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

Synovial membrane-derived mesenchymal stem cells, their production method, and their use

[0001] The present invention relates to synovial membrane-derived mesenchymal stem cells that contain molecules essential for joint treatment. The present invention further relates to a therapeutic agent for arthropathy containing the synovial membrane-derived mesenchymal stem cells, a method for producing the synovial membrane-derived mesenchymal stem cells, and a method for selecting synovial membrane-derived mesenchymal stem cells for use as a therapeutic agent for arthropathy.

[0002] In recent years, advances in regenerative medicine and cell therapy technologies have led to the active development of various cell therapies and research cell products using autologous, allogeneic, and xenogeneic cells. Among these, mesenchymal stem cells (MSCs) are expected to be a useful cell source for cell therapy. Mesenchymal stem cells can be collected from various body tissues, and it has been reported that they can be isolated from bone marrow, adipose tissue, muscle tissue, synovial tissue, periosteal tissue, and other tissues. In particular, synovial membrane-derived mesenchymal stem cells have been reported to have higher proliferation and chondrogenic potential than mesenchymal stem cells derived from various mesenchymal tissues, such as bone marrow. Furthermore, Patent Documents 1 to 3 disclose methods for treating articular cartilage damage and meniscus damage using synovial membrane-derived mesenchymal stem cells. Patent Document 4 describes a therapeutic agent for arthropathy containing synovial membrane-derived mesenchymal stem cells carrying molecules essential for joint treatment, and a method for producing the same. More specifically, Patent Document 4 describes a method in which synovial tissue is treated with an enzyme, washed, and cultured on a substrate, and the resulting cells are selected based on the expression of integrin β1 or platelet-derived growth factor receptor β to ensure quality. Patent Document 5 describes the expression of hepatocyte growth factor (HGF) in adipose tissue-derived multi-lineage progenitor cells treated with IL-1β. Non-Patent Document 1 describes the expression of genes present in chromatin 7, including HGF, in synovial stem cells.

[0003] Stem Cells Translational Medicine, Volume 10, Issue 11, November 2021, Pages 1530-1543

[0004] Japanese Patent No. 5928961 Japanese Patent No. 5656183 Japanese Patent No. 6864302 International Publication WO23 / 032945 Japanese Patent Application Laid-Open No. 2019-218271

[0005] In the quality control of cell products, ensuring the equivalence and identity of each lot is a challenge. However, since the cells that make up the product are not completely uniform and their characteristics are difficult to identify, ensuring the equivalence and identity of each lot is generally difficult. Therefore, in order to control product quality, not only have the quality tests of the final product been carried out, but also the QMS (Quality Management System) concept that has been applied to medical devices has been adopted, and management of the entire process has been carried out by recording and controlling manufacturing raw materials, material management, manufacturing process management, and process control tests. However, with the advancement of science and technology, the importance of identifying the characteristics of the cells themselves, which are the final product, has increased.

[0006] As a method for controlling the quality of cells, for example, cell type-specific surface markers are used as indicators to identify the cell type of the desired final product (e.g., to identify that it is a mesenchymal stem cell), but it is desirable to provide cell products with more stable therapeutic effects.

[0007] An object of the present invention is to provide synovium-derived mesenchymal stem cells that exhibit stable and excellent therapeutic effects against joint diseases. A further object of the present invention is to provide a therapeutic agent for arthropathy containing 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 a therapeutic agent for arthropathy.

[0008] As a result of extensive research to solve the above problems, the present inventors have found that one or more of hepatocyte growth factor, transforming growth factor beta induced protein (TGFBI; also known as BIGH3), and tumor necrosis factor superfamily 15 are quality control markers essential for the effectiveness of synovial stem cell treatment of joint diseases. The present invention was completed based on the above findings.

[0009] That is, the present invention provides the following inventions: <1> Synovium-derived mesenchymal stem cells positive for one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15. <2> Synovium-derived mesenchymal stem cells according to <1>, which are positive for all of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15. <3> Synovium-derived mesenchymal stem cells according to <1> or <2>, which are positive for integrin β1. <4> A cell population comprising the synovium-derived mesenchymal stem cells according to any one of <1> to <3>, wherein the positive rate for platelet-derived growth factor receptor β is less than 50%. <5> The synovium-derived mesenchymal stem cells according to any one of <1> to <4>, obtained by a method comprising the steps of: Step A of treating synovial tissue with an enzyme; Step B of culturing in a culture medium the synovium-derived mesenchymal stem cells contained in the mixture obtained after washing the mixture after the enzyme treatment; and Step C of cryopreserving the cultured synovium-derived mesenchymal stem cells. <6> The synovium-derived mesenchymal stem cells according to <5>, wherein Step A of treating the synovial tissue with an enzyme comprises treating with 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 cell population according to any one of <1> to <6>. <8> A method for producing synovium-derived mesenchymal stem cells according to any one of <1> to <6>, comprising the steps of: Step A of treating synovial tissue with an enzyme; Step B of culturing in a culture medium the synovium-derived mesenchymal stem cells contained in the mixture obtained after washing the mixture after the enzyme treatment; and Step C of cryopreserving the cultured synovium-derived mesenchymal stem cells. <9> The method according to <8>, wherein step A of treating the synovial tissue with an enzyme comprises treating the synovial tissue with a solution containing the enzyme and autologous serum or non-autologous serum. <10> The method according to <8> or <9>, wherein step B comprises performing the treatment using a medium containing an ascorbic acid derivative. <11> The method according to <8> or <9>, wherein in step C, the cell concentration during the cell freezing treatment is 1 x 10 ６<12> The method according to any one of <8> to <11>, wherein in step C, after the cells have been subjected to a freezing treatment, the cells are stored in a frozen state for 2 days or more. <13> The method according to any one of <8> to <12>, wherein step C comprises 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 the expression of one or more of hepatocyte growth factor, transforming growth factor-β-induced protein, or tumor necrosis factor superfamily 15, and selecting positive cells.

[0010] The synovium-derived mesenchymal stem cells and arthropathy therapeutic agent of the present invention can exhibit stable and excellent therapeutic effects against joint diseases by being positive for one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15. The method for producing synovium-derived mesenchymal stem cells and the method for selecting synovium-derived mesenchymal stem cells of the present invention can suppress fluctuations in the therapeutic effects of the obtained synovium-derived mesenchymal stem cells, enabling quality control of the therapeutic effects of synovium-derived mesenchymal stem cells.

[0011] FIG. 1 shows the results of examining the chondrogenic differentiation ability of rat synovial membrane-derived stem cells in which HGF secretion was inhibited. FIG. 2 shows the results of examining the meniscus regeneration effect of rat synovial membrane-derived stem cells in which HGF secretion was inhibited. FIG. 3 shows the results of examining the knee cartilage repair effect of rat synovial membrane-derived stem cells in which HGF secretion was inhibited in a rat knee OA model. FIG. 4 shows the results of examining the knee cartilage repair effect of rat synovial membrane-derived stem cells in which HGF secretion was inhibited in a rat knee OA model. FIG. 5 shows the results of examining the meniscus regeneration effect of rat synovial membrane-derived stem cells in which TGFBI secretion was inhibited. FIG. 6 shows the results of examining the meniscus regeneration effect of rat synovial membrane-derived stem cells in which TNFSF15 gene expression was inhibited. FIG. 7 shows the results of analyzing the CD29 (Integrin β1) and CD140b (PDGFRβ) positivity rates of rat synovial membrane-derived stem cells. FIG. 8 shows the results of examining the suppression of extracellular matrix adhesion ability of rat synovial membrane-derived stem cells by CD29 inhibition. Figure 9 shows the results of examining the meniscus regeneration effect of rat synovium-derived stem cells in which CD29 was inhibited. Figure 10 shows the results of examining the meniscus regeneration effect of rat synovium-derived stem cells in which CD140b was inhibited. Figure 11 shows the results of examining the meniscus regeneration effect of rat synovium-derived stem cells in which TSP2 secretion was inhibited.

[0012] The present invention will be described in detail below. In this specification, the word "to" is used to mean that the numerical values ​​before and after it are included as the lower limit and upper limit.

[0013] The synovium-derived mesenchymal stem cells of the present invention are positive for one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and 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 β-induced protein, and tumor necrosis factor superfamily 15, or may be positive for two of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15, or may be positive for all of hepatocyte growth factor, transforming growth factor β-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 β-induced protein, and tumor necrosis factor superfamily 15.

[0015] The synovium-derived mesenchymal stem cells are preferably positive for integrin β1. In a cell population containing synovium-derived mesenchymal stem cells, the positive rate for platelet-derived growth factor receptor β is preferably less than 50%, more preferably 47% or less, and even more preferably 45% or less. The positive rate for platelet-derived growth factor receptor β (PDGFRβ) can be measured using a flow cytometer with an anti-PDGFRβ antibody.

