7%) within 30 days, 158 mice (88.3%) within 60 days, and 168 mice (93.9%) within 100 days. If Strensiq was not administered, all individuals died within two weeks (Millan JL, et al. J Bone Miner Res. 2008), so administration of Strensiq alone showed some life-prolonging effect, but the effect on life prognosis is thought to be limited. EXAMPLES

[0053] Transplantation study of highly purified mesenchymal stem cells (REC) into ALP(- / -) mice In this example, the efficacy of REC-01 transplantation was evaluated using Alpl- / - mice, a hypophosphatasia model mouse.

[0054] 1. Method (1) Administration of REC-01 to recipient mice Genomic DNA was extracted from the tails of pups on days 0 to 5 obtained by mating Alpl heterozygous (Alpl+ / -) mice, and the genotypes were determined by PCR. After birth, Alpl- / - mice were continuously administered 8 mg / kg of Strensiq subcutaneously two or three times a week (Figure 1).

[0055] On the day before REC-01 transplantation, donor wild-type mice (B6 females) and recipient Alpl- / - mice were administered 0.1 mL of an immunosuppressant (0.5 mg / 1 mL Graceptor) per 10 g body weight. On the day of transplantation, Alpl- / - mice aged 33-159 days (n=9) were administered 0.1 mL of a triple anesthetic mixture (0.75 mg / kg Domitor, 4 mg / kg Midazolam, 5 mg / kg Betorfal) per 10 g body weight to completely sedate them. They were then irradiated with 8 Gy of radiation, followed immediately by 5 × 106 B6 mouse-derived bone marrow cells (5 × 10 6 The REC of cells was suspended and administered to the tail vein of mice (total volume of 200 μL). The same number of bone marrow cells alone was administered to control Alpl- / - mice (Figure 1).

[0056] (2) Maintenance after cell administration After cell transplantation, the mice were administered an immunosuppressant (0.5 mg / 1 mL Graceptor) at a dose of 0.1 mL per 10 g body weight almost daily. In addition, ALpl- / - mice, regardless of whether they had received cell transplantation, were continuously administered 8 mg / kg of Strensic subcutaneously 2-3 times a week until the Alpl- / - mice died or were euthanized for the experiment.

[0057] (3) Recipient mouse survival curve analysis Kaplan-Meier survival curves were analyzed using the statistical analysis software EZR. The analysis method was based on various existing manuals (Department of Hematology, Saitama Medical Center, Jichi Medical University).

[0058] 2.Results (1) Changes in body weight of each mouse after cell transplantation After REC-01 cell transplantation, the body weight of Alpl- / - (n=4) and the normal group, Alp+ / - (n=5), was measured periodically and the changes over time were observed. The body weight of each mouse on the day of cell transplantation was set as 1, and the changes in body weight ratio after transplantation are shown in a graph (Figure 3). As a result, differences were observed between recipients in the Alpl- / - transplant groups, and while Homo-1 (#1233) showed weight gain similar to that of heterozygous mice, Homo-2 (#1313) began to lose weight one month after transplantation and died on day 50. On the other hand, Homo-3 (#1375) and Homo-4 (#1399) showed no significant weight gain or loss.

[0059] (2) Examination of intrabone marrow ALP activity after REC-01 transplantation In normal mice (#1237, hetero-2) prepared as a positive control for ALP activity, ALP activity (light purple area) was detected in the cancellous bone (near the growth plate) and cortical bone surface of the femoral head, where osteoblasts are usually abundant (Figure 4, right). In addition, in mouse #1313 (homo-2, Figure 4, center right), which lost weight during the observation period after REC-01 transplantation, ALP activity was detected in a very small area of ​​the femur. Since ALP activity is a marker for osteoblasts, it can be said that REC-01 differentiated into osteoblasts. On the other hand, in the femur of #1359, which was transplanted with only bone marrow cells (BM) as a negative control, ALP activity was not detected in any area (Figure 4, left).

[0060] Notably, among Alpl- / - mice that had received hematopoietic stem cell transplantation in combination with REC-01, #1233 (homo-1, Fig. 4, center left), which showed normal weight gain, had ALP activity detected in the cancellous bone of the femoral head and on the surface of the cortical bone, just like normal mice.

[0061] All recipient ALP- / - mice were administered Strensiq continuously from immediately after birth until death or sacrifice as specimens. In the group that received only bone marrow transplants, no ALP activity was detected, suggesting that the administered ALP preparation, Strensiq, is capable of increasing blood ALP concentrations but does not remain in the bone marrow, and that in individuals that gained weight after REC-01 administration, REC-01 that had taken root in the bone marrow differentiated into osteoblasts and produced ALP, contributing to weight gain (growth).

[0062] Next, ALP staining was performed on frozen sections of the mouse femurs and heads 100 days after transplantation, which is the technically limiting period. In Alpl- / - mice (#1375 and #1399) transplanted with BM+REC-01, ALP activity was observed in the epiphysis, cortical bone, and cancellous bone, similar to that in normal mice (#1237+ / -,hetero-5). Furthermore, in sections of the head, ALP activity was detected in parts of the skull and other areas thought to be brain tissue, although it was slightly weaker than in the femur (Figure 5).

