Synovium-derived mesenchymal stem cell, method for producing same, and use thereof

Abstract

The present invention addresses the problem of providing: synovium-derived mesenchymal stem cells that stably exhibit an excellent therapeutic effect on joint diseases; an arthropathy therapeutic agent that contains the synovium-derived mesenchymal stem cells; a method for producing the synovium-derived mesenchymal stem cells; and a method for selecting synovium-derived mesenchymal stem cells for use as an arthropathy therapeutic agent. The present invention provides synovium-derived mesenchymal stem cells that express at least one of a hepatocyte growth factor, a transforming growth factor β-inducing protein, and tumor necrosis factor superfamily 15.

Description

Title of Invention: SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS, METHOD FOR PRODUCING THE SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS, AND USE OF THE SYNOVIUM-DERIVED MESENCHYMAL STEM CELLS Technical Field

[0001] The present invention relates to synovium-derived mesenchymal stem cells having molecules essential for joint treatment. The present invention further relates to a therapeutic agent for arthropathy containing the above-described synovium-derived mesenchymal stem cells, a method for producing the above-described synovium-derived mesenchymal stem cells, and a method for selecting synovium-derived mesenchymal stem cells for use as a therapeutic agent for arthropathy. 2. Description of the Related Art

[0002] In recent years, with advances in regenerative medicine and cell therapy technologies, various cell therapies and the development of cell products for research use using autologous, allogeneic, or xenogeneic cells have been actively pursued. Among them, mesenchymal stem cells (MSCs) are expected to serve as a cell source for useful cell therapy. It has been reported that mesenchymal stem cells can be harvested from various body tissues and can be isolated from bone marrow tissue, adipose tissue, muscle tissue, synovial tissue, periosteal tissue, and the like. In particular, it has been reported that synovium-derived mesenchymal stem cells have higher proliferative capacity and chondrogenic potential than mesenchymal stem cells derived from various mesenchymal tissues such as bone marrow. In addition, Patent Documents 1 to 3 disclose a method for treating articular cartilage damage or meniscal damage by using synovium-derived mesenchymal stem cells. Patent Document 4 discloses a therapeutic agent for arthropathy comprising synovium-derived mesenchymal stem cells having molecules essential for joint treatment, and a method for producing the therapeutic agent for arthropathy. More specifically, Patent Document 4 discloses that synovial tissue is treated with an enzyme, a mixture after the enzyme treatment is washed, cells contained in the washed mixture are cultured on a substrate, the obtained cells are selected based on expression of integrin 01 or platelet-derived growth factor receptor 0, and the quality of the cells is ensured. In addition, Patent Document 5 describes that hepatocyte growth factor (HGF) is expressed in adipose tissue-derived multilineage progenitor cells treated with IL-10. Non-Patent Document 1 describes expression of genes located on chromatin 7, including HGF and other genes in synovial stem cells. Background Art Prior Art Documents Non-Patent Documents

[0003] Non-Patent Document 1: Stem Cells Translational Medicine, Volume 10, Issue 11, November 2021, Pages 1530 to 1543 Patent Documents

[0004] Patent Document 1: JP5928961B Patent Document 2: JP5656183B Patent Document 3: JP6864302B Patent Document 4: 1WO23 / 032945A Patent Document 5: JP2019-218271A Summary of Invention Object to be solved by the invention

[0005] In quality control of cell products, ensuring the equivalence and identity of each lot is an issue. However, since the cells themselves constituting a product are not completely homogeneous, and it is difficult to specify the characteristics of the cells, it is generally difficult to ensure the equivalence and identity of each lot. Therefore, in order to control the quality of the product, not only quality testing of the final product, but also the concept of a quality management system (QMS) that has been applied to medical devices has been introduced, and control throughout the entire process has been implemented by recording and controlling manufacturing raw materials, material management, manufacturing process management, and in-process control tests. However, with the advancement of science and technology, the importance of identifying the characteristics of the cells themselves serving as the final product has been increasing.

[0006] As a quality control method for cells, for example, the cell type of an intended final product is identified using a cell type-specific surface marker as an indicator, for example, identifying that the cells are mesenchymal stem cells. However, it is desired to provide a cell product having a more stable therapeutic effect.

[0007] An object of the present invention is to provide synovium-derived mesenchymal stem cells that stably exert an excellent therapeutic effect on joint diseases. A further object of the present invention is to provide a therapeutic agent for arthropathy comprising the above-described synovium-derived mesenchymal stem cells, a method for producing the above-described synovium-derived mesenchymal stem cells, and a method for selecting synovium-derived mesenchymal stem cells for use as a therapeutic agent for arthropathy. Means for solving the object

[0008] As a result of intensive studies to solve the above-described problem, the present inventors have found that any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein (TGFBI; also known as BIGH3), or tumor necrosis factor superfamily 15 is a quality control marker essential for the effectiveness of treatment of joint diseases with synovial stem cells. The present invention has been completed based on the above findings.

[0009] That is, according to the present invention, the following inventions are provided. <1> Synovium-derived mesenchymal stem cells that are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15. <2> The synovium-derived mesenchymal stem cells according to <1>, in which the synovium-derived mesenchymal stem cells are positive for all of the hepatocyte growth factor, the transforming growth factor beta-induced protein, and the tumor necrosis factor superfamily 15. <3> The synovium-derived mesenchymal stem cells according to <1> or <2>, in which the synovium-derived mesenchymal stem cells are positive for integrin 01. <4> A cell population comprising the synovium-derived mesenchymal stem cells according to any one of <1> to <3>, in which a percentage of cells positive for platelet-derived growth factor receptor 0 is less than 50%. <5> The synovium-derived mesenchymal stem cells according to any one of <1> to <4>, in which the synovium-derived mesenchymal stem cells are obtained by a method including a step A of treating synovial tissue with an enzyme, a step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture obtained after washing the mixture after the enzyme treatment, and a step C of cryopreserving the synovium-derived mesenchymal stem cells after the culturing. <6> The synovium-derived mesenchymal stem cells according to <5>, in which the step A of treating the synovial tissue with an enzyme includes a step of treating the synovial tissue using a solution containing the enzyme and autologous serum or non-autologous serum. <7> A therapeutic agent for arthropathy, comprising: the synovium-derived mesenchymal stem cells or the cell population according to any one of <1> to <6>. <8> A method for producing the synovium-derived mesenchymal stem cells according to any one of <1> to <6>, the method comprising: a step A of treating synovial tissue with an enzyme; a step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture obtained after washing the mixture after the enzyme treatment; and a step C of cryopreserving the synovium-derived mesenchymal stem cells after the culturing. <9> The method according to <8>, in which the step A of treating the synovial tissue with an enzyme includes a step of treating the synovial tissue using a solution containing the enzyme and autologous serum or non-autologous serum. <10> The method according to <8> or <9>, in which step B includes a step performed using a culture medium containing an ascorbic acid derivative. <11> The method according to any one of <8> to <10>, in which, in the step C, a cell concentration at the time of performing a freezing treatment on cells is 1M06 cells / mL or more. <12> The method according to any one of <8> to <11>, in which, in the step C, after a freezing treatment is performed on cells, a period during which the cells are stored in a frozen state is 2 days or more. <13> The method according to any one of <8> to <12>, in which the step C includes a step of freezing the cells at -70°C to -90°C and then freezing the cells at -140°C to -160°C. <14> A method for selecting synovium-derived mesenchymal stem cells to be used as a therapeutic agent for arthropathy, the method comprising measuring expression of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, and selecting positive cells. Effect of the invention

[0010] The synovium-derived mesenchymal stem cell and the therapeutic agent for arthropathy according to the embodiment of the present invention can stably exert an excellent therapeutic effect on joint diseases by being positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15. According to the method for producing the synovium-derived mesenchymal stem cells and the method for selecting synovium-derived mesenchymal stem cells according to the embodiment of the present invention, variation in the therapeutic effect of the obtained synovium-derived mesenchymal stem cells can be suppressed, and quality control for the therapeutic effect of the synovium-derived mesenchymal stem cells can be performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] [Fig.1] FIG. 1 shows the results of confirming the chondrogenic differentiation potential of rat synovium-derived stem cells in which HGF secretion is inhibited. [Fig.2] FIG. 2 shows the results of confirming the meniscus regeneration effect of rat synovium-derived stem cells in which HGF secretion is inhibited. [Fig.3]  FIG. 3 shows the results of confirming the knee cartilage repair effect of rat synovium-derived stem cells in which HGF secretion is inhibited in a rat knee OA model. [Fig.4]  FIG. 4 shows the results of confirming the knee cartilage repair effect of rat synovium-derived stem cells in which HGF secretion is inhibited in a rat knee OA model. [Fig.5] FIG. 5 shows the results of confirming the meniscus regeneration effect of rat synovium-derived stem cells in which TGFBI secretion is inhibited. [Fig.6] FIG. 6 shows the results of confirming the meniscus regeneration effect of rat synovium-derived stem cells in which TNFSF15 gene expression is inhibited. [Fig.7] FIG. 7 shows the analysis results of percentages of CD29 (integrin pi)-positive cells and CD140b (PDGFRP)-positive cells in rat synovium-derived stem cells. [Fig.8] FIG. 8 shows the results of confirming suppression of extracellular matrix adhesion ability by CD29 inhibition in rat synovium-derived stem cells. [Fig.9] FIG. 9 shows the results of confirming the meniscus regeneration effect of rat synovium-derived stem cells in which CD29 is inhibited. [Fig.10] FIG. 10 shows the results of confirming the meniscus regeneration effect of rat synovium-derived stem cells in which CD140b is inhibited. [Fig.11] FIG. 11 shows the results of confirming the meniscus regeneration effect of rat synovium-derived stem cells in which TSP2 secretion is inhibited. Embodiments for carrying out the invention

[0012] Hereinafter, the contents of the present invention will be described in detail. In the present specification, the term “to” is used to mean that numerical values described before and after the term are included as a lower limit value and an upper limit value, respectively.

[0013] The synovium-derived mesenchymal stem cells according to the embodiment of the present invention are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15.

[0014] The synovium-derived mesenchymal stem cells may be positive for any one of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, may be positive for two of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, or may be positive for all of hepatocyte growth factor, transforming growth factor beta-induced protein, and tumor necrosis factor superfamily 15. Preferably, the synovium-derived mesenchymal stem cells are positive for all of hepatocyte growth factor, transforming growth factor beta-induced protein, and tumor necrosis factor superfamily 15.

[0015] The synovium-derived mesenchymal stem cells are preferably positive for integrin 01. In a cell population including the synovium-derived mesenchymal stem cells, a percentage of cells positive for platelet-derived growth factor receptor 0 is preferably less than 50%, more preferably 47% or less, and still more preferably 45% or less. The percentage of cells positive for platelet-derived growth factor receptor 0 (PDGFR0) can be measured by flow cytometry using an anti-PDGFR0 antibody.

[0016] The synovium-derived mesenchymal stem cells according to the embodiment of the present invention can be obtained by a method comprising a step A of treating synovial tissue with an enzyme, a step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture obtained after washing a mixture after the enzyme treatment, and a step C of cryopreserving the synovium-derived mesenchymal stem cells after the culturing.

[0017] According to the present invention, there is provided a method for producing the synovium-derived mesenchymal stem cells of the present invention, the method including: a step A of treating synovial tissue with an enzyme; a step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture obtained after washing a mixture after the enzyme treatment; and a step C of cryopreserving the synovium-derived mesenchymal stem cells after the culturing.

[0018] <Step A of treating synovial tissue with an enzyme> Synovial tissue can be collected from a non-load-bearing portion of a joint under anesthesia. The biological origin of synovial tissue is not particularly limited, and synovial tissue derived from any organism, preferably a mammal, can be used. For example, synovial tissue derived from a primate (for example, a chimpanzee, a Japanese monkey, or a human) can be used, and synovial tissue derived from a human can be particularly preferably used.

[0019] The synovial tissue may be synovial tissue derived from a single donor or synovial tissue derived from a plurality of donors, but is preferably synovial tissue derived from a single donor.

[0020] In a case of producing synovium-derived mesenchymal stem cells for the purpose of administration to a human, synovial tissue collected from a donor whose histocompatibility antigen type matches or is similar to that of a recipient may preferably be used. The subject from which the synovium is collected and the subject to which the synovium-derived mesenchymal stem cells are transplanted may be the same subject, that is, synovial tissue collected from the recipient himself or herself may be used (autologous transplantation). Synovial tissue collected from a subject different from the recipient may be used (allogeneic transplantation). More preferably, the subject from which the synovium is collected and the subject to which the synovium-derived mesenchymal stem cells are transplanted are different.