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

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

[0018] <Step A: Treating synovial tissue with enzymes> Synovial tissue can be collected from the non-weight-bearing part of a joint under anesthesia. The origin of the synovial tissue is not particularly limited, and synovial tissue from any organism, preferably from a mammal, can be used. For example, synovial tissue from primates (e.g., chimpanzees, Japanese monkeys, and humans) can be used, and particularly preferably, synovial tissue from humans can be used.

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

[0020] When synovial membrane-derived mesenchymal stem cells are produced for administration to humans, synovial tissue collected from a donor whose histocompatibility antigen type is matched or similar to that of the recipient may be used. The subject from which the synovial membrane is collected and the subject into which the synovial membrane-derived mesenchymal stem cells are transplanted may be the same subject, i.e., synovial tissue collected from the recipient himself / herself may be used (autologous transplant). Synovial tissue collected from a subject different from the recipient may be used (allogeneic transplant). More preferably, the subject from which the synovial membrane is collected and the subject into which the synovial membrane-derived mesenchymal stem cells are transplanted are different.

[0021] The amount of synovial tissue to be collected can be determined taking into consideration the type of donor or the amount of synovial-derived mesenchymal stem cells required. For example, synovial-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 even more preferably 0.1 g to 1.0 g of synovial tissue. The collected synovial tissue can be shredded with scissors or the like as needed, and then subjected to the enzymatic treatment described below.

[0022] The synovial tissue is treated with an enzyme. The enzyme is not particularly limited as long as it contains a protease, but a mixed enzyme containing one or more types of collagenase and one or more types of neutral protease is preferred. 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 (thermocillin).

[0023] The enzymatic reaction can be carried out in an aqueous solution containing an enzyme. An aqueous solution containing serum (e.g., human serum, fetal bovine serum, etc.) or a serum substitute (e.g., human platelet lysate) can be used. When serum is used, it may be autologous or non-autologous serum. Non-autologous serum includes allogeneic serum and xenogeneic serum. When human serum is used, it may be autologous or allogeneic serum. Preferably, step A of treating synovial tissue with an enzyme includes a step of treating the synovial tissue with an enzyme and a solution containing non-autologous serum or a serum substitute. Furthermore, for ease of treatment, step A of treating synovial tissue with an enzyme preferably uses a solution containing an enzyme and a serum substitute. When an aqueous solution containing serum is used, the serum concentration is not particularly limited, but is generally 10% to 40% by volume, preferably 15% to 35% by volume, and more preferably 20% to 30% by volume. When a serum substitute is used, the concentration of the serum substitute is not particularly limited, but is 10% to 40% by volume, preferably 15% to 35% by volume, and more preferably 20% to 30% by volume. Examples of solvents for the aqueous solution containing the enzyme include water for injection, physiological saline, and phosphate buffer solution (PBS).

[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, even more preferably 0.5 mg / ml to 10 mg / ml, still 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 enzyme is preferably 1000:1 to 10:1, more preferably 500:1 to 20:1, even more preferably 100:1 to 20:1, and even more preferably 50:1 to 20:1.

[0026] The enzyme reaction can be carried out at a temperature of preferably 15°C to 40°C, more preferably 25°C to 40°C, and even more preferably 35°C to 40°C. The reaction time is sufficient as long as it is 10 minutes or longer, preferably 30 minutes or longer, more preferably 1 hour or longer, even 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 within 10 hours, 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 enzyme-treated mixture contains synovium-derived mesenchymal stem cells. The enzyme-treated mixture can be transferred to a centrifuge tube through a cell strainer and centrifuged to recover synovium-derived mesenchymal stem cells.

[0027] <Step B: Culturing the synovium-derived mesenchymal stem cells contained in the mixture after washing the mixture after enzyme treatment in a culture medium> The mixture after enzyme treatment as described above is washed. In washing, the mixture can be washed preferably 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, even more preferably 0.2 ng / mL or less, and particularly preferably 0.1 ng / mL or less.

[0028] Washing can be performed by resuspending the synovium-derived mesenchymal stem cells recovered by the above-mentioned centrifugation in a medium and centrifuging them again (e.g., at 400 g for 5 minutes). The medium that can be used is, but is not limited to, α-modified Eagle's minimum essential medium (α-MEM). Washing can be performed multiple times (two or more times) using the medium described above.

[0029] The synovium-derived mesenchymal stem cells contained in the mixture after washing the mixture after the enzyme treatment are cultured in a culture medium. The substrate can be, but is not limited to, a flat plastic substrate such as a culture plate, or a three-dimensional substrate such as a culture bag, microcarrier, or gel.

[0030] The medium used for culturing can be prepared using a medium used for culturing ordinary animal cells as a basal medium. Examples of media used for culturing ordinary animal cells include αMEM, DMEM (Dulbecco Modified Eagle Medium), a mixed medium of DMEM and F12 (DMEM:F12=1:1), RPMI medium (GIBCO (registered trademark) RPMI1640 medium, etc.), a mixed medium of DMEM / F12 and RPMI (DMEM / F12:RPMI=1:1), and ROOSTERNOURISH. TM -MSC-XF (ROOSTERBIO), PRCROOSTERNOURISH TM Examples of suitable medium include, but are not limited to, MSC-CC (ROOSTERBIO), PRIME-XV MSC EXPANSION XSFM (FUJIFILM IRVINE SCIENTIFIC, INC.), and MEM A (FUJIFILM Wako Pure Chemical Industries, Ltd.). The medium may also contain an antibiotic or antimycotic agent (e.g., penicillin, streptomycin, amphotericin B, etc.).

[0031] The medium may be supplemented with HGF, IGF-II, or SCF, or a combination thereof. The concentrations of HGF, IGF-II, and SCF in the medium are not particularly limited, but are preferably 0.01 to 10,000 ng / mL, more preferably 0.01 to 1,000 ng / mL, even more preferably 0.1 to 1,000 ng / mL, and even more preferably 1 to 100 ng / mL.

[0032] The medium may be a medium containing serum, a medium containing a serum substitute, or a medium without serum. When synovium-derived mesenchymal stem cells are produced from autologous tissue for administration to the body, the medium may contain allogeneic serum. That is, when synovium-derived mesenchymal stem cells are produced from human tissue for administration to humans, a medium containing human serum may be used. When serum is used, it may be autologous serum or allogeneic serum, but allogeneic serum is preferred. When serum is used, the amount of serum added to the medium is, for example, 20% by volume or less, 10% by volume or less, or 5% by volume or less. For ease of operation, it is preferable to use a serum substitute. When a serum substitute is used, 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, even more preferably 1% by volume to 15% by volume, and even more preferably 5% by volume to 15% by volume.

[0033] Preferably, step B includes a step carried out using a medium containing an ascorbic acid derivative. Examples of ascorbic derivatives include, but are not limited to, ascorbic acid 2-phosphate, trisodium ascorbic acid 2-phosphate, magnesium ascorbic acid 2-phosphate, and ascorbic acid 2-glycoside. When a medium containing an ascorbic acid derivative is used, the concentration of the ascorbic acid derivative in the medium is not particularly limited, but is preferably 0.03 mmol / L or more, more preferably 0.1 mmol / L or more, and even more preferably 0.14 mmol / L or more. The upper limit is preferably 5.0 mmol / L or less, more preferably 1.0 mmol / L or less, and even more preferably 0.57 mmol / L or less.

[0034] The cell culture conditions are not particularly limited, and ordinary cell culture conditions can be used, for example, at a temperature of 30 to 40°C and 3 to 7% CO ２ The incubation temperature can be, but is not limited to, 37°C, 5% CO. ２ Examples of such methods include culturing in a medium containing lactic acid bacteria.

[0035] In the present invention, the culture may be performed without or with medium exchange, but medium exchange is preferred. When medium exchange is performed, the first half of step B may be cultured in a medium containing an ascorbic acid derivative, and the second half of step B may be cultured in a medium not containing an ascorbic acid derivative. Furthermore, in the above-mentioned culture, it is preferred that the synovium-derived mesenchymal stem cells are produced without being co-cultured with cells other than the synovium-derived mesenchymal stem cells.

[0036] It is known that the differentiation of synovium-derived mesenchymal stem cells into chondrocytes progresses more as the culture period is longer, and therefore, the in situ chondrogenic ability of synovium-derived mesenchymal stem cells decreases if the culture period exceeds a certain 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 in which they have good in situ chondrogenic ability. In step B, it is preferable that the period for culturing synovium-derived mesenchymal stem cells is 28 days or less.

[0037] Furthermore, in the present invention, it is necessary to consider the need to prepare a sufficient number of undifferentiated synovial stem cells for treatment, or to prepare a sufficient number of undifferentiated synovial stem cells for creating 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 cartilage tissue can be produced in vitro by culturing them in a chondrogenic medium supplemented with transforming growth factor β3 (TGF-β3), dexamethasone, and bone morphogenetic protein 2 (BMP-2). Therefore, in the present invention, to prevent synovium-derived mesenchymal stem cells from differentiating into chondrocytes, it is preferable to culture isolated synovium-derived mesenchymal stem cells in the absence of TGF-β3, dexamethasone, or BMP-2.