[0063] Although the survival period of mesenchymal stem cells in the bone marrow has not been experimentally proven at all, in hematopoietic stem cell transplantation, long-term engraftment is defined as when donor cells are detected after 3 months (Osawa et al. Science 273(5272):242-5, 1996), and by applying this definition, it can be determined that long-term engraftment of REC-01 in the bone marrow has been observed.

[0064] (3) Improved survival rate with or without REC-01 transplantation (Figure 6) The survival curves of the non-transplant group (n=67, with enzyme, right panel: black line) and the transplant group (n=9, with BM+REC+enzyme, right panel: red line) were evaluated using the Kaplan-Meier method with the log-rank test.

[0065] As a result, REC-01 transplantation significantly improved the survival rate of Alpl- / - mice (Figure 6, p=0.00 045). The enzyme (Strensic) cannot pass through the blood-brain barrier, and so it has been pointed out that it is not possible to suppress epileptic seizures, one of the main symptoms of hypophosphatasia. In fact, in the observation group without transplants (with the enzyme), a high frequency of mice was observed to have epileptic seizures and die.

[0066] In contrast, in the REC-01 transplant group, epileptic seizures were hardly observed, and ALP-positive cells were detected in the skull as shown in Figure 5, indicating that administration of REC-01 was also effective in suppressing epileptic seizures.

[0067] 3. Summary In Alpl- / - recipient mice transplanted with REC-01, normal ALP production in the bone marrow (ALP activity confirmed by ALP staining) and long-term engraftment of transplanted REC-01-derived human cells in the mouse femur (ALP, STEM121 antibody positive) were confirmed. Furthermore, a significant increase in survival rate was observed in the REC-01 transplant group compared to the non-transplant group.

[0068] Based on the above results, in addition to the clinical outcomes (increased survival rate) achieved by administering Strensiq alone, transplantation of REC-01 is expected to result in the normalization of the patient's bone tissue through the transplanted REC-01 taking root in the femur of patients with hypophosphatasia, differentiating into osteoblasts, and producing normal bone-type ALP, and further improving functionality such as suppressing epileptic seizures, thereby bringing about greater benefits to the patient's QOL.

[0069] There have been no reports of successful long-term engraftment of mesenchymal stem cells after intravenous administration in either humans or mice. In contrast, in the present invention, a genetically mutated animal with a normal immune system was used as the recipient, and immunosuppressants were used in combination with xenotransplantation, resulting in extremely low tissue compatibility. Therefore, it is a surprising result that transplanted REC-01 was detectable for up to three months.

[0070] The present invention represents a successful combination of xenogeneic human mesenchymal stem cell transplantation by intravenous administration and hematopoietic stem cell transplantation, and is extremely useful as a medicine for bone diseases.

Claims

1. A pharmaceutical composition for increasing osteoblasts in a subject, comprising mesenchymal stem cells, The mesenchymal stem cells are a cell population of stem cell clones that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and satisfy the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less; The pharmaceutical composition is used in combination with hematopoietic stem cell transplantation into the subject.

2. The pharmaceutical composition according to claim 1, wherein the hematopoietic stem cells are derived from umbilical cord blood.

3. 3. The pharmaceutical composition according to claim 1 or 2, wherein the hematopoietic stem cells are derived from the bone marrow of a donor other than the subject.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject is a patient with a congenital bone disease.

5. The pharmaceutical composition according to claim 4, wherein the congenital bone disease is hypophosphatasia.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the mesenchymal stem cells are fast-proliferating mesenchymal stem cells derived from human bone marrow.

7. The composition according to claim 6, wherein the human bone marrow-derived fast-proliferating mesenchymal stem cells are a cell population of fast-proliferating mesenchymal stem cell clones isolated based on the positivity of LNGFR (CD271) or co-positivity of LNGFR (CD271) and Thy-1 (CD90), and the cell population satisfies the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less;

8. The composition according to claim 6, wherein the human bone marrow-derived fast-proliferating mesenchymal stem cells are a cell population of fast-proliferating mesenchymal stem cell clones derived from cells that are positive for LNGFR (CD271) or co-positive for LNGFR (CD271) and Thy-1 (CD90), and which satisfy the following characteristics (a) and (b): (a) The coefficient of variation of forward scattered light in flow cytometry is 40% or less. (b) the average cell size is 20 μm or less;

9. Cells were administered at a dose of 1x10 per kg of subject body weight. ７ The pharmaceutical composition according to any one of claims 1 to 8, wherein the cells are administered once a week, and the administration is repeated four times.

10. At least 1x10 cells ６ The pharmaceutical composition according to claim 9, which is used at a concentration of cells / ml.

11. The composition according to any one of claims 1 to 10, wherein the HLA of the donor of the mesenchymal stem cells is mismatched with the HLA of the subject.

12. The composition according to any one of claims 1 to 10, wherein the HLA of the donor of the mesenchymal stem cells matches at least 4 of the 6 antigens at 3 loci of the HLA of the subject.

13. The composition according to any one of claims 1 to 10, wherein the HLA of a donor of mesenchymal stem cells matches at least three of the six antigens at three loci of the HLA of the subject.

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