[0021] The amount of synovial tissue to be collected can be determined in consideration of the type of donor or the required amount of synovium-derived mesenchymal stem cells. For example, synovium-derived mesenchymal stem cells can be obtained from 0.1 g to 10 g, preferably 0.1 g to 2.0 g, more preferably 0.1 g to 1.5 g, and still more preferably 0.1 g to 1.0 g of synovial tissue. The collected synovial tissue is minced with scissors or the like as necessary, and then subjected to an enzyme treatment described later.

[0022] The synovial tissue is treated with an enzyme. The enzyme is not particularly limited as long as the enzyme contains a protease, and is preferably a mixed enzyme containing one or more collagenases and one or more neutral proteases. A particularly preferred enzyme is Liberase (registered trademark). As Liberase (registered trademark), for example, Liberase MNP-S (manufactured by Roche) can be used, which is an enzyme containing collagenase class I, collagenase class II, and a neutral protease (thermolysin).

[0023] The enzyme reaction can be carried out in an aqueous solution containing an enzyme, and an aqueous solution containing serum (for example, human serum, fetal bovine serum, or the like) may be used, or an aqueous solution containing a serum substitute (for example, human platelet lysate) may be used. In a case of using serum, the serum may be autologous serum or non-autologous serum. The non-autologous serum includes allogeneic serum and xenogeneic serum. In a case of using human serum, the serum may be autologous serum or allogeneic serum. Preferably, the step A of treating synovial tissue with an enzyme includes a step of treating the synovial tissue with a solution containing an enzyme and either non-autologous serum or a serum substitute. In addition, from the viewpoint of simplicity of the treatment, it is preferable that the step A of treating synovial tissue with an enzyme be carried out using a solution containing an enzyme and a serum substitute. In a case of using an aqueous solution containing a serum, the concentration of the serum is not particularly limited, but is generally 10% by volume to 40% by volume, preferably 15% by volume to 35% by volume, and more preferably 20% by volume to 30% by volume. In a case of using a serum substitute, the concentration of the serum substitute is not particularly limited, but is 10% by volume to 40% by volume, preferably 15% by volume to 35% by volume, and more preferably 20% by volume to 30% by volume. As a solvent of the aqueous solution containing an enzyme, water for injection, physiological saline, phosphate-buffered saline (PBS), or the like can be used.

[0024] The enzyme concentration in the enzyme treatment is preferably 0.01 mg / ml to 10 mg / ml, more preferably 0.1 mg / ml to 10 mg / ml, still more preferably 0.5 mg / ml to 10 mg / ml, yet more preferably 0.5 mg / ml to 5.0 mg / ml, particularly preferably 0.5 mg / ml to 2.0 mg / ml, and most preferably 0.7 mg / ml to 2.0 mg / ml.

[0025] The mass ratio of synovial tissue to the enzyme is preferably 1000:1 to 10:1, more preferably 500:1 to 20:1, still more preferably 100:1 to 20:1, and yet more preferably 50:1 to 20:1.

[0026] The enzyme reaction can be performed at a temperature of preferably 15°C to 40°C, more preferably 25°C to 40°C, and still more preferably 35°C to 40°C. The reaction time may be 10 minutes or longer, and is preferably 30 minutes or longer, more preferably 1 hour or longer, still more preferably 1.5 hours or longer, and may be 2 hours or longer. The upper limit of the reaction time is not particularly limited, but may be 10 hours or shorter, 9 hours or shorter, 8 hours or shorter, 7 hours or shorter, 6 hours or shorter, 5 hours or shorter, 4 hours or shorter, or 3 hours or shorter. The mixture after the enzyme treatment contains synovium-derived mesenchymal stem cells. The mixture after the enzyme treatment can be transferred to a centrifuge tube through a cell strainer and subjected to centrifugation to recover the synovium-derived mesenchymal stem cells.

[0027] <Step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture after washing the mixture after the enzyme treatment> The mixture after the enzyme treatment is washed as described above. In the washing, washing can preferably be carried out until the residual enzyme concentration in the supernatant is 0.5 ng / mL or less. The residual enzyme concentration in the supernatant is more preferably 0.3 ng / mL or less, still more preferably 0.2 ng / mL or less, and particularly preferably 0.1 ng / mL or less.

[0028] The washing can be carried out by resuspending the synovium-derived mesenchymal stem cells recovered by the above-described centrifugation in a medium and performing centrifugation again (for example, at 400 x g for 5 minutes). As the culture medium, a-modified Eagle's Minimum Essential Medium (aMEM) can be used, but the medium is not particularly limited. The washing may be carried out a plurality of times (twice or more) using the culture medium as described above.

[0029] Synovium-derived mesenchymal stem cells contained in a mixture obtained after washing a mixture after the enzyme treatment are cultured in a culture medium. Examples of the substrate include a planar plastic substrate such as a culture plate, and a three-dimensional substrate such as a culture bag, a microcarrier, or a gel, but the substrate is not particularly limited.

[0030] The culture medium used in the culture can be prepared using a culture medium used for culturing ordinary animal cells as a basal medium. Examples of the culture medium used for culturing ordinary animal cells include aMEM, DMEM (Dulbecco Modified Eagle Medium), a mixed medium of DMEM and F12 (DMEM:F12 = 1:1), an RPMI medium such as GIBCO (registered trademark) RPMI 1640 medium, a mixed medium of DMEM / F12 and RPMI (DMEM / F12:RPMI = 1:1), ROOSTERNOURISHTM-MSC-XF (ROOSTERBIO), PRCROOSTERNOURISHTM-MSC-CC (ROOSTERBIO), PRIME-XV MSC EXPANSION XSFM (FUJIFILM IRVINE SCIENTIFIC, INC.), and MEMa (FUJIFILM Wako Pure Chemical Corporation), but the culture medium is not particularly limited. An antibiotic or an antifungal agent (for example, penicillin, streptomycin, amphotericin B, or the like) may be added to the culture medium.

[0031] HGF, IGF-II, or SCF may be added to the culture medium, or a combination thereof may be added to the culture medium. The concentrations of HGF, IGF-II, and SCF in the culture medium are not particularly limited, but are preferably 0.01 to 10000 ng / mL, more preferably 0.01 to 1000 ng / mL, still more preferably 0.1 to 1000 ng / mL, and yet more preferably 1 to 100 ng / mL.

[0032] The culture medium may be a serum-containing culture medium, a serum substitute-containing culture medium, or a serum-free culture medium. In a case of producing synovium-derived mesenchymal stem cells from autologous tissue for the purpose of administration to a living body, the culture medium may contain autologous serum. That is, in a case of producing synovium-derived mesenchymal stem cells from human tissue for the purpose of administration to a human, a culture medium containing human serum may be used. In a case of using serum, the serum may be autologous serum or allogeneic serum, but is preferably allogeneic serum. In a case of using a serum, the amount of the serum to be added to the culture medium is, for example, 20% by volume or less, 10% by volume or less, or 5% by volume or less. From the viewpoint of simplicity of the operation, it is preferable to use a serum substitute. In a case of using a serum substitute, the concentration of the serum substitute is not particularly limited, but is preferably 0.5% by volume to 20% by volume, more preferably 0.5% by volume to 15% by volume, still more preferably 1% by volume to 15% by volume, and yet more preferably 5% by volume to 15% by volume.

[0033] Preferably, the step B includes a step of carrying out the culture using a culture medium containing an ascorbic acid derivative. Examples of the ascorbic acid derivative include ascorbic acid 2-phosphate, trisodium ascorbic acid 2-phosphate, magnesium ascorbic acid 2-phosphate, and ascorbic acid 2-glucoside, but the ascorbic acid derivative is not particularly limited. In a case of using a culture medium containing an ascorbic acid derivative, the concentration of the ascorbic acid derivative in the culture medium is not particularly limited, but is preferably 0.03 mmol / L or more, more preferably 0.1 mmol / L or more, and still more preferably 0.14 mmol / L or more. The upper limit value thereof is preferably 5.0 mmol / L or less, more preferably 1.0 mmol / L or less, and still more preferably 0.57 mmol / L or less.

[0034] The culture conditions for the cells are not particularly limited, and ordinary cell culture conditions can be adopted. Examples include culture at a temperature of 30°C to 40°C and 3% to 7% CO2, but the culture conditions are not particularly limited. An example includes culture at a temperature of 37°C and 5% CO2.

[0035] In the present invention, the culture may be carried out without medium exchange or with medium exchange, and the culture is preferably carried out with medium exchange. In a case of exchanging the culture medium, the culture may be carried out in a culture medium containing an ascorbic acid derivative in the first half of the step B, and the culture may be carried out in a culture medium containing no ascorbic acid derivative in the second half of the step B. In addition, in the above-described culture, it is preferable that synovium-derived mesenchymal stem cells be produced without being co-cultured with cells other than synovium-derived mesenchymal stem cells.

[0036] It is known that differentiation of synovium-derived mesenchymal stem cells into chondrocytes progresses as the culture period becomes longer, and therefore the in situ chondrogenic potential of synovium-derived mesenchymal stem cells decreases when the culture period exceeds a specific length. Therefore, in the present invention, it is preferable to adjust the culture period in order to proliferate synovium-derived mesenchymal stem cells in an undifferentiated state and in a state of having good in situ chondrogenic potential. In the step B, the period of culturing synovium-derived mesenchymal stem cells is preferably 28 days or less.

[0037] In addition, in the present invention, it is necessary to consider the necessity of preparing a sufficient number of undifferentiated synovial stem cells for treatment. Alternatively, it is necessary to consider the necessity of preparing a sufficient number of undifferentiated synovial stem cells for preparing a master cell bank. Therefore, the culture period is preferably 5 days or more, 7 days or more, or 8 days or more, and may be 8 to 14 days, 8 to 21 days, or 8 to 28 days, or may be 5 to 8 days, 5 to 10 days, or 5 to 14 days.

[0038] It is known that mesenchymal stem cells can be differentiated into chondrocytes and can produce cartilage tissue in vitro by culturing mesenchymal stem cells in a chondrogenic culture medium to which transforming growth factor 03 (TGF-03), dexamethasone, and bone morphogenetic factor 2 (BMP-2) are added. Therefore, in the present invention, in order to prevent synovium-derived mesenchymal stem cells from differentiating into chondrocytes, it is preferable to culture the isolated synovium-derived mesenchymal stem cells in the absence of TGF-03, dexamethasone, or BMP-2.

[0039] In the present invention, since a certain or higher cell density is required from the viewpoint of proliferation of synovium-derived mesenchymal stem cells, it is preferable to seed synovium-derived mesenchymal stem cells after the enzyme treatment at a cell density of 100 cells / cm2 or more and 5,000 cells / cm2 or less, 200 cells / cm2 or more and 5,000 cells / cm2 or less, 500 cells / cm2 or more and 5,000 cells / cm2 or less, 500 cells / cm2 or more and 2,500 cells / cm2 or less, or 500 cells / cm2 or more and 2,000 cells / cm2 or less, and culture the synovium-derived mesenchymal stem cells. In addition, in order to proliferate synovium-derived mesenchymal stem cells after the enzyme treatment, it is more preferable to culture the synovium-derived mesenchymal stem cells for 5 days or more.

[0040] The number of cells obtained at the end of the culture is preferably 1.0 x 107 cells or more, 2.0 x 107 cells or more, 2.5 x 107 cells or more, or 3.0 x 107 cells or more, more preferably 4.0 x 107 cells or more, still more preferably 5.0 x 107 cells or more, and particularly preferably 6.0 x 107 cells or more.

[0041] <Separation of synovium-derived mesenchymal stem cells from a substrate> It is preferable that the synovium-derived mesenchymal stem cells after culturing in the step B be separated from the substrate before the step C. Preferably, separation from the substrate can be carried out by allowing a cell detachment solution to act on the mesenchymal stem cells for a period of 120 minutes or less. Examples of the cell detachment solution include a solution containing a trypsin-like enzyme and EDTA. A particularly preferred enzyme is TrypLE. As TrypLE, for example, TrypLE Express (manufactured by Gibco), TrypLE Select (manufactured by Gibco), or the like can be used.