[0039] In the present invention, a certain cell density or more is required from the viewpoint of proliferation of synovial membrane-derived mesenchymal stem cells, so the synovial membrane-derived mesenchymal stem cells after enzyme treatment are cultured at a density of 100 cells / cm. ２ More than 5000 cells / cm ２ Below, 200 cells / cm ２ More than 5000 cells / cm ２ Below, 500 cells / cm ２ More than 5000 cells / cm ２ Below, 500 cells / cm ２ More than 2500 cells / cm ２ or less than 500 cells / cm ２ More than 2000 cells / cm ２ It is preferable to seed and culture at the following cell densities: Furthermore, in order to proliferate the synovium-derived mesenchymal stem cells after the enzyme treatment, it is more preferable to culture for 5 days or more.

[0040] The number of cells obtained at the end of the culture was 1.0 x 10 ７ More than cells, 2.0 x 10 ７ More than cells, 2.5 x 10 ７ cells or more, or 3.0 x 10７ Preferably, the number of cells is 4.0 x 10 or more. ７ More preferably, it is 5.0 x 10 cells or more. ７ More preferably, the number of cells is 6.0 x 10 or more. ７ Cells or larger are particularly preferred.

[0041] <Separation of synovium-derived mesenchymal stem cells from the substrate> After the culture in step B, the synovium-derived mesenchymal stem cells are preferably separated from the substrate prior to step C. Preferably, separation from the substrate can be achieved by allowing a cell detachment solution to act on the mesenchymal stem cells for up to 120 minutes. The cell detachment solution is a solution containing a trypsin-like enzyme and EDTA. A particularly preferred enzyme is TrypLE. Examples of TrypLE that can be used include TrypL Express (Gibco) and TrypLE Select (Gibco).

[0042] The time for which the cell detachment solution is allowed to act on the mesenchymal stem cells is preferably 5 minutes or longer in order to sufficiently detach the cells. The time for which the cell detachment solution is allowed to act on the mesenchymal stem cells is preferably 5 to 120 minutes, and more preferably 5 to 60 minutes. It may be 5 to 50 minutes, 5 to 40 minutes, 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, or 10 to 40 minutes.

[0043] <Step C of cryopreserving cultured synovium-derived mesenchymal stem cells> The cultured synovium-derived mesenchymal stem cells can be cryopreserved. In step C, the cell concentration during the cell freezing treatment is 1 x 10 ６ cells / mL or more, preferably 2 x 10 ７ cells / mL or more, more preferably 1 x 10 ８ cells / mL, more preferably 1.1 x 10 ８ cells / mL or more, more preferably 1.2 x 10 ８ cells / mL or more, 1.5 x 10 ８ cells / mL or more, or 2.0 x 10 ８ It may be more than 100 cells / mL.

[0044] In step C, the cryopreservation solution used when freezing the cells is preferably a cryopreservation solution containing DMSO. Specific examples include, but are not limited to, CTS Synth-a-Freeze Medium (Thermo Fisher Scientific), STEM-CELLBANKER GMP grade (Xenogen Pharma), and CP-1 High Grade (Kyokuto Pharmaceutical Industries Co., Ltd.). When using CP-1 High Grade, albumin may be added. Albumin derived from humans, monkeys, cows, pigs, rabbits, mice, or rats can be used, but it is preferable to add human serum albumin (HSA). The HSA to be added may be prepared from human plasma, or recombinant HSA expressed in animals or plants. The final concentration of HSA to be added can be adjusted appropriately, but may be 1 to 5%, preferably 3 to 5%.

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

[0046] Step C preferably comprises a step of freezing the cells at −70° C. to −90° C., followed by freezing at −140° C. to −160° C. Step C more preferably comprises a step of freezing the cells at −70° C. to −90° C. for one day or more, followed by freezing at −140° C. to −160° C. for one day or more.

[0047] In step C, the cells are subjected to a freezing treatment and then stored in a frozen state as a master cell bank, and then a portion of the master cell bank may be frozen, thawed, and expanded to produce synovial mesenchymal stem cells as a therapeutic agent for arthropathy.

[0048] Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyme). Mesenchymal stem cells are known to exist in bone marrow, synovium, periosteum, adipose tissue, and muscle tissue, and are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle cells. In relation to the differentiation of mesenchymal stem cells into chondrocytes, it is known that the addition of BMP or TGF-β to the culture medium promotes the differentiation of undifferentiated mesenchymal stem cells into chondrocytes, and cartilage tissue can be regenerated under in vitro conditions.

[0049] Mesenchymal stem cells can be identified by detecting molecules characteristic of mesenchymal stem cells, such as enzymes, receptors, and low-molecular-weight compounds. Molecules characteristic of mesenchymal stem cells include, but are not limited to, cell surface markers (positive markers), such as CD73, CD90, CD105, and CD166. Negative markers not expressed in mesenchymal stem cells include, but are not limited to, CD19, CD34, CD45, HLA-DR, CD11b, and CD14. CD stands for Clusters of Differentiation, and HLA-DR stands for Human Leukocyte Antigen-D-Related. These positive and negative markers can be used to identify mesenchymal stem cells. These markers can be detected using immunological methods, but detection can also be performed by quantifying the amount of mRNA for each molecule.

[0050] As used herein, synovial membrane-derived mesenchymal stem cells are stem cells contained in the synovial membrane. Synovial membrane-derived mesenchymal stem cells are a type of mesenchymal stem cell. Synovial membrane-derived mesenchymal stem cells can be detected, for example, by detecting CD90 positivity, CD45 negativity, and chondrogenic differentiation ability, but the detection method is not particularly limited.

[0051] As used herein, "positive" refers to the expression of the gene, the expression of the protein, or both. Protein expression includes expression on the cell surface, secretion from the cell, or both.

[0052] As used herein, the positive rate refers to the ratio of positive cells in a cell population containing synovium-derived mesenchymal stem cells.

[0053] The present invention provides a therapeutic agent for arthropathy, comprising the synovium-derived mesenchymal stem cells of the present invention. When using synovium-derived mesenchymal stem cells as a therapeutic agent for arthropathy, the cells can be prepared into a formulation suitable for administration to an individual by, for example, mixing the cells with a pharmaceutically acceptable carrier in a conventional manner. Examples of the carrier include saline, distilled water for injection made isotonic by adding glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Additionally, the agent may contain buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0054] The method for producing the arthropathy therapeutic agent of the present invention may preferably further comprise a step of selecting synovium-derived mesenchymal stem cells that are positive for one or more of hepatocyte growth factor, transforming growth factor β-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 to be used as a therapeutic agent for arthropathy, the method comprising measuring the expression of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15, and selecting positive cells.

[0056] A process for selecting synovium-derived mesenchymal stem cells that are positive for one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15 includes controlling the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15.

[0057] The expression level of any one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 refers to the expression level of any one gene or protein of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15. The expression level of any one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 can be calculated as an absolute value or a relative value (e.g., a ratio or difference from a comparative control or standard expression level).

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

[0059] The primers and probes used in the above measurement methods can be labeled, and the amount of mRNA can be measured by examining the signal intensity of the label. Real-time RT-PCR is preferable because it allows RNA to be used directly as a sample and enables gene quantification based on the number of temperature cycles required for amplification by optically measuring the gene amplification process. Furthermore, the expression levels of mRNAs of housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin can be used as controls to standardize the expression levels of genes encoding one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15. The primers and probes used in the above measurement methods can be appropriately designed and prepared by those skilled in the art based on information on the nucleotide sequences of genes encoding one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15.

[0060] The expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15 can be measured, for example, by immunological measurement using an antibody or antibody fragment against one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15. Specific examples include flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent antibody assay, cell array assay, etc. These measurement methods can also be performed using standard protocols or protocols that are appropriately modified or altered from standard protocols.

[0061] For example, when the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 in cells is measured by flow cytometry, if the positive rate of one or more of hepatocyte growth factor, transforming growth factor β-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 positive for one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15.

[0062] Selection of synovium-derived mesenchymal stem cells positive for one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15 can be carried out, for example, by comparing the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15 in cells measured by the above-mentioned method with a predetermined reference expression level. The reference expression level may be, for example, the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15 in cells already confirmed to have a certain quality (positive control), or the expression level in cells already confirmed to not have a certain quality (negative control).

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

[0064] Alternatively, a cutoff value for the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 may be preset, and the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 measured in cells may be compared with the cutoff value. The cutoff value may be, for example, based on a regression line showing the correlation between the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 and the therapeutic effect, and may be set to the expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 that provides the desired therapeutic effect. For example, cells whose expression level of one or more of hepatocyte growth factor, transforming growth factor β-induced protein, or tumor necrosis factor superfamily 15 in the cells is equal to or greater than the cutoff value can be selected and used as a therapeutic agent for arthropathy.

[0065] The positive rate of platelet-derived growth factor receptor β (PDGFRβ) is preferably less than 50% of the cell population containing synovium-derived stem cells, and may be 10% or more but less than 50%, 20% or more but less than 50%, 30% or more but less than 50%, 35% or more but less than 50%, or 40% or more but less than 50%.

[0066] The therapeutic agent for arthropathy of the present invention can be used for joint treatment. Examples of joint treatment include treatment of diseases accompanied by joint injury, damage, or inflammation, including joint diseases caused by degeneration and / or inflammation of connective tissues such as cartilage, and non-inflammatory joint diseases. Examples of joint treatment include, but are not limited to, treatment of diseases selected from the group consisting of meniscus injury, traumatic cartilage injury, osteochondritis dissecans, avascular necrosis, osteoarthritis (e.g., knee osteoarthritis), rheumatoid arthritis (e.g., rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defects. Target sites for joint treatment include, but are not limited to, the knee joint, shoulder joint, hand joint, hip joint, elbow joint, finger joint, spine, intervertebral disc, foot joint, toe joint, ankle joint, and temporomandibular joint.

[0067] A method for treating a joint using the arthropathy therapeutic agent of the present invention comprises the steps of: transplanting the arthropathy therapeutic agent of the present invention so that the cartilage damaged area or meniscus damaged area is covered with synovium-derived mesenchymal stem cells; and regenerating cartilage tissue in situ at the cartilage damaged area or meniscus damaged area by differentiating the synovium-derived mesenchymal stem cells contained in the arthropathy therapeutic agent into chondrocytes.

[0068] When the therapeutic agent for arthropathy of the present invention is transplanted into a patient, in order to efficiently treat the cartilage damaged area or meniscus damaged area, 2.0 × 10 ７ ~1.0 x 10 １１ pieces, or 2.5 x 10 ７ ~1.0 x 10 １１ pieces, or 3.0 x 10 ７ ~1.0 x 10 １１ pieces, 4.0×10 ７ ~1.0 x 10 １１ pieces, or 2.5 x 10 ７ ~1.0 x 10 １０ pieces, or 2.5 x 10 ７ ~1.0 x 10 ９ pieces, or 2.5 x 10 ７ ~1.0 x 10 ８synovial membrane-derived mesenchymal stem cells or 2.0 x 10 ７ ~1.0 x 10 ８ It is preferable to apply synovium-derived mesenchymal stem cells.

[0069] By transplanting synovium-derived mesenchymal stem cells into a cartilage injury site or a meniscus injury site, the cartilage injury site or the meniscus injury site is covered with synovium-derived mesenchymal stem cells. Transplantation of synovium-derived mesenchymal stem cells can be performed by open surgery or arthroscopic surgery. To minimize invasion as much as possible, it is preferable to transplant synovium-derived mesenchymal stem cells under arthroscopic surgery.

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

[0071] In the case of treating cartilage damage, the minimally invasive procedure of the present invention is characterized by covering the cartilage damage with synovium-derived mesenchymal stem cells, and includes the following steps: maintaining the body position so that the cartilage damage 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 the surface of the cartilage damage; and maintaining the body position for a specific period of time, thereby allowing the synovium-derived mesenchymal stem cells to adhere to the surface of the cartilage damage.

[0072] In the case of treating meniscus injuries, the minimally invasive procedure of the present invention is characterized by covering the damaged meniscus with synovium-derived mesenchymal stem cells, and includes the following steps: maintaining the body position so that the damaged meniscus faces downward; injecting a suspension of synovium-derived mesenchymal stem cells into the knee joint; and maintaining the body position for a specific period of time to allow the synovium-derived mesenchymal stem cells to adhere to the damaged meniscus.

[0073] In order to ensure that synovium-derived MSCs adhere to the surface of the cartilage injury or meniscus injury, it is preferable to maintain the transplanted synovium-derived MSCs on the surface of the cartilage injury or meniscus injury for at least 10 minutes, preferably 15 minutes. To achieve this, the cartilage injury or meniscus injury is oriented upward, and the body position is maintained for at least 10 minutes, preferably 15 minutes, with the aim of maintaining the synovium-derived MSCs on the upward-oriented cartilage injury or meniscus injury.

[0074] The cartilage injury or meniscus injury accompanied by synovium-derived mesenchymal stem cells can be further covered with periosteum to further strengthen the adhesion of the synovium-derived mesenchymal stem cells to the cartilage injury or meniscus injury. The synovium-derived mesenchymal stem cells are left on the surface of the cartilage injury or meniscus injury for at least 10 minutes before the surgery is completed.

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

[0076] During the in situ chondrogenesis process of synovium-derived mesenchymal stem cells, cartilage tissue is regenerated according to the local microenvironment (nutrient supply, cytokine environment, etc.), and no external manipulation is required. As a result of in situ chondrogenesis of synovium-derived mesenchymal stem cells, cartilage tissue is regenerated at the cartilage injury site or meniscus injury site to repair the injury, and in the case of cartilage injury, the bone region, the boundary between cartilage and bone, the cartilage center, the surface region, and the region adjacent to the original cartilage are formed as original cartilage tissue, or in the case of meniscus injury, meniscus cartilage is formed.

[0077] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0078] Example 1: Preparation of rat synovium-derived mesenchymal stem cells Ten ACI / NSlc rats (male, 3 weeks old at time of arrival, 4 weeks old at time of tissue collection) (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.

[0079] A solution for digestion of synovial tissue was prepared by adding water for injection (Otsuka Pharmaceutical) to Liberase MNP-S (Roche Cat. No. 5578566001) to a concentration of 1.25 mg / mL. 1 mL of heat-inactivated fetal bovine serum (Gibco Cat. No. 10500064) was further mixed with 4 mL of the liberase solution to prepare a digestion solution.

[0080] 153.68 mg of the collected synovial tissue was immersed in the digestion solution and reacted for 2 hours at 37 ° C. The cell culture medium was prepared by adding heat-inactivated fetal bovine serum to a final concentration of 20% and penicillin-streptomycin-amphotericin B suspension (x 100) (antibiotic-antimycotic solution) (Fujifilm Wako Pure Chemical Industries, Ltd. 161-23181) to a final concentration of 1% to a basal medium of MEMα with L-alanyl-L-glutamine, pheno; red and sodium pyruvate (Fujifilm Wako Pure Chemical Industries, Ltd. Cat. No. 289-33365) containing 0.28 mM trisodium ascorbic acid phosphate, an ascorbic acid derivative, to prepare a culture medium.

[0081] To the digestion solution in which the synovial tissue had been reacted for 2 hours, two volumes of ice-cold culture medium were added, and the mixture was passed through a 40 μm cell strainer (FACLON Cat. No. 352340) to remove residual tissue. The collected cells were plated in ten T225 cell culture flasks at 1000 cells / cm. ２ The cells were seeded at a density of 1000 x g / ml, and the culture medium was incubated at 25°C for 1 hour. ２After culturing for 8 days, the medium in the flask was discarded and the flask was washed twice with PBS. TrypLE Express (Gibco Cat. No. 12604-013) was added and the flask was left to stand in a 37°C incubator for 5 minutes. The cells were cultured at a concentration of 4.54 x 10 as synovial membrane-derived mesenchymal stem cells. ７ The cells were collected. The supernatant was discarded by centrifugation and replaced with a cryopreservation solution prepared by adding HSA (human albumin, plant-expressed recombinant) (Fujifilm Wako Pure Chemical Industries, Ltd.) to CP-1 High Grade (Kyokuto Pharmaceutical Industries, Ltd., Cat. No. 27207) to a final concentration of 4%. The cells were then frozen at a concentration of 1.2 x 10 ６ The mixture was frozen at 1000x1000 cells / mL in a -80°C freezer for at least one day, and then frozen in a -150°C freezer for at least one day to prepare a frozen stock (Passage 0) of rat synovium-derived mesenchymal stem cells. The stock was stored at the same temperature until use.

[0082] Example 2 Confirmation of Hepatocyte Growth Factor (HGF) Secretion from Rat Synovium-Derived Stem Cells and Treatment for Inhibition The frozen stock (Passage 0) of rat synovium-derived stem cells prepared in Example 1 was put to sleep and incubated in the same culture medium as in Example 1 under CO ２The cells were cultured at a concentration of 5% at 37°C. On day 6 of culture, Silencer Select Pre-designed siRNA (Thermofisher Scientific Cat. No. 4390816 IDs127876) (hereinafter referred to as Hgf siRNA) that inhibits the gene expression of hepatocyte growth factor (HGF) was transfected into the cells using Lipofectamine RNAiMAX Transfection Reagent (Thermofisher Scientific Cat. No. 13778150) and Opti-MEMI Reduced Serum Medium (Thermofisher Scientific Cat. No. 13778150). Cat. No. 31985070) and added to the cells in culture (final siRNA concentration 11.76 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermofisher Scientific Cat. No. 4390844) (hereinafter referred to as NC siRNA), which does not inhibit the expression of any genes including HGF, was similarly treated to the cells. One day after Hgf or NC siRNA treatment, the medium in the flask was discarded and the cells were washed twice with PBS, after which TrypLE Express was added and the cells were left to stand in a 37°C incubator for 5 minutes, and the cells were collected as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation and replaced with CP-1 High Grade. Then, the cells were frozen at a concentration of 2 x 10 ８ Frozen stocks of rat synovium-derived mesenchymal stem cells treated with Hgf or NC siRNA were prepared by freezing at 1000x1000 cells / mL in a -80°C freezer for at least one day, and then freezing in a -150°C freezer for at least one day.