[0042] From the viewpoint of sufficiently detaching the cells, the time for allowing the cell detachment solution to act on the mesenchymal stem cells is preferably 5 minutes or longer. The time for allowing the cell detachment solution to act on the mesenchymal stem cells is preferably 5 minutes to 120 minutes, and more preferably 5 minutes to 60 minutes. The time may be 5 minutes to 50 minutes, 5 minutes to 40 minutes, 5 minutes to 60 minutes, 5 minutes to 50 minutes, or 5 minutes to 40 minutes, and may be 10 minutes to 40 minutes.

[0043] <Step C of cryopreserving synovium-derived mesenchymal stem cells after culturing> The synovium-derived mesenchymal stem cells after culturing can be cryopreserved. In the step C, the cell concentration at the time of performing a freezing treatment on the cells is 1 x 106 cells / mL or more, preferably 2 x 107 cells / mL or more, more preferably more than 1 x 108 cells / mL, more preferably 1.1 x 108 cells / mL or more, still more preferably 1.2 x 108 cells / mL or more, and may be 1.5 x 108 cells / mL or more or 2.0 x 108 cells / mL or more.

[0044] In the step C, as the cryopreservation solution used when performing a freezing treatment on the cells, a cryopreservation solution containing DMSO is preferably used. Specifically, CTS Synth-a-Freeze Medium (Thermo Fisher Scientific), STEM-CELLBANKER GMP grade (XenoGen Pharma), CP-1 High Grade (Kyokuto Pharmaceutical Industrial Co., Ltd.), or the like can be used, but is not limited thereto. In a case of using CP-1 High Grade, CP-1 High Grade may be used with albumin added thereto. As the albumin, albumin derived from a human, monkey, bovine, pig, rabbit, mouse, or rat can be used, but human serum albumin (HSA) is preferably added and used. As the HSA to be added, HSA prepared from human blood plasma may be used, or an animal-expressed recombinant or a plant-expressed recombinant may also be used. In addition, the final concentration of the HSA to be added can be appropriately adjusted, and may be 1% to 5%, and is preferably 3% to 5%.

[0045] In the step C, after a freezing treatment is performed on the cells, the period during which the cells are stored in a frozen state is preferably 2 days or longer, more preferably 10 days or longer, and still more preferably 1 month or longer. The cells can be stored in a frozen state for 3 months or longer, 4 months or longer, 5 months or longer, 6 months or longer, 7 months or longer, 8 months or longer, 9 months or longer, 10 months or longer, 11 months or longer, 12 months or longer, 16 months or longer, 20 months or longer, or 24 months or longer.

[0046] The step C preferably includes a step of freezing the cells at -70°C to -90°C and then freezing the cells at -140°C to -160°C. The step C more preferably includes a step of freezing the cells at -70°C to -90°C for 1 day or longer and then freezing the cells at -140°C to -160°C for 1 day or longer.

[0047] In the step C, after a freezing treatment is performed on the cells, the cells may be stored in a frozen state as a master cell bank, and then a part of the master cell bank may be thawed and subjected to expansion culture to produce synovial-derived mesenchymal stem cells as a therapeutic agent for arthropathy.

[0048] Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme). It is known that mesenchymal stem cells are present in bone marrow, synovium, periosteum, adipose tissue, and muscle tissue, and that mesenchymal stem cells have an ability to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle cells. It is known that, in relation to differentiation of mesenchymal stem cells into chondrocytes, differentiation of undifferentiated mesenchymal stem cells into chondrocytes is promoted by adding BMP or TGF-P to a culture solution, and cartilage tissue can be regenerated under in vitro conditions.

[0049] Mesenchymal stem cells can be confirmed by detecting molecules characteristic of mesenchymal stem cells, for example, enzymes, receptors, low-molecular-weight compounds, or the like. Examples of molecules characteristic of mesenchymal stem cells include CD73, CD90, CD105, CD166, and the like, which are cell surface markers (positive markers), but are not limited thereto. In addition, examples of negative markers that are not expressed in mesenchymal stem cells include CD19, CD34, CD45, HLA-DR, CD11b, CD14, and the like, but are not limited thereto. It is noted that CD is an abbreviation for clusters of differentiation, and HLA-DR is an abbreviation for human leukocyte antigen-D-related. Mesenchymal stem cells can be confirmed by using these positive markers and negative markers. An immunological method can be used for the detection of these markers, but the detection may be performed by quantifying the amount of mRNA of each molecule.

[0050] In the present specification, synovium-derived mesenchymal stem cells are stem cells included in the synovium. Synovium-derived mesenchymal stem cells are a type of mesenchymal stem cells. Synovium-derived mesenchymal stem cells can be detected, for example, by detecting CD90 positivity, CD45 negativity, and chondrogenic differentiation potential, but the detection method is not particularly limited.

[0051] In the present specification, “positive” means expressing the corresponding gene, expressing the corresponding protein, or expressing both the corresponding gene and the corresponding protein. The expression of the protein includes a case where the protein is expressed on a cell surface, a case where the protein is secreted from cells, or both cases.

[0052] In the present specification, the positive rate means a ratio of the number of positive cells in a cell population containing synovium-derived mesenchymal stem cells.

[0053] According to the present invention, there is provided a therapeutic agent for arthropathy comprising the above-described synovium-derived mesenchymal stem cells of the present invention. In a case where synovium-derived mesenchymal stem cells are used as a therapeutic agent for arthropathy, the cells may be mixed with a pharmaceutically acceptable carrier by a conventional method to form a preparation in a form suitable for administration to an individual. Examples of the carrier include physiological saline, and distilled water for injection made isotonic by adding glucose or other auxiliary agents (for example, D-sorbitol, D-mannitol, sodium chloride, and the like). Furthermore, a buffering agent (for example, a phosphate buffer solution or a sodium acetate buffer solution), a pain-reducing agent (for example, benzalkonium chloride or procaine hydrochloride), a stabilizer (for example, human serum albumin or polyethylene glycol), a preservative, an antioxidant, and the like may be blended.

[0054] The method for producing the therapeutic agent for arthropathy of the present invention may preferably further include a step of selecting synovium-derived mesenchymal stem cells that are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15.

[0055] According to one aspect of the present invention, there is provided a method for selecting synovium-derived mesenchymal stem cells for use as a therapeutic agent for arthropathy, the method comprising measuring expression of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, and selecting cells that are positive.

[0056] Examples of the step of selecting synovium-derived mesenchymal stem cells that are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 include managing an expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15.

[0057] The expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 refers to an expression amount of a gene or protein corresponding to any one of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15. The expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, and tumor necrosis factor superfamily 15 can be calculated as an absolute value or a relative value, such as a ratio to or a difference from a comparative control or a reference expression level.

[0058] The expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 can be measured by any method known to those skilled in the art, and the measurement can be carried out according to a conventional method. As the measurement of the expression level, the amount of mRNA, which is a transcript of a gene, may be measured. The measurement of the amount of mRNA is not particularly limited as long as it is a method capable of measuring a desired amount of mRNA, and a known method can be appropriately selected and used. For example, a gene amplification method using, as a primer, an oligonucleotide that hybridizes to a gene encoding any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, or a hybridization method using, as a probe, an oligo(poly)nucleotide that hybridizes to a gene encoding a specific protein molecule can be used. Specific examples thereof include a reverse transcription polymerase chain reaction (RT-PCR) method, a real-time RT-PCR method, a DNA microarray method, a cell array method, a Northern blotting method, a dot blotting method, and an RNase protection assay method.

[0059] The primer or probe used in the above-described measurement method can be labeled, and the amount of mRNA can be measured by examining the signal intensity of the label. The real-time RT-PCR method is preferable in that RNA can be directly used as a sample, and gene quantification is possible based on the number of temperature cycles required for amplification by optically measuring the gene amplification process. In addition, as a control, the expression level of a gene encoding any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 can be normalized using the expression level of mRNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is a housekeeping gene, beta-actin, or the like. The primer and probe used in the above-described measurement method can be appropriately designed and prepared by those skilled in the art based on information on the nucleotide sequence of a gene encoding any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15.

[0060] For measurement of the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, for example, a method of immunologically measuring the expression level using an antibody or antibody fragment against any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 can be used. Specific examples thereof include flow cytometry, a Western blotting method, an enzyme immunoassay (ELISA), a radioimmunoassay (RIA), a fluorescent antibody method, a cell array method, and the like. These measurement methods can also be carried out according to a protocol of a conventional method or a protocol obtained by appropriately modifying or changing the protocol of the conventional method.

[0061] For example, in a case where the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, and tumor necrosis factor superfamily 15 in cells is measured by flow cytometry, when the positive rate for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 is preferably 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, the cells can be selected as synovium-derived mesenchymal stem cells that are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15.

[0062] In a case of selecting synovium-derived mesenchymal stem cells that are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, for example, the selection can be carried out by comparing the expression level of any one of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 in cells measured by the above-described method with a predetermined reference expression amount. The reference expression amount may be, for example, an expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, and tumor necrosis factor superfamily 15 in cells that have already been confirmed to have a certain quality (positive control), or may be an expression level in cells that have already been confirmed not to have a certain quality (negative control).

[0063] By comparing the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 with the reference expression level, cells in which the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 is equal to or higher than the expression amount of the positive control can be selected and used as a therapeutic agent for arthropathy.

[0064] In addition, a cut-off value of the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 may be set in advance, and the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 measured in cells may be compared with the cut-off value. The cut-off value can be, for example, an expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 that gives a desired therapeutic effect, based on a regression line showing a correlation between the expression amount of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 and a therapeutic effect. For example, cells in which the expression level of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15 is equal to or higher than the cut-off value can be selected and used as a therapeutic agent for arthropathy.

[0065] The positive rate for platelet-derived growth factor receptor P (PDGFRP) in a cell population including synovium-derived stem cells is preferably less than 50%, and may be 10% or more and less than 50%, 20% or more and less than 50%, 30% or more and less than 50%, 35% or more and less than 50%, or 40% or more and less than 50%.

[0066] The therapeutic agent for arthropathy according to the embodiment of the present invention can be used for joint treatment. Examples of the joint treatment include treatment of a disease accompanied by damage, injury, or inflammation of a joint, and examples thereof include treatment of a joint disease caused by degeneration and / or inflammation of a connective tissue such as cartilage, or a non-inflammatory joint disease. Examples of joint treatment include treatment of a disease selected from the group consisting of meniscal damage, traumatic cartilage damage, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis, for example, knee osteoarthritis, rheumatoid arthritis, for example, chronic rheumatoid arthritis, gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defect, but the disease is not limited to these diseases. Examples of a target site of the joint treatment include a knee joint, a shoulder joint, a hand joint, a hip joint, an elbow joint, a finger joint, a spine, an intervertebral disc, an ankle joint, a toe joint, a tarsal joint, and a jaw joint, but the present invention is not limited thereto.

[0067] A method for treating a joint using the therapeutic agent for arthropathy according to the embodiment of the present invention includes: a step of transplanting the therapeutic agent for arthropathy of the present invention such that a cartilage damage site or a meniscal damage site is covered with synovium-derived mesenchymal stem cells; and a step of regenerating cartilage tissue in situ at the cartilage damage site or the meniscal damage site by differentiating synovium-derived mesenchymal stem cells contained in the therapeutic agent for arthropathy into chondrocytes.

[0068] In a case of transplanting the therapeutic agent for arthropathy of the present invention to a patient, in order to efficiently treat a cartilage damage site or a meniscal damage site, it is preferable to apply 2.0 x 107 to 1.0 x 1011, 2.5 x 107 to 1.0 x 1011, 3.0 x 107 to 1.0 x 1011, 4.0 x 107 to 1.0 x 1011, 2.5 x 107 to 1.0 x 1010, 2.5 x 107 to 1.0 x 109, 2.5 x 107 to 1.0 x 108, or 2.0 x 107 to 1.0 x 108 synovium-derived mesenchymal stem cells per cartilage damage site or meniscal damage site.

[0069] By transplanting synovium-derived mesenchymal stem cells to a cartilage damage site or a meniscal damage site, the cartilage damage site or the meniscal damage site is covered with the synovium-derived mesenchymal stem cells.     The transplantation of synovium-derived mesenchymal stem cells can be performed by open surgery or arthroscopic surgery. In order to minimize invasiveness, it is preferable to transplant synovium-derived mesenchymal stem cells under arthroscopy.