[0083] Frozen stocks of rat synovium-derived mesenchymal stem cells treated with Hgf or NC siRNA were awakened and cultured in culture medium. Portions of the culture supernatant were collected on days 1, 3, and 7 after the start of culture. The culture medium was not replaced during these periods. The HGF concentration 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 control rat synovium-derived mesenchymal cells treated with NC siRNA (labeled NC in the table), HGF concentrations increased over time, and on days 3 and 7, HGF levels of 170.79 pg / mL and 198.16 pg / mL were detected, exceeding the lower limit of quantitation. On the other hand, in rat synovial membrane-derived mesenchymal cells treated with Hgf siRNA (labeled HGFkd in the table), no increase in HGF concentration over time was observed, and the concentration was below the lower limit of quantification (156 pg / mL) and was similar to the HGF concentration in the culture medium (labeled GM in the table). This indicates that rat synovial membrane-derived mesenchymal cells secrete HGF, and that rat synovial membrane-derived mesenchymal cells in which HGF secretion was inhibited by Hgf siRNA treatment were prepared.

[0084]

[0085] Example 3 Evaluation of the chondrogenic differentiation potential of rat synovial membrane-derived stem cells in which HGF secretion was inhibited To investigate the relationship between hepatocyte growth factor and the cartilage repair function of rat synovial membrane-derived stem cells, the chondrogenic differentiation potential of rat synovial membrane-derived stem cells in which HGF secretion was inhibited (hereinafter referred to as HGFkd-SMSC) was evaluated. 2.5 × 10 HGFkd-SMSC or NC-SMSC prepared in Example 2 were used. ５　The cells were diluted with TGF-β3 (R&D Systems Cat. No. 243-B3-002) to a final concentration of 10 ng / mL, dexamethasone (Fujifilm Wako Pure Chemical Cat. No. 041-18861) to a final concentration of 3.92 μg / mL, L-Ascorbic Acid 2-phosphate (Cayman Chemical Cat. No. 16457) to a final concentration of 50 μg / mL, L-proline (MP Biomedicals Cat. No. 194728) to a final concentration of 40 μg / mL, and sodium pyruvate (Invitrogen) to a final concentration of 1 μg / mL. The cells were suspended in DMEM high glucose (Thermofisher Scientific Cat. No. 11965092) containing ITS-X supplement (Fujifilm Wako Pure Chemical Industries, Ltd. Cat. No. 094-06761) to a final concentration of 1%, BMP-2 (R&D Systems Cat. No. 355-BM-010) to a final concentration of 0.5 μg / mL, centrifuged at 450 g for 10 minutes, and then defrosted in CO ２ Culture was initiated at a concentration of 5% and 37°C to induce chondrogenesis. Control cells, rat synovium-derived mesenchymal cells treated with NC siRNA (hereafter referred to as NC-SMSCs), were also induced to differentiate into cartilage in the same manner as HGFkd-SMSCs. After 3 weeks of culture, chondrogenesis potential was assessed based on the diameter, weight, and histological staining of the cell clusters. The results are shown in Table 2 and Figure 1. NC-SMSCs weighed 2.33 ± 0.06 mg and had a major axis of 1.77 ± 0.06 mm, while HGFkd-SMSCs weighed 0.77 ± 0.06 mg and had a major axis of 1.17 ± 0.06 mm, demonstrating a decrease in both cartilage weight and size due to the inhibition of HGF secretion. Furthermore, examination of cartilage matrix staining in pathological specimens revealed areas of loss of staining with toluidine blue (TB), safranin O (SafO), and type II collagen immunostaining (Col II) in HGFkd-SMSCs. This confirmed that HGF is an important molecule that contributes to the chondrogenic differentiation ability of cells.

[0086]

[0087] Example 4 Meniscus Regeneration Effect of Rat Synovium-Derived Stem Cells with HGF Secretion Inhibited Rat Synovium-Derived Stem Cells HGF-inhibited rat synovium-derived stem cells were prepared as described in Example 2. Female LEW / CrlCrlj rats were used to create a meniscus injury model to evaluate the meniscus regeneration effect. The meniscus injury and mesenchymal stem cell transplantation method involved incising the skin over the knee joint under isoflurane anesthesia to expose the knee joint. The medial infrapatellar joint capsule was exposed and incised longitudinally with a scalpel to expose the cartilage of the distal femur. The medial meniscus was detached from the synovium, exposing the medial meniscus, and approximately two-thirds of the meniscus was resected. The patellar tendon and synovium were sutured, followed by muscle sutures to create a meniscus injury model. The treated animals were then divided into three groups, and 1 x 10 HGFkd-SMSCs were injected into the knee joint. ７ 1 × 10 cells, NC-SMSCs ７ After the treatment, all rats were returned to their cages and allowed to move and eat and drink freely.

[0088] Three weeks after treatment, the animals were euthanized by exsanguination via transection of the inferior aorta under isoflurane anesthesia. The meniscus was then exposed from the knee joint, and the medial meniscus was excised and photographed. Images of the excised medial meniscus from both knee joints are shown in Figure 2. The regenerated area was identified based on differences in color and shape from the normal meniscus and is enclosed by a dashed line. In the negative control Vehicle group, many animals had menisci extending up to the mid-segment. In the positive control NC-SMSC group, many menisci had regenerated up to the anterior segment, and the regenerated meniscus was large. In the HGFkd-SMSC group, the regenerated meniscus area appeared smaller than in the positive control NC-SMSC group, and was comparable to the Vehicle group, which did not receive cells.

[0089] To quantitatively evaluate the macroscopic findings of the regenerated meniscus in Figure 2, the area of ​​the regenerated meniscus (within the dashed line) was calculated using Image J (version 1.52) according to the following formula:

[0090] Meniscal regeneration area (mm ２ ) = Number of pixels in the meniscus regeneration area / 1 mm ２ Pixels per

[0091] The mean values, standard deviations, and statistical analyses of the regenerated areas of each group were all performed using Microsoft Excel 2007 (Microsoft Corp.). Statistical analyses were performed using Graph Pad Prism 5.04 software, with a round-robin test between all groups using Bonferroni's Multiple Comparison Test. The significance level was less than 5%, with a difference considered to exist, and the results are shown in Table 3.

[0092] Regarding the area of ​​the regenerated meniscus shown in Table 3, the HGFkd-SMSC group had a 0.69 mm ２ is 1.22 mm of IgG-rSMSCs ２ On the other hand, the HGFkd-SMSC group showed a significant decrease compared to the Vehicle group, which was 0.64 mm ２ This confirmed that the HGF molecule secreted by synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0093]

[0094] Example 5: Knee cartilage repair effect of rat synovium-derived stem cells with inhibited HGF secretion in a rat knee OA model. Rat synovium-derived stem cells with inhibited HGF secretion were prepared as described in Example 2. Female LEW / CrlCrlj rats were used to create a knee OA model to evaluate the cartilage repair effect. Anterior cruciate ligament transection (ACLT) and rat synovial stem cell transplantation for knee OA induction were performed on both knees. Under isoflurane anesthesia, the skin over the knee joint was incised to expose the knee joint. The medial infrapatellar joint capsule was exposed, and the joint capsule was incised longitudinally with a scalpel to expose and transcribe the anterior cruciate ligament. The joint capsule, muscle, and skin were then sutured. The ACLT-treated animals were divided into three groups, and 1 x 10 HGF-inhibited synovial stem cells (HGFkd-SMSCs) were transplanted into the rats. ６ cells, and 1x10 control-treated synovial stem cells (NC-SMSCs) ６ The cells and the vehicle alone were administered intra-articularly seven times every week starting one week after ACLT treatment.

[0095] Eight weeks after ACLT, the animals were euthanized by exsanguination via infraaortic section under isoflurane anesthesia. The femoral and tibial cartilage were then exposed from the knee joint, and the cartilage was photographed after being smeared with India ink. Images of the femoral and tibial cartilage were used to evaluate cartilage damage using a macroscopic cartilage damage score (Yanagisawa K. BMC Musculoskeletal Disorders (2016) 17:188). Representative images of femoral and tibial cartilage are shown in Figure 3. Table 4 lists the macroscopic cartilage damage scores. The scores obtained for each group were statistically analyzed using the Mann-Whitney test with GraphPad Prism 5.04 software. A significance level of less than 5% was considered significant, and the results are listed in Table 4.