[0070] The cartilage damage site or the meniscal damage site may be covered with a suspension of synovium-derived mesenchymal stem cells or may be covered with a cell sheet of synovium-derived mesenchymal stem cells. For example, a bioabsorbable gel such as gelatin or collagen can be used as a gel-like substance. Synovium-derived mesenchymal stem cells have a high ability to adhere to a cartilage damage site or a meniscal damage site.

[0071] In the case of treatment of cartilage damage, the minimally invasive procedure of the present invention is characterized by covering a cartilage damage site with synovium-derived mesenchymal stem cells, and includes the following steps: maintaining a body position such that the cartilage damage site faces upward; placing a cell sheet of synovium-derived mesenchymal stem cells, a suspension of synovium-derived mesenchymal stem cells, or a gel-like substance containing synovium-derived mesenchymal stem cells on a surface of the cartilage damage site; and maintaining the body position for a specific time, thereby causing the synovium-derived mesenchymal stem cells to adhere to the surface of the cartilage damage site.

[0072] In the case of treatment of meniscal damage, the minimally invasive procedure of the present invention is characterized by covering a meniscal damage site with synovium-derived mesenchymal stem cells, and includes the following steps: maintaining a body position such that the meniscal damage site faces downward; injecting a suspension of synovium-derived mesenchymal stem cells into a knee joint; and maintaining the body position for a specific time to cause the synovium-derived mesenchymal stem cells to adhere to the meniscal damage site.

[0073] In order to reliably adhere synovium-derived mesenchymal stem cells to the surface of a cartilage damage site or a meniscal damage site, it is preferable to hold the transplanted synovium-derived mesenchymal stem cells on the surface of the cartilage damage site or the meniscal damage site for at least 10 minutes, preferably 15 minutes. In order to realize this, the body position is maintained for at least 10 minutes, preferably 15 minutes, for the purpose of orienting a cartilage damage site or a meniscal damage site upward and holding synovium-derived mesenchymal stem cells on the upward-facing cartilage damage site or meniscal damage site.

[0074] The cartilage damage site or the meniscal damage site with synovium-derived mesenchymal stem cells can be further covered with periosteum in order to further strengthen adhesion of the synovium-derived mesenchymal stem cells to the cartilage damage site or the meniscal damage site. The surgery is completed after holding synovium-derived mesenchymal stem cells on the surface of the cartilage damage site or the surface of the meniscal damage site for at least 10 minutes.

[0075] In the present invention, the transplanted synovium-derived mesenchymal stem cells differentiate into chondrocytes at the cartilage damage site or the meniscal damage site, and regenerate cartilage tissue in situ at the cartilage damage site or the meniscal damage site.

[0076] During the in situ chondrogenesis process of synovium-derived mesenchymal stem cells, cartilage tissue is regenerated according to the local microenvironment, such as nutrient supply and the cytokine environment, and thus external manipulation is not required. As a result of in situ chondrogenesis of synovium-derived mesenchymal stem cells, cartilage tissue is regenerated at the cartilage damage site or the meniscal damage site to repair the damage, and in a case of cartilage damage, a bone region, a boundary between cartilage and bone, a central cartilage region, a surface region, and a region adjacent to original cartilage are formed as original cartilage tissue, or in a case of meniscal damage, meniscal cartilage is formed.

[0077] The present invention will be described more specifically by the following examples, but the present invention is not limited to the examples. Examples

[0078] <Example 1> Preparation of rat synovium-derived mesenchymal stem cells Ten ACI / NSlc rats (male, 3 weeks old at arrival, 4 weeks old at tissue sampling) (Japan SLC) were used to establish rat synovium-derived mesenchymal stem cells. After euthanasia under isoflurane anesthesia, synovial tissue was collected from both knees and stored in ice-cold PBS until digestion treatment.

[0079] As a solution for digestion treatment of synovial tissue, water for injection (Otsuka Pharmaceutical Co., Ltd.) was added to Liberase MNP-S (Roche Cat. No. 5578566001) so as to give a concentration of 1.25 mg / mL, thereby preparing a Liberase solution. Furthermore, 1 mL of heat-inactivated fetal bovine serum (Gibco Cat. No. 10500064) was mixed with 4 mL of the Liberase solution to obtain a digestion solution.

[0080] 153.68 mg of the collected synovial tissue was immersed in the digestion solution and reacted at 37°C for 2 hours. As a culture medium for cell culture, a culture medium was prepared by adding heat-inactivated fetal bovine serum to a basal medium of MEMa with L-Alanyl-L-Glutamine, Phenol Red and Sodium Pyruvate (FUJIFILM Wako Pure Chemical Corporation, Cat. No. 289-33365) containing 0.28 mM trisodium ascorbic acid 2-phosphate, which is an ascorbic acid derivative, so that the final concentration was 20%, and adding a penicillin-streptomycin-amphotericin B suspension (x100) (antibiotic-antimycotic solution) (FUJIFILM Wako Pure Chemical Corporation, Cat. No. 161-23181) so that the final concentration was 1%.

[0081] A two-fold volume of ice-cold culture medium was added to the digestion solution in which the synovial tissue had been reacted for 2 hours, and the resulting mixture was passed through a 40 urn cell strainer (FALCON, Cat. No. 352340) to remove residual tissue. The recovered cells were seeded in ten T225 cell culture flasks at a density of 1000 cells / cm2, and cultured in the culture medium at a CO2 concentration of 5% and 37°C. After 8 days of culture, the culture medium in the flasks was discarded, the cells were washed twice with PBS, TrypLE Express (Gibco, Cat. No. 12604-013) was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and 4.54 x 107 cells were recovered as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation, and the cells were replaced with a cryopreservation solution prepared by adding HSA (albumin, human, plant-expressed recombinant) (FUJIFILM Wako Pure Chemical Corporation) to CP-1 High Grade (Kyokuto Pharmaceutical Industrial Co., Ltd., Cat. No. 27207) so that the final concentration was 4%. Thereafter, the cells were frozen in a -80°C freezer for 1 day or more at a cell freezing concentration of 1.2 x 106 cells / mL, and then frozen in a -150°C freezer for 1 day or more to prepare a frozen stock (Passage 0) of the rat synovium-derived mesenchymal stem cells. The cells were stored at the same temperature until use.

[0082] <Example 2> Confirmation of hepatocyte growth factor (HGF) secretion from rat synovium-derived stem cells and inhibition treatment thereof The frozen stock (Passage 0) of the rat synovium-derived stem cells prepared in Example 1 was thawed and cultured at a CO2 concentration of 5% and 37°C using the culture medium used in Example 1. On day 6 of culture, Silencer Select Pre-designed siRNA (Thermo Fisher Scientific, Cat. No. 4390816, ID s127876) (hereinafter, Hgf siRNA) for inhibiting gene expression of hepatocyte growth factor (HGF) was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Cat. No. 31985070), and added to the cells during culture (final siRNA concentration: 11.76 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermo Fisher Scientific, Cat. No. 4390844) (hereinafter, NC siRNA), which does not inhibit expression of any gene including HGF, was used to treat the cells in the same manner. One day after the Hgf or NC siRNA treatment, the culture medium in the flask was discarded, the cells were washed twice with PBS, TrypLE Express was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and the cells were recovered as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation, and the cells were replaced with CP-1 High Grade. Thereafter, the cells were frozen in a -80°C freezer for 1 day or more at a cell freezing concentration of 2 x 108 cells / mL, and then frozen in a -150°C freezer for 1 day or more to prepare a frozen stock of the rat synovium-derived mesenchymal stem cells treated with Hgf or NC siRNA.

[0083] The frozen stock of the rat synovium-derived mesenchymal stem cells treated with Hgf or NC siRNA was thawed and cultured in the culture medium. A portion of the culture supernatant was collected on days 1, 3, and 7 from the start of culture. During this period, the culture medium was not exchanged. The concentration of HGF in the collected culture supernatant was measured using an Hgf (Rat) ELISA Kit (Abnova, Cat. No. KA4322). The results are shown in Table 1. In the rat synovium-derived mesenchymal cells treated with NC siRNA, which is a control treatment (indicated as NC in the table), the HGF concentration increased over time, and HGF was detected at 170.79 pg / mL and 198.16 pg / mL, which exceeded the lower limit of quantification, on Day 3 and Day 7, respectively. On the other hand, in the rat synovium-derived mesenchymal cells treated with Hgf siRNA (indicated as HGFkd in the table), an increase in the HGF concentration over time was not observed, and the HGF concentration was equal to or lower than the lower limit of quantification (156 pg / mL) and was comparable to the HGF concentration in the culture medium (indicated as GM in the table). From these results, it was found that the rat synovium-derived mesenchymal cells secrete HGF, and rat synovium-derived mesenchymal cells in which HGF secretion was inhibited by Hgf siRNA treatment were prepared.

[0084] [Table 1] Treatment Sampling day HGF concentration (pg / mL) Average ± S.D. NC 1d 82.19 ± 28.22 3d 170.79 ± 14.44 7d 198.16 ± 18.00 HGFkd 1d 80.08 ± 13.53 3d 69.75 ± 18.15 7d 67.69 ± 21.68 GM 1d 92.63 ± 48.50 3d 50.15 ± 19.85 7d 64.57 ± 9.29 Lower limit of quantification: 156 pg / mL

[0085] <Example 3> Evaluation of chondrogenic differentiation potential in rat synovium-derived stem cells in which HGF secretion is inhibited In order to examine the relationship between hepatocyte growth factor and cartilage repair function in rat synovium-derived stem cells, the chondrogenic differentiation potential of rat synovium-derived stem cells in which HGF secretion was inhibited (hereinafter referred to as HGFkd-SMSC) was evaluated. 2.5 x 105 cells of HGFkd-SMSC or NC-SMSC prepared in Example 2 were suspended in DMEM high glucose (Thermo Fisher Scientific, Cat. No. 11965092) containing TGF-P3 (R&D Systems, Cat. No. 243-B3-002) at a final concentration of 10 ng / mL, dexamethasone (FUJIFILM Wako Pure Chemical Corporation, Cat. No. 041-18861) at a final concentration of 3.92 pg / mL, L-ascorbic acid 2-phosphate (Cayman Chemical, Cat. No. 16457) at a final concentration of 50 pg / mL, L-proline (MP Biomedicals, Cat. No. 194728) at a final concentration of 40 pg / mL, sodium pyruvate (Invitrogen, Cat. No. 11360070) at a final concentration of 1 pg / mL, ITS-X supplement (x100) (FUJIFILM Wako Pure Chemical Corporation, Cat. No. 094-06761) at a final concentration of 1%, and BMP-2 (R&D Systems, Cat. No. 355-BM-010) at a final concentration of 0.5 pg / mL, centrifuged at 450 x g for 10 minutes, and then cultured at a CO2 concentration of 5% and 37°C to induce chondrogenic differentiation. In addition, rat synovium-derived mesenchymal cells treated with NC siRNA, which were control cells (hereinafter referred to as NC-SMSC), were also subjected to chondrogenic differentiation induction in the same manner as HGFkd-SMSC. After 3 weeks of culture, the chondrogenic differentiation potential was evaluated based on the diameter and weight of the cell aggregates and histological staining of the cell aggregates, and the results are shown in Table 2 and FIG. 1. NC-SMSC had a weight of 2.33 ± 0.06 mg and a major axis of 1.77 ± 0.06 mm, whereas HGFkd-SMSC had a weight of 0.77 ± 0.06 mg and a major axis of 1.17 ± 0.06 mm, and it was found that both the cartilage weight and the size were reduced by the inhibition of HGF secretion. In addition, when the stainability of the cartilage matrix was confirmed in histological specimens, regions where staining with toluidine blue staining (TB), safranin O staining (SafO), and type II collagen immunostaining (Col II) was lost were observed in HGFkd-SMSC. From these results, it was confirmed that HGF is an important molecule that contributes to the chondrogenic differentiation potential of cells.