[0096] Table 4 shows the macroscopic cartilage damage scores. The superficial cartilage damage scores of 3.0 and 2.9 in the NC-SMSC group were significantly reduced compared to 3.9 and 3.6 in the Vehicle group for both tibial and femoral cartilage, demonstrating that administration of synovial stem cells alleviated knee cartilage damage. Meanwhile, the cartilage damage scores of the HGFkd-SMSC group were 3.7 and 3.3 in both tibial and femoral cartilage, both higher than the scores of the NC-SMSC group, and the score in the tibia was significantly elevated and comparable to that of the Vehicle group.

[0097]

[0098] In addition to macroscopic evaluation, pathological specimens were evaluated for femoral and tibial cartilage damage scores (OARSI scores). To prepare pathological specimens (Pritzker KPH, Osteoarthritis and Cartilage (2006) 14, 13), the excised femoral and tibial cartilage tissues were formalin-fixed and decalcified. The specimens were then prepared in sagittal sections containing the most severely damaged areas macroscopically, and stained with Safranin O. Figure 4 shows representative images of femoral and tibial cartilage pathological specimens. Table 5 lists the OARSI scores for the pathological specimens. The obtained scores for each group were statistically analyzed using GraphPad Prism 5.04 software with the Mann-Whitney test to compare the differences between groups. A significance level of less than 5% was considered significant, and the results are shown in Table 5.

[0099] Table 5 shows the OARSI scores under pathological specimens. Regarding the cartilage damage scores under pathological specimens, the NC-SMSC group had scores of 7.4 and 4.8 for both tibial and femoral cartilage, which were reduced compared to the vehicle group's scores of 11.8 and 5.6. This difference was significant in the tibia, demonstrating that administration of synovial stem cells reduced knee cartilage damage. Meanwhile, the cartilage damage scores of the HGFkd-SMSC group were 9.7 and 5.9 for both tibial and femoral cartilage, both higher than the scores of the NC-SMSC group, and significantly increased in the tibia, comparable to the scores of the vehicle group.

[0100]

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

[0102] Example 6 Confirmation of TGFBI Secretion from Rat Synovial Membrane-Derived Stem Cells and Treatment for Inhibition Thereof Frozen stocks of rat synovial membrane-derived stem cells prepared in Example 1 were put to sleep, and the same culture medium as in Example 1 was used to incubate the cells in CO ２The cells were cultured at a concentration of 5% at 37°C. On day 4 of culture, Silencer Select Pre-designed siRNA (Thermofisher scientific Cat. No. 4390816 IDs137979) that inhibits TGFBI gene expression (hereinafter referred to as Tgfbi siRNA) was transfected into the cells using Lipofectamine RNAiMAX Transfection Reagent (Thermofisher scientific Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermofisher scientific Cat. No. 13778150). Cat. No. 31985070) and added to the cells in culture (final siRNA concentration 20.7 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermofisher Scientific Cat. No. 4390844) (hereinafter referred to as NC siRNA), which does not inhibit the expression of any genes including TGFBI, was similarly treated to the cells. Three days after TGFBI or NC siRNA treatment, the medium in the flask was discarded and washed twice with PBS, after which TrypLE Express was added and the cells were left to stand in a 37°C incubator for 5 minutes, and the cells were collected as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation and replaced with CP-1 High Grade. Then, cells were frozen at a concentration of 2 x 10 ８ The cells / mL were frozen in a -80°C freezer for at least one day, and then frozen in a -150°C freezer for at least one day to prepare frozen stocks of rat synovium-derived mesenchymal stem cells treated with Tgfbi or NC siRNA.

[0103] Frozen stocks of rat synovium-derived mesenchymal stem cells treated with TGFBI or NC siRNA were awakened and cultured in culture medium. Portions of the culture supernatant were collected on days 1, 3, and 7 after the start of culture. The culture medium was not replaced during this period. The TGFBI concentration in the collected culture supernatant was measured using a beta IG-H3 / TGFBI PicoKine ELISA Kit (Boster Immunoleader. Cat. No. EK1571). The results are shown in Table 6.

[0104]

[0105] For untreated synovial stem cells (labeled SMSC (no treated) in the table), TGFBI secretion in the culture supernatant was observed at 676, 2070, and 2142 pg / mL on Days 1, 3, and 7. For rat synovial-derived mesenchymal cells treated with control NC siRNA (labeled NC-SMSC in the table), TGFBI secretion in the culture supernatant was also observed at 298, 1185, and 1721 pg / mL on Days 1, 3, and 7. On the other hand, for rat synovial-derived mesenchymal cells treated with Tgfbi siRNA (labeled TGFBIkd-SMSC in the table), no increase in TGFBI concentration over time was observed, and the concentration was below the lower limit of quantification (156 pg / mL) and was similar to the HGF concentration in the culture medium (labeled GM in the table). These results indicate that rat synovial membrane-derived mesenchymal cells secrete TGFBI, and that rat synovial membrane-derived mesenchymal cells in which TGFBI secretion was inhibited by TGFBI siRNA treatment could be prepared.

[0106] Example 7 Meniscus Regeneration Effect of Rat Synovium-Derived Stem Cells with TGFBI Secretion Inhibited Rat synovium-derived stem cells with TGFBI secretion inhibited were prepared as described in Example 6. A meniscus injury model for evaluating meniscus regeneration effect was created in the same manner as in Example 4. Treated animals were divided into three groups, and TGFBIkd-SMSCs were injected at 1 x 10 ７ 1x10 NC-SMSCs ７ Cells and the freezing solvent CP1 (Vehicle) alone were each administered into the joint capsule.

[0107] Three weeks after treatment, the animals were euthanized by exsanguination via infra-aortic section under isoflurane anesthesia. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial meniscus from both knee joints are shown in Figure 5. The regenerated area was identified based on differences in color and shape from the normal meniscus and is enclosed by a dashed line. In the positive control NC-SMSC group, the regenerated area appeared slightly larger than in the negative control Vehicle and TGFBIkd-SMSC groups, but no clear difference was observed.

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

[0109] Regarding the area of ​​the regenerated meniscus shown in Table 7, the TGFBIkd-SMSC group had a 0.78 mm ２ is NC-SMSC 1.14 mm ２ On the other hand, the TGFBIkd-SMSC group showed a significant decrease compared to the Vehicle group, which was 0.77 mm ２ This confirmed that the TGFBI molecule in synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0110]

[0111] Example 8 Confirmation of TNFSF15 secretion from rat synovial membrane-derived stem cells and treatment for inhibiting it. The frozen stock of rat synovial membrane-derived stem cells prepared in Example 1 was put to sleep, and the same culture medium as in Example 1 was used to incubate the cells in CO ２The cells were cultured at a concentration of 5% at 37°C. On day 4 of culture, Silencer Select Pre-designed siRNA (Thermofisher Scientific Cat. No. 4390816 ID s141123) (hereinafter referred to as Tnfsf15 siRNA) that inhibits TNFSF15 gene expression was transfected into the cells using Lipofectamine RNAiMAX Transfection Reagent (Thermofisher Scientific Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermofisher Scientific Cat. No. 13778150). Cat. No. 31985070) and added to the cells in culture (final siRNA concentration 11.2 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermofisher Scientific Cat. No. 4390844) (hereinafter referred to as NC siRNA), which does not inhibit the expression of any genes including TNFSF15, was similarly treated to the cells. Three days after Tnfsf15 or NC siRNA treatment, the medium in the flask was discarded and washed twice with PBS, after which TrypLE Express was added and the cells were left to stand in a 37°C incubator for 5 minutes, and the cells were collected as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation and replaced with CP-1 High Grade. Then, cells were frozen at a concentration of 2 x 10 ８ The cells / mL were frozen in a -80°C freezer for at least one day, and then frozen in a -150°C freezer for at least one day to prepare frozen stocks of rat synovium-derived mesenchymal stem cells treated with Tnfsf15 or NC siRNA.