[0086] [Table 2] Treatment Weight (mg) Major axis (mm) Average ± S.D. Average ± S.D. NC-SMSC 2.33 ± 0.06 1.77 ± 0.06 HGFkd-SMSC 0.77 ± 0.06 1.17 ± 0.06

[0087] <Example 4> Meniscus regeneration effect of rat synovium-derived stem cells in which HGF secretion is inhibited Rat synovium-derived stem cells in which HGF secretion was inhibited were prepared as described in Example 2. Female LEW / CrlCrlj rats were used to prepare a meniscal injury model for evaluating the meniscus regeneration effect. In the method for producing meniscal injury and transplanting mesenchymal stem cells, the skin of the knee joint region was incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule under the patella was exposed and incised longitudinally with a scalpel to expose the cartilage of the distal femur. The medial meniscus was peeled off from the synovium, the medial meniscus was exposed, and about 2 / 3 of the entire meniscus was resected. The patellar tendon and the synovium were sutured, and then the muscle was sutured to prepare a meniscal injury model. Thereafter, the treated animals were divided into three groups, and 1 x 10A7 HGFkd-SMSC cells, 1 x 10A7 NC-SMSC cells, or only the cryopreservation medium CP1 (Vehicle) was administered into the joint capsule. After the treatment, all the rats were returned to their cages and allowed free movement and access to food and water.

[0088] Three weeks after the treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, the meniscus was exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the medial menisci excised from both knee joints are shown in FIG. 2. The regenerated portion was identified based on differences in color tone and shape from the normal meniscus and surrounded by a broken line. In the Vehicle group, which was a negative control, many of the menisci had a shape extending to the middle segment. In the NC-SMSC group, which was a positive control, many of the menisci were regenerated up to the anterior segment, and large meniscus regenerated portions were observed. In the HGFkd-SMSC group, the meniscus regenerated portion appeared smaller than that in the NC-SMSC group, which was a positive control, and was comparable to that in the Vehicle group, to which cells were not administered.

[0089] In order to quantitatively evaluate the macroscopic findings of the meniscus regenerated portion in Fig. 2, the area of the meniscus regenerated portion (inside the broken line) was calculated by the following expression using Image J (version 1.52).

[0090] Area of meniscus regenerated portion (mm2) = number of pixels in meniscus regenerated portion / number of pixels per 1 mm2

[0091] The mean value, standard deviation, and statistical analysis of the area of the regenerated portion in each group were all calculated or performed using Microsoft Excel 2007 (Microsoft Corporation). Statistical analysis was performed by Bonferroni’s multiple comparison test using GraphPad Prism 5.04 software, and all pairwise comparisons among all groups were performed. A significance level of less than 5% was regarded as indicating a significant difference, and the results are shown in Table 3.

[0092] Regarding the area of the meniscus regenerated portion shown in Table 3, the area of 0.69 mm2 in the HGFkd-SMSC group was significantly decreased as compared with the area of 1.22 mm2 in NC-SMSC. On the other hand, the HGFkd-SMSC group had a regeneration area comparable to 0.64 mm2 in the Vehicle group. From these results, it was confirmed that HGF molecules secreted by synovial stem cells are important molecules that contribute to meniscus regeneration.

[0093] [Table 3] Group Meniscus regenerative area (mm2) Average ± S.D. Vehicle (CP1) 0.64 ± 0.31 NC-SMSC 1.22*** ± 0.30 HGFkd-SMSC 0.69ttt ± 0.37 ****P < 0.0001 vs Vehicle, tttp < 0.001 vs NC-SMSC

[0094] <Example 5> Knee cartilage repair effect of rat synovium-derived stem cells in which HGF secretion is inhibited in a rat knee OA model Rat synovium-derived stem cells in which HGF secretion was inhibited were prepared as described in Example 2. Female LEW / CrlCrlj rats were used to prepare a knee OA model for evaluating the cartilage repair effect. In the method for performing anterior cruciate ligament transection (ACLT) for inducing knee OA and transplanting rat synovial stem cells, the skin of the knee joint region was incised under isoflurane anesthesia to expose the knee joint, with both knees being targeted. The medial joint capsule under the patella was exposed, the joint capsule was incised longitudinally with a scalpel, and the anterior cruciate ligament was exposed and cut. Thereafter, the joint capsule, the muscle, and the skin were sutured. The animals subjected to the ACLT treatment were divided into three groups, and 1 x 106 HGF-inhibited synovial stem cells (HGFkd-SMSC), 1 x 106 synovial stem cells subjected to control treatment without inhibition (NC-SMSC), or solvent alone was administered into the joint, for a total of seven times once a week from one week after the ACLT treatment.

[0095] Eight weeks after the ACLT treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, femoral and tibial cartilage was exposed from the knee joint, and the cartilage portion was coated with India ink and then photographed. Cartilage damage was evaluated from images of femoral and tibial cartilage using a macroscopic cartilage damage score (Yanagisawa K., BMC Musculoskeletal Disorders (2016) 17:188). FIG. 3 shows representative images of femoral and tibial cartilage. In addition, Table 4 shows the macroscopic cartilage damage score. As statistical analysis, the obtained scores of each group were subjected to tests between groups by a Mann-Whitney test using GraphPad Prism 5.04 software. A significance level of less than 5% was regarded as indicating a significant difference, and the results are shown in Table 4.

[0096] Table 4 shows the macroscopic cartilage damage score. Regarding the cartilage damage score, the scores of 3.0 and 2.9 for tibial and femoral cartilage, respectively, in the NC-SMSC group were significantly decreased as compared with the scores of 3.9 and 3.6, respectively, in the Vehicle group, and thus it was found that knee cartilage damage was reduced by administration of synovial stem cells. On the other hand, the cartilage damage scores in the HGFkd-SMSC group were 3.7 and 3.3 for tibial and femoral cartilage, respectively, which were higher than the scores in the NC-SMSC group, and the score for the tibia was significantly increased and was comparable to the score in the Vehicle group.

[0097] [Table 4] Group n = 20 knees Tibia (both knees) Femur (both knees) Average ± S.D. Average ± S.D. Vehicle (CP1) 3.9 ± 0.5 3.6 ± 0.6 NC-SMSC 3.0*** ± 0.0 2.9* ± 1.1 HGFkd-SMSC 3.7™ ± 0.5 3.3 ± 0.9

[0098] In addition to the macroscopic evaluation, cartilage damage scores (OARSI scores) of femoral and tibial cartilage were evaluated in histological specimens. For preparation of histological specimens (Pritzker KPH, Osteoarthritis and Cartilage (2006) 14, 13), the excised femoral and tibial cartilage tissues were subjected to formalin fixation and decalcification treatment, and then a specimen plane was prepared in a sagittal section including a region with the most severe damage in the macroscopic findings, and safranin O staining was performed. FIG. 4 shows representative histological specimen images of femoral and tibial cartilage. In addition, Table 5 shows the OARSI scores in histological specimens. As statistical analysis, the obtained scores of each group were subjected to tests between groups by a Mann-Whitney test using GraphPad Prism 5.04 software. A significance level of less than 5% was regarded as indicating a significant difference, and the results are shown in Table 5.

[0099] Table 5 shows the OARSI scores in histological specimens. Regarding the cartilage damage score under the histological specimen, both the tibial and femoral cartilage of the NC-SMSC group of 7.4 and 4.8 were decreased as compared with 11.8 and 5.6 of the Vehicle group, and there was a significant difference in the tibia, and thus it was found that the knee cartilage damage was reduced by the administration of the synovial stem cell. On the other hand, the cartilage damage scores in the HGFkd-SMSC group were 9.7 and 5.9 for tibial and femoral cartilage, respectively, which were higher than the scores in the NC-SMSC group, and the score for the tibia was significantly increased and was comparable to the score in the Vehicle group. Group n = 20 knees Tibia (both knees) Femur (both knees) Average ± S.D. Average ± S.D. Vehicle (CP1) 11.8 ± 5.8 5.6 ± 2.4 NC-SMSC 7.4*** ± 1.9 4.8 ± 2.1 HGFkd-SMSC 9.7™ ± 2.4 5.9 ± 2.4 ***P < 0.001 vs Vehicle, W < 0.001 vs NC-SMSC

[0101] From the above, it was confirmed that synovial stem cells have a therapeutic effect on cartilage damage associated with knee OA, and that HGF is an essential molecule for the therapeutic effect.

[0102] <Example 6> Confirmation of TGFBI secretion from rat synovium-derived stem cells and inhibition treatment thereof The frozen stock of the rat synovium-derived stem cells prepared in Example 1 was thawed and cultured at a CO2 concentration of 5% and 37°C using the culture medium used in Example 1. On day 4 of culture, Silencer Select Pre-designed siRNA (Thermo Fisher Scientific, Cat. No. 4390816, ID s137979) (hereinafter, Tgfbi siRNA) for inhibiting gene expression of TGFBI was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Cat. No. 31985070), and added to the cells during culture (final siRNA concentration: 20.7 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermo Fisher Scientific, Cat. No. 4390844) (hereinafter, NC siRNA), which does not inhibit gene expression of any genes including TGFBI, was used to treat the cells in the same manner. Three days after the Tgfbi siRNA or NC siRNA treatment, the culture medium in the flask was discarded, the cells were washed twice with PBS, TrypLE Express was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and the cells were recovered as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation, and the cells were replaced with CP-1 High Grade. Thereafter, the cells were frozen in a -80°C freezer for 1 day or more at a cell freezing concentration of 2 x 108 cells / mL, and then frozen in a -150°C freezer for 1 day or more to prepare a frozen stock of the rat synovium-derived mesenchymal stem cells treated with Tgfbi siRNA or NC siRNA.

[0103] The frozen stock of the rat synovium-derived mesenchymal stem cells treated with Tgfbi siRNA or NC siRNA was thawed and cultured in the culture medium. A portion of the culture supernatant was collected on days 1, 3, and 7 from the start of culture. During this period, the culture medium was not exchanged. The concentration of TGFBI in the collected culture supernatant was measured using beta IG-H3 / TGFBI PicoKine ELISA Kit (Boster Immunoleader, Cat. No. EK1571). The results are shown in Table 6. Treatment Sampling day TGFBI conc. (pg / mL) Average ± S.D. SMSC (no treated) 1d 676 ± 102 3d 2070 ± 493 7d 2142 ± 93 NC-SMSC 1d 298 ± 64 3d 1185 ± 316 7d 1721 ± 108 TGFBIkd-SMSC 1d 0 ± 0 3d 28 ± 49 7d 126 ± 63 GM 1d 60 ± 91 3d 47 ± 33 7d 13 ± 22 Lower limit of quantification: 156 pg / mL

[0105] In the untreated synovial stem cells (indicated as SMSC (not treated) in the table), TGFBI secretion in the culture supernatant was observed at 676, 2070, and 2142 pg / mL on Day 1, Day 3, and Day 7, respectively. In the rat synovium-derived mesenchymal cells treated with NC siRNA, which is a control treatment (indicated as NC-SMSC in the table), TGFBI secretion in the culture supernatant was observed at 298, 1185, and 1721 pg / mL on Day 1, Day 3, and Day 7, respectively. On the other hand, in the rat synovium-derived mesenchymal cells treated with Tgfbi siRNA (indicated as TGFBIkd-SMSC in the table), an increase in the TGFBI concentration over time was not observed, and the TGFBI concentration was equal to or lower than the lower limit of quantification (156 pg / mL) and was comparable to the TGFBI concentration in the culture medium (indicated as GM in the table). From these results, it was found that the rat synovium-derived mesenchymal cells secrete TGFBI, and rat synovium-derived mesenchymal cells in which TGFBI secretion was inhibited by Tgfbi siRNA treatment were prepared.

[0106] <Example 7> Meniscus regeneration effect of rat synovium-derived stem cells in which TGFBI secretion is inhibited Rat synovium-derived stem cells in which TGFBI secretion was inhibited were prepared as described in Example 6. The preparation of the meniscal injury model for evaluating the meniscus regeneration effect was carried out in the same manner as in Example 4. The treated animals were divided into three groups, and 1 x 107 TGFBIkd-SMSC cells, 1 x 107 NC-SMSC cells, or only the cryopreservation medium CP1 (Vehicle) was administered into the joint capsule.

[0107] Three weeks after the treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, the meniscus was exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the medial menisci excised from both knee joints are shown in FIG. 5. The regenerated portion was identified based on differences in color tone and shape from the normal meniscus and surrounded by a broken line. In the NC-SMSC group, which was a positive control, the regenerated portion appeared slightly larger than those in the Vehicle group, which was a negative control, and the TGFBIkd-SMSC group, but no clear difference was observed.

[0108] In order to quantitatively evaluate the macroscopic findings of the meniscus regenerated portion in FIG. 5, the area was calculated as described in Example 4 and evaluated by statistical processing.