[0112] Frozen stocks of rat synovial mesenchymal stem cells treated with Tnfsf15 or NC siRNA were awakened and cultured in culture medium. Cells were harvested on days 1, 3, and 7 after the start of culture, and total RNA was extracted from the cells and RT-PCR was performed using the TaqMan Gene Expression Cells-to-CT Kit (Thermofisher Scientific Cat. No. AM1728) to obtain cDNA. Thereafter, the target gene TNFSF15 was amplified using TaqMan Gene Expression Assay (FAM) Assay ID: Rn00595596_m1 Tnfsf15 (Thermofisher scientific Cat. No. 4331182), and the housekeeping gene was amplified using TaqMan Gene Expression Assay (FAM) Assay ID: Rn00667869_m1 Actb (Thermofisher scientific Cat. No. 4331182) as primer probes. The genes were amplified using a real-time PCR instrument, QuantStudio 7 flex, and the respective Ct values ​​were obtained. The deltaCt value was calculated by subtracting the Ct value of the housekeeping gene ACTB from the Ct value of TNFSF15 obtained, and the values ​​at each level were compared. The results are shown in Table 8. On Day 1, the deltaCt values ​​of non-treated ACI-SMSC and NC-SMSC were 14.05 and 14.71, respectively. In contrast, the deltaCt value of TNFSF15kd SMSC was 15.64, confirming a relative decrease in gene expression. On Day 3, the deltaCt values ​​of non-treated ACI-SMSC and NC-SMSC were 12.13 and 12.51, respectively. In contrast, the deltaCt value of TNFSF15kd SMSC was 13.22, confirming a relative decrease in gene expression. On the other hand, on Day 7, the deltaCt values ​​of the non-treated ACI-SMSCs and NC-SMSCs were 10.56 and 9.46, respectively, while that of the TNFSF15kd SMSCs was 10.58, and no decrease in the relative gene expression level was observed.This indicates that rat synovium-derived mesenchymal cells express the TNFSF15 gene, and that rat synovium-derived mesenchymal cells in which TNFSF15 gene expression was inhibited until Day 3 by treatment with Tnfsf15 siRNA were prepared.

[0113]

[0114] To examine the protein expression of TNFSF15 in rat synovial mesenchymal cells in which TNFSF15 gene expression was inhibited by Tnfsf15 siRNA treatment, 1 × 10 ５ To the remaining cells, 1 mL of cell lysis buffer M (Fujifilm Wako Pure Chemical Industries, Ltd. 038-21141) was added, the cells were suspended by pipetting, and then the mixture was left on ice for 10 minutes. The mixture was centrifuged at 4°C for 10 minutes at a maximum of 20,000 x g, and the supernatant was collected as cell lysate (protein extract). The TNFSF15 concentration 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. For untreated synovial stem cells (labeled SMSC (no treated) in the table), TNFSF15 protein expression was observed at 29.43, 42.62, and 37.28 pg / mL on Days 1, 3, and 7. Similar TNFSF15 protein expression was observed in rat synovial mesenchymal cells treated with control NC siRNA (labeled NC-SMSC in the table), at 29.13, 51.23, and 43.13 pg / mL on Days 1, 3, and 7. On the other hand, in rat synovial mesenchymal cells treated with TNFSF15 siRNA (labeled TNFSF15kd-SMSC in the table), the TNFSF15 concentration on Day 3 was reduced to 27.22 pg / mL, a decrease compared to SMSC (no treated) and NC-SMSC. The concentrations were similar for all three treatments on Day 7. This confirmed that rat synovial membrane-derived mesenchymal cells expressed TNFSF15 protein, and that TNFSF15 siRNA treatment was able to inhibit TNFSF15 protein expression up to Day 3.

[0115]

[0116] Example 9: Meniscus regeneration effect of rat synovium-derived stem cells with inhibited TNFSF15 gene expression Rat synovium-derived stem cells with inhibited TNFSF15 gene expression were prepared as described in Example 6. A meniscus injury model for evaluating meniscus regeneration effect was created in the same manner as in Example 4. Treated animals were divided into three groups, and TNFSF15kd-SMSCs were injected at 1 x 10 ７ 1x10 NC-SMSCs ７ Cells and the freezing solvent CP1 (Vehicle) alone were each administered into the joint capsule.

[0117] Three weeks after treatment, the animals were euthanized by exsanguination via infra-aortic section under isoflurane anesthesia. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial meniscus from both knee joints are shown in Figure 6. The regenerated area was identified based on differences in color and shape from the normal meniscus and is enclosed by a dashed line. In the positive control NC-SMSC group, the regenerated area appears slightly larger than in the negative control Vehicle and TNFSF15kd-SMSC groups.

[0118] To quantitatively evaluate the macroscopic findings of the regenerated meniscus in Figure 6, the area was calculated as described in Example 4 and evaluated by statistical processing. The results are shown in Table 10. The area of ​​the regenerated meniscus shown in Table 10 was 0.84 mm in the TNFSF15kd-SMSC group. ２ is NC-SMSC 1.29 mm ２ On the other hand, the TNFSF15kd-SMSC group showed a significant decrease compared to the Vehicle group, which was 0.87 mm ２ This confirmed that the TGFBI molecule in synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0119]

[0120] Example 10: Positive rates of CD29 (Integrin β1) and CD140b (PDGFRβ) in rat synovial membrane-derived stem cells. The rat synovial membrane-derived stem cells obtained in Example 1 were expanded and cultured for 7 days under the same conditions as in Example 1 to prepare frozen stocks. The frozen stocks were thawed, and 10 cells were cultured. ６The cells were suspended in 500 μL of PBS. For dead cell staining, 0.5 μL of LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Cat. No. L34957) was added to the cell suspension and incubated at room temperature for 30 minutes. After centrifugation, the supernatant was discarded, and 1 mL of FACS buffer (PBS containing 2 mM EDTA 2Na and 1% bovine serum albumin) was added to suspend the cells. To measure the positive rate of Integrin β1, 5 μL of PE anti-mouse / rat Integrin β1 Antibody (Biolegend Cat. No. 102207) or PE Armenian Hamster IgG Isotype Control Antibody (Biolegend Cat. No. 400907) was added and reacted for 30 minutes at 4°C. After centrifugation, the supernatant was discarded and the cells were suspended in 1 mL of FACS buffer, centrifuged again, the supernatant was discarded, and the cells were suspended in 500 μL of FACS buffer for measurement. To measure the positive rate of CD140b, 5 μL of Anti-PDGF Receptor β, Human, Goat-Poly (R&D Systems Cat. No. AF385) or Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was added and incubated at 4°C for 30 minutes. After centrifugation, the supernatant was discarded and the cells were suspended in 1 mL of FACS buffer. 1 μL of Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC (Invitrogen Cat. No. A16006) was added and incubated at 4°C for 30 minutes. The cells were then centrifuged, the supernatant discarded, and the cells suspended in 1 mL of FACS buffer. The cells were then centrifuged again, the supernatant discarded, and the cells suspended in 500 μL of FACS buffer. The CD29 and CD140b positivity rates were measured using a flow cytometer. The analysis results for the CD29 and CD140b positivity rates of synovial membrane-derived stem cells are shown in Figure 7. CD29 showed a positivity rate of 91.6%. Meanwhile, CD140b showed a positivity rate of 44.4%, less than half the positivity rate of CD29.

[0121] Example 11: Suppression of extracellular matrix adhesion ability of rat synovium-derived stem cells by CD29 inhibition To confirm that CD29 is inhibited, one of its functions, adhesion to extracellular matrix, was examined. To prepare rat synovium-derived stem cells with CD29 inhibited, the frozen stock of rat synovium-derived stem cells prepared in Example 1 was put to sleep and expanded under the same conditions as in Example 1. After 7 days of culture, the medium in the flask was discarded and the cells were washed twice with PBS, after which TrypLE Express (Gibco Cat. No. 12604-013) was added and the cells were allowed to stand in a 37°C incubator for 5 minutes to recover the cells. The supernatant was discarded by centrifugation, and the cells were then incubated in a CO ２ The cells were cultured at a concentration of 5% at 37°C for 1 week, and the collected cells were suspended in a reaction solvent, PBS containing 2% FBS. ６ Purified anti-mouse / rat Integrin β1 antibody (BioLegend Cat. No. 102202) was added at 12 μg per cell, and the cells were incubated on ice for 1 hour before harvesting (CD29ab-SMSC). Purified Armenian Hamster IgG Isotype Control (BioLegend Cat. No. 400902) was added as a control treatment without Integrin β1 inhibition, and the cells were incubated on ice for 1 hour before harvesting (IgG-SMSC). Untreated cells (SMSC) were also incubated in the same manner using the reaction solvent alone. The collected cells were centrifuged to discard the supernatant, and the supernatant was replaced with CP-1 High Grade (Kyokuto Pharmaceuticals, Cat. No. 27207). ８ The cells / mL were frozen in a -80°C freezer for at least one day, and then frozen in a -150°C freezer for at least one day, and then subjected to the following adhesion treatment.

[0122] 10 mM MgCl for extracellular matrix adhesion reaction ２ ・6H ２ The cells were washed with PBS containing 0 (hereinafter referred to as PBS(+)), and then suspended again in PBS(+) at 1 × 10 ５　The solution was adjusted to 10 μL / well and seeded onto a collagen I-coated chamber slide II with 8 chambers (IWAKI Cat. No. 4732-010), with 6 wells seeded per treatment (n=6). After leaving the slides at room temperature for 10 minutes, the slides were washed with PBS(+). Then, the slides were observed under a microscope (OLIMPUS Cat. No. IX71) with a 10x objective. Images of the three fields of view where the most adherent cells were observed per well were also taken, and the number of adherent cells was counted. The average number of cells per well was calculated as the number of adherent cells.