[0109] Regarding the area of the meniscus regenerated portion shown in Table 7, the area of 0.78 mm2 in the TGFBIkd-SMSC group was significantly decreased as compared with the area of 1.14 mm2 in the NC-SMSC group. On the other hand, the TGFBIkd-SMSC group had a regeneration area comparable to 0.77 mm2 in the Vehicle group. From these results, it was confirmed that TGFBI molecules in synovial stem cells are important molecules that contribute to meniscus regeneration. Group Meniscus regenerative area (mm2) Average ± S.D. Vehicle (CP1) 0.77 ± 0.36 NC-SMSC 1.14* ± 0.31 TGFBIkd-SMSC 0.78t ± 0.34 *P < 0.05 vs Vehicle, tP < 0.05 vs NC-SMSC

[0111] <Example 8> Confirmation of TNFSF15 secretion from rat synovium-derived stem cells and inhibition treatment thereof The frozen stock of the rat synovium-derived stem cells prepared in Example 1 was thawed and cultured at a CO2 concentration of 5% and 37°C using the culture medium used in Example 1. On day 4 of culture, Silencer Select Pre-designed siRNA (Thermo Fisher Scientific, Cat. No. 4390816, ID s141123) (hereinafter, Tnfsf15 siRNA) for inhibiting gene expression of TNFSF15 was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Cat. No. 31985070), and added to the cells during culture (final siRNA concentration: 11.2 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermo Fisher Scientific, Cat. No. 4390844) (hereinafter, NC siRNA), which does not inhibit gene expression of any genes including TNFSF15, was used to treat the cells in the same manner. Three days after the Tnfsf15 siRNA or NC siRNA treatment, the culture medium in the flask was discarded, the cells were washed twice with PBS, TrypLE Express was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and the cells were recovered as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation, and the cells were replaced with CP-1 High Grade. Thereafter, the cells were frozen in a -80°C freezer for 1 day or more at a cell freezing concentration of 2 x 108 cells / mL, and then frozen in a -150°C freezer for 1 day or more to prepare a frozen stock of the rat synovium-derived mesenchymal stem cells treated with Tnfsf15 siRNA or NC siRNA.

[0112] The frozen stock of the rat synovium-derived mesenchymal stem cells treated with Tnfsf15 siRNA or NC siRNA was thawed and cultured in the culture medium. The cells were recovered on days 1, 3, and 7 from the start of culture, and total RNA in the cells was extracted and RT-PCR was performed using a TaqMan Gene Expression Cells-to-CT Kit (Thermo Fisher Scientific, Cat. No. AM1728) to obtain cDNA. Thereafter, TaqMan Gene Expression Assay (FAM) Assay ID: Rn00595596_m1 Tnfsf15 (Thermo Fisher Scientific, Cat. No. 4331182) was used as a primer-probe for amplification of the target gene TNFSF15, and TaqMan Gene Expression Assay (FAM) Assay ID: Rn00667869_m1 Actb (Thermo Fisher Scientific, Cat. No. 4331182) was used as a primer-probe for amplification of the housekeeping gene, and gene amplification was performed using a real-time PCR instrument, QuantStudio 7 Flex, to obtain respective Ct values. A value obtained by subtracting the Ct value of the housekeeping gene ACTB from the obtained Ct value of TNFSF15 was used as a delta Ct value, and the values for the respective conditions were compared. The results are shown in Table 8. On Day 1, the delta Ct values of untreated ACI-SMSC and NC-SMSC were 14.05 and 14.71, respectively. In contrast, the delta Ct value of TNFSF15kd-SMSC was 15.64, and a relative decrease in the gene expression level was confirmed. On Day 3, the delta Ct values of untreated ACI-SMSC and NC-SMSC were 12.13 and 12.51, respectively. In contrast, the delta Ct value of TNFSF15kd-SMSC was 13.22, and a relative decrease in the gene expression level was confirmed. On the other hand, on Day 7, the delta Ct values of untreated ACI-SMSC and NC-SMSC were 10.56 and 9.46, respectively, whereas the delta Ct value of TNFSF15kd-SMSC was 10.58, and a relative decrease in the gene expression level was no longer observed. From these results, it was found that rat synovium-derived mesenchymal cells express the TNFSF15 gene, and rat synovium-derived mesenchymal cells in which TNFSF15 gene expression was inhibited up to Day 3 by Tnfsf15 siRNA treatment were prepared.

[0113] [Table 8] Target TNFSF15 delta Ct value (CtTNFSF15-CtACTB) Day 1 Day 3 Day 7 No treated ACI-SMSC 14.05 12.13 10.56 Control siRNA NC-SMSC 14.71 12.51 9.46 TNFSFkd SMSC 15.64 13.22 10.58

[0114] In order to examine TNFSF15 protein expression in rat synovium-derived mesenchymal cells in which TNFSF15 gene expression was inhibited by Tnfsf15 siRNA treatment, 1 mL of Cell Lysis Buffer M (FUJIFILM Wako Pure Chemical Corporation, Cat. No. 038-21141) was added to the remaining cells excluding 1 * 105 cells used for the above-described RNA extraction, and the cells were suspended by pipetting and allowed to stand on ice for 10 minutes. The cells were centrifuged at a maximum of 20,000 * g at 4°C for 10 minutes, and the supernatant was collected as a cell lysate (protein extract). The concentration of TNFSF15 in the collected cell lysate was measured using a Rat Tumor Necrosis Factor Ligand Superfamily Member 15 (TNFSF15) ELISA Kit (Signalway Antibody, EK6199). The results are shown in Table 9. In the untreated synovial stem cells (indicated as SMSC (no treated) in the table), TNFSF15 protein expression was observed at 29.43, 42.62, and 37.28 pg / mL on Day 1, Day 3, and Day 7, respectively. In the rat synovium-derived mesenchymal cells treated with NC siRNA as a control treatment (indicated as NC-SMSC in the table), similar TNFSF15 protein expression was observed at 29.13, 51.23, and 43.13 pg / mL on Day 1, Day 3, and Day 7, respectively. On the other hand, in the rat synovium-derived mesenchymal cells treated with TNFSF15 siRNA (indicated as TNFSF15kd-SMSC in the table), the TNFSF15 concentration on Day 3 was 27.22 pg / mL, and a decrease was observed as compared with SMSC (no treated) and NC-SMSC. On Day 7, the concentration was comparable among all three treatment groups. From these results, it was confirmed that rat synovium-derived mesenchymal cells express the TNFSF15 protein, and that TNFSF15 protein expression was inhibited up to Day 3 by TNFSF15 siRNA treatment.

[0115] [Table 9] Treatment Sampling day TNFSF15 conc. (pg / mL) in cell lysate Average ± S.D. SMSC (no treated) 1d 29.43 ± 3.87 3d 42.62 ± 6.26 7d 37.28 ± 4.54 NC-SMSC 1d 29.13 ± 6.14 3d 51.23 ± 9.94 7d 43.13 ± 7.66 TGFBIkd-SMSC 1d 34.46 ± 5.63 3d 27.22 ± 14.19 7d 32.92 ± 3.73 Cell Lysis buffer - 12.91 ± 2.68 Lower limit of quantification: 15.6 pg / mL

[0116] <Example 9> Meniscus regeneration effect of rat synovium-derived stem cells in which TNFSF15 gene expression is inhibited Rat synovium-derived stem cells in which TNFSF15 gene expression was inhibited were prepared as described in Example 8. The preparation of the meniscal injury model for evaluating the meniscus regeneration effect was carried out in the same manner as in Example 4. The treated animals were divided into three groups, and 1 x 107 TNFSF15kd-SMSC cells, 1 x 107 NC-SMSC cells, or only the cryopreservation medium CP1 (Vehicle) was administered into the joint capsule.

[0117] Three weeks after the treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, the meniscus was exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the medial menisci excised from both knee joints are shown in FIG. 6. The regenerated portion was identified based on differences in color tone and shape from the normal meniscus and surrounded by a broken line. In the NC-SMSC group, which was a positive control, the regenerated portion appeared slightly larger than those in the Vehicle group, which was a negative control, and the TNFSF15kd-SMSC group.

[0118] In order to quantitatively evaluate the macroscopic findings of the meniscus regenerated portion in FIG. 6, the area was calculated as described in Example 4 and evaluated by statistical processing. The results are shown in Table 10. Regarding the area of the meniscus regenerated portion shown in Table 10, the area of 0.84 mm2 in the TNFSF15kd-SMSC group was significantly decreased as compared with the area of 1.29 mm2 in the NC-SMSC group. On the other hand, the TNFSF15kd-SMSC group had a regeneration area comparable to 0.87 mm2 in the Vehicle group. From these results, it was confirmed that the TNFSF15 molecule in synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0119] [Table 10] Group Meniscus regenerative area (mm2) Average ± S.D. Vehicle (CP1) 0.87 ± 0.31 NC-SMSC 1.29* ± 0.55 TNFSF15kd-SMSC 0.84t ± 0.27 *P < 0.05 vs Vehicle, tP < 0.05 vs NC-SMSC

[0120] <Example 10> Positive rates for CD29 (integrin pi) and CD140b (PDGFRP) in rat synovium-derived stem cells The rat synovium-derived stem cells obtained in Example 1 were subjected to expansion culture for 7 days under the same conditions as in Example 1 to prepare a frozen stock. The frozen stock was thawed, and 106 cells were suspended in 500 pL of PBS. For dead cell staining, 0.5 pL of LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Cat. No. L34957) was added to the cell suspension, and the mixture was incubated at room temperature for 30 minutes. After centrifugation, the supernatant was discarded, 1 mL of FACS buffer (PBS containing 2 mM EDTA^2Na and 1% bovine serum albumin at final concentrations) was added, and the cells were suspended. For measurement of the positive rate for integrin p1, 5 pL of PE anti-mouse / rat Integrin p1 Antibody (BioLegend, Cat. No. 102207) or 5 pL of PE Armenian Hamster IgG Isotype Ctrl Antibody (BioLegend, Cat. No. 400907) was added, and the mixture was allowed to react at 4°C for 30 minutes. Thereafter, the mixture was centrifuged, the supernatant was discarded, the cells were suspended in 1 mL of FACS buffer, the mixture was centrifuged again, the supernatant was discarded, the cells were suspended in 500 pL of FACS buffer, and the cells were subjected to measurement. For measurement of the positive rate for CD140b, 5 pL of Anti-PDGF Receptor p, Human, Goat-Poly (R&D Systems, Cat. No. AF385) or 5 pL of Normal Goat IgG Control (R&D Systems, Cat. No. AB-108-C) was added, and the mixture was allowed to react at 4°C for 30 minutes. Thereafter, the mixture was centrifuged, the supernatant was discarded, and the cells were suspended in 1 mL of FACS buffer. Furthermore, 1 liL of Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC (Invitrogen, Cat. No. A16006) was added, and the mixture was allowed to react at 4°C for 30 minutes. Thereafter, the mixture was centrifuged, the supernatant was discarded, the cells were suspended in 1 mL of FACS buffer, the mixture was centrifuged again, the supernatant was discarded, the cells were suspended in 500 lL of FACS buffer, and the cells were subjected to flow cytometry to measure the positive rates for CD29 and CD140b. The analysis results of the positive rates for CD29 and CD140b in synovium-derived stem cells are shown in FIG. 7. CD29 showed a positive rate of 91.6%. On the other hand, CD140b showed a positive rate of 44.4%, which was less than half the positive rate of CD29.

[0121] <Example 11> Suppression of extracellular matrix adhesion ability by CD29 inhibition in rat synovium-derived stem cells In order to confirm that CD29 was inhibited, adhesion function to the extracellular matrix, which is one of the functions of CD29, was confirmed. As preparation of rat synovium-derived stem cells in which CD29 was inhibited, the frozen stock of rat synovium-derived stem cells prepared in Example 1 was thawed and subjected to expansion culture under the same conditions as in Example 1. After 7 days of culture, the culture medium in the flask was discarded, the cells were washed twice with PBS, TrypLE Express (Gibco, Cat. No. 12604-013) was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and the cells were recovered. The supernatant was discarded by centrifugation, the cells were cultured at a CO2 concentration of 5% and 37°C for 1 week, and the recovered cells were suspended in PBS containing 2% FBS as a reaction solvent. 12 pg of Purified anti-mouse / rat Integrin pi Antibody (BioLegend, Cat. No. 102202) was added per 5 x 106 cells, and the cells were allowed to react on ice for 1 hour and then recovered (CD29ab-SMSC). As a control treatment in which Integrin p1 inhibition was not performed, Purified Armenian Hamster IgG Isotype Ctrl (BioLegend, Cat. No. 400902) was allowed to react with the cells on ice for 1 hour, and then the cells were recovered (IgG-SMSC). In addition, untreated cells (SMSC) that had undergone the same reaction only with the reaction solvent were provided. For each recovered cell sample, the supernatant was discarded by centrifugation, and the cells were replaced with CP-1 High Grade (Kyokuto Pharmaceutical Industrial Co., Ltd., Cat. No. 27207). Thereafter, the cells were frozen in a -80°C freezer for 1 day or more at a cell freezing concentration of 2 x 108 cells / mL, then frozen in a -150°C freezer for 1 day or more, and then subjected to the following adhesion treatment.

[0122] For the extracellular matrix adhesion reaction, the cells were washed with PBS containing 10 mM MgCh^6H2O (hereinafter, PBS (+)), suspended again in PBS (+), adjusted to 1 x 105 cells / 10 gL / well, and seeded in six wells per treatment in a collagen I-coated Chamber Slide II 8-chamber (IWAKI, Cat. No. 4732-010) (n = 6). After allowing to stand at room temperature for 10 minutes, the cells were washed with PBS (+). Thereafter, the cells were observed with a microscope (OLYMPUS, Cat. No. IX71) using a 10x objective lens. In addition, images of three fields of view having the largest number of adherent cells per well were acquired, and the number of adherent cells was measured. The average value of the number of cells in the three measured fields of view was defined as the number of adherent cells per well.

[0123] The results are shown in Table 11 and FIG. 8. As the number of adherent cells, the number of cells treated with the CD29 neutralizing antibody (CD29ab-SMSC) was 253 cells, showing a marked decrease as compared with 472 cells of antibody-untreated cells (SMSC) and 396 cells of IgG-treated cells (IgG-SMSC). From these results, it was confirmed that the function of CD29 in rat synovial stem cells can be inhibited by treatment with Purified anti-mouse / rat Integrin pi Antibody.

[0124] [Table 11] Treatment Adherent cells Average ± S.D. SMSC (no treatment) 472 ± 99 IgG-SMSC 396 ± 105 CD29ab-SMSC 253***' tt ± 60 ***P < 0.001 vs SMSC group, ttp < 0.01 vs IgG-SMSC group

[0125] <Example 12> Meniscus regeneration effect of rat synovium-derived stem cells in which CD29 is inhibited Frozen rat synovium-derived stem cells in which CD29 was inhibited were prepared as described in Example 3. The frozen cell stock (Passage 0) prepared in Example 1 was thawed and cultured for 1 week, and the recovered cells were suspended in PBS containing 2% FBS as a reaction solvent. 24 gg of Purified anti-mouse / rat Integrin p1 Antibody was added per 1 x 107 cells, and the cells were allowed to react on ice for 1 hour and then cryopreserved at a concentration of 2 x 107 cells / 100 gL per cryovial using cryopreservation solution CP-1 High Grade. In addition, as a control treatment in which inhibition was not performed, Purified Armenian Hamster IgG Isotype Ctrl was allowed to react with the cells on ice for 1 hour, and then the cells were cryopreserved at a concentration of 2 x 107 cells / 100 gL per cryovial using cryopreservation solution CP-1 High Grade.

[0126] Female LEW / CrlCrlj rats were used to prepare a meniscal injury model for evaluating the meniscus regeneration effect. In the method for producing meniscal injury and transplanting mesenchymal stem cells, the skin of the knee joint region was incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule under the patella was exposed and incised longitudinally with a scalpel to expose the cartilage of the distal femur. The medial meniscus was peeled off from the synovium, the medial meniscus was exposed, and about 2 / 3 of the entire meniscus was resected. The patellar tendon and the synovium were sutured, and then the muscle was sutured to prepare a meniscal injury model. Thereafter, the treated animals were divided into three groups, and 1 x 107 CD29-inhibited synovial stem cells (CD29ab-SMSC), 1 x 107 synovial stem cells subjected to control treatment without inhibition (IgG-SMSC), or solvent alone was administered into the joint capsule of each group. After the treatment, all the rats were returned to their cages and allowed free movement and access to food and water.

[0127] Three weeks after the treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, the meniscus was exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the medial menisci excised from both knee joints are shown in FIG. 9. The regenerated portion was identified based on differences in color tone and shape from the normal meniscus and surrounded by a broken line. In the solvent group, which was a negative control, many of the menisci had a shape extending only to the middle segment. In the IgG-SMSC group, which was a positive control, many of the menisci were regenerated up to the anterior segment, and large meniscus regenerated portions were observed. On the other hand, in the CD29ab-SMSC group, which was a group of cells in which the molecule was inhibited or deleted, the meniscus regenerated portion appeared smaller than that in the positive control.

[0128] In order to quantitatively evaluate the macroscopic findings of the meniscus regenerated portion in FIG. 9, the area of the meniscus regenerated portion (inside the broken line) was calculated by the following expression using ImageJ (version 1.52).

[0129] Area of meniscus regenerated portion (mm2) = number of pixels in meniscus regenerated portion / number of pixels per 1 mm2

[0130] The mean value, standard deviation, and statistical analysis of the area of the regenerated portion in each group were all calculated or performed using Microsoft Excel 2007 (Microsoft Corporation). Statistical analysis was performed by Bonferroni’s multiple comparison test using GraphPad Prism 5.04 software, and all pairwise comparisons among all groups were performed. A significance level of less than 5% was regarded as indicating a significant difference, and the results are shown in Table 12.

[0131] Regarding the area of the meniscus regenerated portion shown in Table 12, the area of 0.69 mm2 in the CD29ab-SMSC group was significantly decreased as compared with the area of 1.44 mm2 in the IgG-SMSC group. On the other hand, the CD29ab-SMSC group had a regeneration area comparable to 0.87 mm2 in the solvent group. From these results, it was confirmed that the CD29 molecule is an essential molecule for meniscus regeneration by synovial stem cells.

[0132] [Table 12] Group Regenerative meniscus (mm2) Average ± S.D. Vehicle 0.87 ± 0.5 IgG-SMSC 1.44** ± 0.79 CD29ab-SMSC 0.6911 ± 0.35 ** P < 0.01 vs Vehicle group, 11 P < 0.01 vs IgG-SMSC group

[0133] <Example 13> Inhibition of cell proliferative capacity by inhibition of platelet-derived growth factor receptor P (PDGFRp, also referred to as CD140b) factor of rat synovium-derived stem cell As preparation of rat synovium-derived stem cells in which CD140b was inhibited, the frozen stock of rat synovium-derived stem cells prepared in Example 1 was thawed and subjected to expansion culture under the same conditions as in Example 1. After 7 days of culture, the culture medium in the flask was discarded, the cells were washed twice with PBS, TrypLE Express (Gibco, Cat. No. 12604-013) was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and the cells were recovered. The supernatant was discarded by centrifugation, the cells were cultured at a CO2 concentration of 5% and 37°C for 1 week, and the recovered cells were suspended in PBS containing 2% FBS as a reaction solvent. Anti-PDGF Receptor P Human Goat-Poly (R&D Systems, Cat. No. AF385) was added in an amount of 20 pg per 1 x 106 cells, and the cells were allowed to react on ice for 1 hour and then recovered (CD140b-SMSC). As a control treatment in which CD140b inhibition was not performed, Normal Goat IgG Control (R&D Systems, Cat. No. AB-108-C) was allowed to react with the cells on ice for 1 hour, and then the cells were recovered (IgG-SMSC). In addition, untreated cells (SMSC) that had undergone the same reaction only with the reaction solvent were provided. For each recovered cell sample, the supernatant was discarded by centrifugation, and the cells were replaced with CP-1 High Grade (Kyokuto Pharmaceutical Industrial Co., Ltd., Cat. No. 27207). Thereafter, the cells were frozen in a -80°C freezer for 1 day or more at a cell freezing concentration of 2 x 108 cells / mL, and then stored frozen in a -150°C freezer for 1 day or more, and then subjected to the following treatment and evaluation.

[0134] In order to confirm that CD140b was inhibited, the cell proliferative capacity, which is a function of CD140b, was confirmed. Table 13 shows the experimental levels in which each treatment is described. After the above treatment, the cryopreserved cells SMSC, IgG-SMSC, and CD140b-SMSC were seeded in a 96-well plate and cultured for 1 day in culture medium alone (levels 1 to 3), or in culture medium containing Anti-PDGF Receptor P Human Goat-Poly at a concentration of 40 gg / mL (level 4) or Normal Goat IgG Control at a concentration of 40 gg / mL (level 5).

[0135] On the day after cell seeding, the culture supernatant was discarded, and the medium was replaced with culture medium (level 1), or with culture medium containing 0.1% FBS at a final concentration (levels 2 to 5), or with culture medium containing PDGF-BB, Rat, Recombinant (R&D Systems, Cat. No. 520-BB-050) at a final concentration of 5 ng / mL (levels 3 to 5). Furthermore, as experimental conditions, Normal Goat IgG Control (level 4) or Anti-PDGF Receptor P Human (level 5) was added so that the final concentration was 40 gg / mL, and the total amount of the culture medium was set to 100 uL. In the levels to which IgG or the antibody was not added, only 100 gL of the culture medium of each composition was added (levels 1 to 3). On day 6 after cell seeding, the number of cells in each level was regarded as the luminescence intensity based on ATP using Cell Titer-Glo (Promega, Cat. No. G7571), and the proliferative ability of the cells was relatively evaluated.

[0136] [Table 13] Level Antibody treatment at cell freezing Day 0 Day 1 Day 6 Seeding Medium replacement,  ligand addition, and antibody addition ATP assay 1 Untreated    (antibody reaction solution only) 20%   FBS   medium (Normal culture medium) 20% FBS medium (Normal culture medium) 2 Untreated    (antibody reaction solution only) 0.1% FBS medium 3 Untreated    (antibody reaction solution only) 0.1% FBS medium + 5 ng / mL PDFG-BB 4 20 ug IgG per 106 cells 20%   FBS   medium containing 40 ug / mL IgG 0.1% FBS medium + 5 ng / mL PDFG-BB + 40 ug / mL IgG 5 20  ug  Anti-PDGF Receptor P Human per 106 cells 20%   FBS   medium (Normal culture medium) containing  40  ug / mL Anti-PDGF Receptor P Human 0.1% FBS medium + 5 ng / mL PDFG-BB  +  40  ug / mL Anti-PDGF Receptor P Human

[0137] The results are shown in Table 14. In culture in ordinary culture medium (level 1), the luminescence intensity was 18.57, whereas in culture in 0.1% FBS culture medium (level 2), the cell proliferation number markedly decreased to 3.69. Since the luminescence intensity was increased to 8.81 by adding PDGF-BB, which is a ligand of PDGFR-P (level 3), it was confirmed that the cell proliferation was dependent on PDGFR-p. On the other hand, by adding Anti-PDGF Receptor P Human Goat-Poly, which is an antibody that inhibits CD140b, in addition to PDGF-BB (level 5), the luminescence intensity was 6.22, which was significantly decreased as compared with the luminescence intensity of 8.25 for IgG (level 4), which was the control treatment. However, the inhibition rate of level 5, calculated based on the values of 4.56 and 2.53 obtained by subtracting the value of level 2 as a baseline from the values of levels 4 and 5, was 44.5%, and not all of the cell proliferation effect induced by PDGF-BB was suppressed. As shown in FIG. 7, since the positive rate for CD140b in the present cells was 44.4%, it is considered that, among the proliferation effects induced by PDGF-BB, the proliferation effect involving CD140b was completely inhibited by the present antibody treatment. From these results, it was confirmed that PDGFRP (CD140b) in rat synovial stem cells can be ligand-specifically inhibited by treatment with Anti-PDGF Receptor P Human Goat-Poly.

[0138] [Table 14] Treatment Luminescence (ATP assay) Average ± SD 1 20% FBS growth medium (GM) 18.57 ± 2.34 2 0.1% FBS GM 3.69 ± 0.49 3 0.1% FBS GM + PDGF - BB 8.81 ± 0.89 4 0.1% FBS GM + PDGF - BB + IgG 8.25 ± 0.78 5 0.1% FBS GM + PDGF - BB + CD140b-ab 6.22 ± 0.31 ****P < 0.0001 Vs Treatment 4 Students' T-test

[0139] <Example 14> Meniscus regeneration effect of rat synovium-derived stem cells in which CD140b is inhibited Frozen rat synovium-derived stem cells in which CD140b was inhibited were prepared as described in Example 13. That is, the frozen cell stock (Passage 0) prepared in Example 1 was thawed and cultured for 1 week, and the recovered cells were suspended in PBS containing 2% FBS as a reaction solvent. 20 pg of Anti-PDGF Receptor P Human Goat-Poly was added per 1 x 106 cells, and the cells were allowed to react on ice for 1 hour and then cryopreserved at a concentration of 2 x 107 cells / 100 pL per cryovial using cryopreservation solution CP-1 High Grade. In addition, as a control treatment in which inhibition was not performed, Purified Armenian Hamster IgG Isotype Ctrl was allowed to react with the cells on ice for 1 hour, and then the cells were cryopreserved at a concentration of 2 x 107 cells / 100 pL per cryovial using cryopreservation solution CP-1 High Grade.

[0140] Female LEW / CrlCrlj rats were used to prepare a meniscal injury model for evaluating the meniscus regeneration effect. In the method for producing meniscal injury and transplanting mesenchymal stem cells, the skin of the knee joint region was incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule under the patella was exposed and incised longitudinally with a scalpel to expose the cartilage of the distal femur. The medial meniscus was peeled off from the synovium, the medial meniscus was exposed, and about 2 / 3 of the entire meniscus was resected. The patellar tendon and the synovium were sutured, and then the muscle was sutured to prepare a meniscal injury model. Thereafter, the treated animals were divided into three groups, and 1 x 107 CD140b-inhibited synovial stem cells (CD140b-ab-SMSC), 1 x 107 synovial stem cells subjected to control treatment without inhibition (IgG-SMSC), or solvent alone was administered into the joint capsule. After the treatment, all the rats were returned to their cages and allowed free movement and access to food and water.

[0141] Three weeks after the treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, the meniscus was exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the medial menisci excised from both knee joints are shown in FIG. 10. The regenerated portion was identified based on differences in color tone and shape from the normal meniscus and surrounded by a broken line. In the solvent group, which was a negative control, many of the menisci had a shape extending only to the middle segment. In the IgG-SMSC group, which was a positive control, many of the menisci were regenerated up to the anterior segment, and large meniscus regenerated portions were observed. On the other hand, in the CD140b-ab-SMSC group, which was a group of cells in which the molecule was inhibited, no difference from the positive control was confirmed, and comparable meniscus regeneration was observed.

[0142] In order to quantitatively evaluate the macroscopic findings of the meniscus regenerated portion in FIG. 10, the area of the meniscus regenerated portion (inside the broken line) was calculated by the following expression using ImageJ (version 1.52).

[0143] Area of meniscus regenerated portion (mm2) = number of pixels in meniscus regenerated portion / number of pixels per 1 mm2

[0144] The mean value, standard deviation, and statistical analysis of the area of the regenerated portion in each group were all calculated or performed using Microsoft Excel 2007 (Microsoft Corporation). Statistical analysis was performed by Bonferroni’s multiple comparison test using GraphPad Prism 5.04 software, and all pairwise comparisons among all groups were performed. A significance level of less than 5% was regarded as indicating a significant difference, and the results are shown in Table 15.

[0145] Regarding the area of the meniscus regenerated portion shown in Table 15, the area of 1.03 mm2 in the Vehicle group was significantly increased as compared with the area of 0.74 mm2 in the CD140b-ab-SMSC group. On the other hand, the CD140b-ab-SMSC group had a regeneration area comparable to that of the IgG-SMSC group. From these results, it was shown that the CD140b molecule is not an essential molecule for meniscus regeneration by synovial stem cells.

[0146] [Table 15] Area of meniscus regenerated portion Group Regenerative meniscus (mm2) Average ± S.D. Vehicle 0.74 ± 0.22 IgG-SMSC 1.03** ± 0.32 CD140b-ab-SMSC 0.93 ± 0.21 ** P < 0.01 vs Vehicle group

[0147] <Reference Example 1> Confirmation of TSP2 secretion from rat synovium-derived stem cells and inhibition treatment thereof The frozen stock of the rat synovium-derived stem cells prepared in Example 1 was thawed and cultured at a CO2 concentration of 5% and 37°C using the culture medium used in Example 1. On day 4 of culture, Silencer Select Pre-designed siRNA (Thermo Fisher Scientific, Cat. No. 4390816, ID s146414) (hereinafter, Tsp2 siRNA) for inhibiting gene expression of TSP2 was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific, Cat. No. 31985070), and added to the cells during culture (final siRNA concentration: 20 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermo Fisher Scientific, Cat. No. 4390844) (hereinafter, NC siRNA), which does not inhibit gene expression of any genes including TSP2, was used to treat the cells in the same manner. Three days after the Tsp2 siRNA or NC siRNA treatment, the culture medium in the flask was discarded, the cells were washed twice with PBS, TrypLE Express was added, the cells were allowed to stand in an incubator at 37°C for 5 minutes, and the cells were recovered as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation, the cells were replaced with CP-1 High Grade, and a frozen stock of the rat synovium-derived mesenchymal stem cells treated with Tsp2 siRNA or NC siRNA was prepared by a freezing treatment.

[0148] The frozen stock of the rat synovium-derived mesenchymal stem cells treated with Tsp2 siRNA or NC siRNA was thawed and cultured in the culture medium. A portion of the culture supernatant was collected on days 1, 3, and 7 from the start of culture. During this period, the culture medium was not exchanged. For evaluation, the collected culture supernatant was diluted 300-fold with PBS, and the TSP2 concentration was measured using a rat Thbs2 ELISA Kit (Cloud-Clone Corp., Cat. No. SED822RA). The concentration obtained after measurement was multiplied by 300 and converted to the calculated original concentration. The results are shown in Table 16.

[0149] [Table 16] Treatment Sampling day TSP2 conc. (ng / mL) Average ± S.D. SMSC (no treated) 1d 1358 ± 183 3d 6102 ± 901 7d 7203 ± 806 NC-SMSC 1d 1848 ± 1427 3d 5817 ± 921 7d 9432 ± 806 TSP2kd-SMSC 1d 133 ± 63 3d 436 ± 58 7d 1630 ± 296 GM 1d 0.13 ± 0.16 3d 0.32 ± 0.13 7d 0.02 ± 0.03 Lower limit of quantification: 0.78 ng / mL

[0150] In the untreated synovial stem cells (indicated as SMSC (no treated) in the table), TSP2 secretion in the culture supernatant was observed at 1,358, 6,102, and 7,203 ng / mL on Day 1, Day 3, and Day 7, respectively. In the rat synovium-derived mesenchymal cells treated with NC siRNA, which was a control treatment (indicated as NC-SMSC in the table), TSP2 secretion in the culture supernatant was observed at 1,848, 5,817, and 9,432 ng / mL on Day 1, Day 3, and Day 7, respectively. On the other hand, in the rat synovium-derived mesenchymal cells treated with Tsp2 siRNA (indicated as TSP2kd-SMSC in the table), TSP2 secretion was 133 and 436 ng / mL on Day 1 and Day 3, respectively, which was one-tenth or less of that in NC-SMSC, but on Day 7, TSP2 secretion was detected at 1,630 ng / mL, which was at the same level as that on Day 1 in other treatments. TSP2 in the culture medium (indicated as GM in the table) was not detected. From these results, it was found that the rat synovium-derived mesenchymal cells secrete TSP2, and rat synovium-derived mesenchymal cells in which TSP2 secretion was inhibited for at least 3 days by Tsp2 siRNA treatment were prepared.

[0151] <Reference Example 2> Meniscus regeneration effect of rat synovium-derived stem cells in which TSP2 secretion is inhibited Rat synovium-derived stem cells in which TSP2 secretion was inhibited were prepared as described in Reference Example 1. The preparation of the meniscal injury model for evaluating the meniscus regeneration effect was carried out in the same manner as in Example 4. The treated animals were divided into three groups, and 1 x 107 TSP2kd-SMSC cells, 1 x 107 NC-SMSC cells, or only the cryopreservation medium CP1 (Vehicle) was administered into the joint capsule of each group.

[0152] Three weeks after the treatment, the animals were exsanguinated by cutting the lower aorta under isoflurane anesthesia and euthanized. Thereafter, the meniscus was exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the medial menisci excised from both knee joints are shown in FIG. 11. The regenerated portion was identified based on differences in color tone and shape from the normal meniscus and surrounded by a broken line. In the NC-SMSC group, which was a positive control, the meniscus regenerated portion appeared larger than that in the Vehicle group, which was a negative control, but no clear difference from the TSP2kd-SMSC group was confirmed.

[0153] In order to quantitatively evaluate the macroscopic findings of the meniscus regenerated portion in FIG. 11, the area was calculated as described in Example 4 and evaluated by statistical processing. Regarding the area of the meniscus regenerated portion shown in Table 17, although the area of 0.83 mm2 in the TSP2kd-SMSC group differed from the area of 1.06 mm2 in NC-SMSC, the difference was not statistically significant. In addition, although the TSP2kd-SMSC group differed from the Vehicle group having an area of 0.65 mm2, the difference was not statistically significant. From these results, it is considered that the TSP2 molecule in synovial stem cells is not an important molecule that contributes to meniscus regeneration.

[0154] [Table 17] Group Meniscus regenerative area (mm2) Average ± S.D. Vehicle (CP1) 0.65 ± 0.44 NC-SMSC 1.06* ± 0.51 TSP2kd-SMSC 0.83 ± 0.34 *P < 0.05 vs Vehicle

Claims

1. Synovium-derived mesenchymal stem cells that are positive for any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15.

2. The synovium-derived mesenchymal stem cells according to claim 1,wherein the synovium-derived mesenchymal stem cells are positive for all of the hepatocyte growth factor, the transforming growth factor beta-induced protein, and the tumor necrosis factor superfamily 15.

3. The synovium-derived mesenchymal stem cells according to claim 1, wherein the synovium-derived mesenchymal stem cells are positive for integrin 01.

4. A cell population comprising: the synovium-derived mesenchymal stem cells according to claim 1,wherein a percentage of cells positive for platelet-derived growth factor receptor 0 is less than 50%.

5. The synovium-derived mesenchymal stem cells according to claim 1,wherein the synovium-derived mesenchymal stem cells are obtained by a method including a step A of treating synovial tissue with an enzyme, a step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture obtained after washing the mixture after the enzyme treatment, and a step C of cryopreserving the synovium-derived mesenchymal stem cells after the culturing.

6. The synovium-derived mesenchymal stem cells according to claim 5,wherein the step A of treating the synovial tissue with an enzyme includes a step of treating the synovial tissue using a solution containing the enzyme and autologous serum or non-autologous serum.

7. A therapeutic agent for arthropathy, comprising:the synovium-derived mesenchymal stem cells or the cell population according to anyone of claims 1 to 6.

8. A method for producing the synovium-derived mesenchymal stem cells according to claim 1, the method comprising:a step A of treating synovial tissue with an enzyme;a step B of culturing, in a culture medium, synovium-derived mesenchymal stem cells contained in a mixture obtained after washing the mixture after the enzyme treatment; anda step C of cryopreserving the synovium-derived mesenchymal stem cells after the culturing.

9. The method according to claim 8,wherein the step A of treating the synovial tissue with an enzyme includes a step of treating the synovial tissue using a solution containing the enzyme and autologous serum or non-autologous serum.

10. The method according to claim 8 or 9,wherein the step B includes a step performed using a culture medium containing an ascorbic acid derivative.

11. The method according to claim 8 or 9,wherein, in the step C, a cell concentration at the time of performing a freezing treatment on cells is 1*106 cells / mL or more.

12. The method according to claim 8 or 9,wherein, in the step C, after a freezing treatment is performed on cells, a period during which the cells are stored in a frozen state is 2 days or more.

13. The method according to claim 8 or 9,wherein the step C includes a step of freezing the cells at -70°C to -90°C and then freezing the cells at -140°C to -160°C.

14. A method for selecting synovium-derived mesenchymal stem cells to be used as a therapeutic agent for arthropathy, the method comprising:measuring expression of any one or more of hepatocyte growth factor, transforming growth factor beta-induced protein, or tumor necrosis factor superfamily 15, and selecting positive cells.

Images