[0123] The results are shown in Table 11 and Figure 8. The number of adherent cells was significantly reduced to 253 cells for CD29 neutralizing antibody-treated cells (CD29ab-SMSC), compared to 472 cells for antibody-untreated cells (SMSC) and 396 cells for IgG-treated cells (IgG-SMSC). This confirmed that treatment with purified anti-mouse / rat integrin β1 antibody can inhibit the function of CD29 in rat synovial stem cells.

[0124]

[0125] Example 12: Meniscus regeneration effect of CD29-inhibited rat synovium-derived stem cells. CD29-inhibited frozen rat synovium-derived stem cells were prepared as described in Example 3. The frozen cell stock (Passage 0) prepared in Example 1 was awake, cultured for 1 week, and the collected cells were suspended in a reaction solvent, PBS containing 2% FBS. The number of cells was 1 x 10 ７ Purified anti-mouse / rat Integrin β1 antibody (24 μg per cell) was added and incubated on ice for 1 hour. After incubation, 2 × 10 cells per cryovial were frozen using CP-1 High Grade cryostat. ７ As a control treatment without inhibition, purified Armenian hamster IgG isotype Ctrl was reacted on ice for 1 hour, and then frozen at a concentration of 2 x 10 cells per cryovial using freezing solution CP-1 High Grade. ７The cells were cryopreserved at a concentration of 100 μL.

[0126] Female LEW / CrlCrlj rats were used to create a meniscus injury model to evaluate the meniscus regeneration effect. The meniscus injury and mesenchymal stem cell transplantation method involved incising the skin over the knee joint under isoflurane anesthesia to expose the knee joint. The medial infrapatellar joint capsule was exposed and then incised longitudinally with a scalpel to expose the cartilage of the distal femur. The medial meniscus was detached from the synovial membrane, exposing the medial meniscus, and approximately two-thirds of the meniscus was resected. The patellar tendon and synovial membrane were sutured, followed by muscle sutures to create a meniscus injury model. The treated animals were then divided into three groups, and 1 x 10 SMSCs with CD29 inhibition (CD29ab-SMSCs) were injected. ７ 1 × 10 cells, and 1 × 10 control-treated synovial stem cells (IgG-SMSCs) ７ After the treatment, all rats were returned to their cages and allowed to move freely and eat and drink.

[0127] Three weeks after treatment, the animals were euthanized by exsanguination via aortic transection under isoflurane anesthesia. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the medial meniscus from both knee joints are shown in Figure 9. The regenerated areas were identified based on differences in color and shape compared to normal meniscus and are enclosed by dashed lines. In the negative control vehicle group, most menisci continued to grow to the mid-segment. In the positive control IgG-SMSC group, many menisci regenerated to the anterior segment, revealing large regenerated areas. In contrast, in the CD29ab-SMSC group, which contained molecules that had been inhibited or deleted, the regenerated areas of the meniscus appeared smaller than in the positive control.

[0128] To quantitatively evaluate the macroscopic findings of the regenerated meniscus in Figure 9, the area of ​​the regenerated meniscus (within the dashed line) was calculated using Image J (version 1.52) according to the following formula:

[0129] Meniscal regeneration area (mm ２ ) = Number of pixels in the meniscus regeneration area / 1 mm ２ Pixels per

[0130] The mean values, standard deviations, and statistical analyses of the regenerated areas of each group were all performed using Microsoft Excel 2007 (Microsoft Corp.). Statistical analyses were performed using Graph Pad Prism 5.04 software, with a round-robin test performed between all groups using Bonferroni's Multiple Comparison Test. The significance level was less than 5%, with a difference being considered significant, and the results are shown in Table 12.

[0131] Regarding the area of ​​the regenerated meniscus shown in Table 11, the CD29ab-SMSC group had a 0.69 mm ２ is 1.44 mm for IgG-SMSCs. ２ On the other hand, the CD29ab-SMSC group showed a significant decrease compared to the vehicle group, with a decrease of 0.87 mm ２ This confirmed that the CD29 molecule is essential for meniscus regeneration by synovial stem cells.

[0132]

[0133] Example 13: Suppression of cell proliferation ability of rat synovium-derived stem cells by inhibiting platelet-derived growth factor receptor β (PDGFRβ, also known as CD140b) and hepatocyte growth factor To prepare rat synovium-derived stem cells with CD140b inhibited, the frozen stock of rat synovium-derived stem cells prepared in Example 1 was put to sleep and expanded under the same conditions as in Example 1. After 7 days of culture, the medium in the flask was discarded and the cells were washed twice with PBS, after which TrypLE Express (Gibco Cat. No. 12604-013) was added and the cells were left to stand in a 37°C incubator for 5 minutes to recover the cells. The supernatant was discarded by centrifugation, and the cells were then incubated in a CO ２ The cells were cultured at a concentration of 5% at 37°C for 1 week, and the collected cells were suspended in a reaction solvent, PBS containing 2% FBS. ６The cells were incubated with 20 μg of Anti-PDGF Receptor β Human Goat Poly (R&D Systems Cat. No. AF385) per cell on ice for 1 hour, and then collected (CD140b-SMSC). As a control treatment without CD140b inhibition, Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was incubated on ice for 1 hour, and then collected (IgG-SMSC). Untreated cells (SMSC) were also incubated in the same manner using only the reaction solvent. The supernatants of the collected cells were discarded by centrifugation, and replaced with CP-1 High Grade (Kyokuto Pharmaceutical Cat. No. 27207). Then, cells were frozen at a concentration of 2 x 10 ８ The cells / mL were frozen in a -80°C freezer for at least one day, and then stored frozen in a -150°C freezer for at least one day, and then subjected to the following treatment and evaluation.

[0134] To confirm that CD140b was inhibited, cell proliferation, a function of CD140b, was examined. Table 13 lists the experimental levels for each treatment. After the above treatments, cryopreserved SMSC, IgG-SMSC, and CD140b-SMSC cells were seeded into 96-well plates and cultured for one day in culture medium alone (Levels 1 to 3), or in culture medium containing 40 μg / mL of Anti-PDGF Receptor β Human Goat-Poly (Level 4) or Normal Goat IgG Control (Level 5).

[0135] The day after cell seeding, the culture supernatant was discarded and replaced with culture medium (Level 1) or culture medium containing 0.1% FBS (Levels 2 to 5), or 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 levels, Normal Goat IgG Control (Level 4) or Anti-PDGF Receptor β Human (Level 5) was added at a final concentration of 40 μg / mL, bringing the total volume of the medium to 100 μL. For levels without IgG or antibody, only 100 μL of the medium of the respective composition was added (Levels 1 to 3). On day 6 after cell seeding, the cell proliferation was relatively evaluated by an ATP assay using Cell Titer Glo (Promega Cat. No. G7571) in which the number of cells at each level was regarded as the luminescence intensity due to ATP.

[0136]

[0137] The results are shown in Table 14. The luminescence intensity was 18.57 when cultured in a normal culture medium (Level 1), but was 3.69 when cultured in a 0.1% FBS culture medium (Level 2), significantly reducing the number of proliferating cells. The addition of PDGF-BB, a PDGFR-β ligand, increased the luminescence intensity to 8.81 (Level 3), confirming PDGFR-β-dependent cell proliferation. Meanwhile, the addition of Anti-PDGF Receptor β Human Goat-Poly, an antibody that inhibits CD140b, in addition to PDGF-BB (Level 5) resulted in a luminescence intensity of 6.22, a significant decrease compared to the luminescence intensity of 8.25 obtained with the control treatment, IgG (Level 4). However, the reduction was 44.5% at level 5, calculated based on the values ​​of 4.56 and 2.53 obtained by subtracting level 2 from levels 4 and 5, using level 2 as the baseline. This indicated that the inhibition rate was 44.5%, and did not completely inhibit the cell proliferation effect induced by PDGF-BB. As shown in Figure 7, the CD140b positivity rate of these cells was 44.4%, suggesting that the CD140b-related proliferation effect of PDGF-BB was completely inhibited by this antibody treatment. This confirmed that treatment with Anti-PDGF Receptor β Human Goat-Poly enabled ligand-specific inhibition of PDGFRβ (CD140b) in rat synovial stem cells.

[0138]

[0139] Example 14: Meniscus regeneration effect of CD140b-inhibited rat synovium-derived stem cells Frozen rat synovium-derived stem cells with CD140b inhibition were prepared as described in Example 13. Specifically, the frozen cell stock (Passage 0) prepared in Example 1 was awake and cultured for one week, and the collected cells were suspended in the reaction solvent PBS containing 2% FBS. The number of cells was 1 x 10 ６ 20 μg of Anti-PDGF Receptor β Human Goat-Poly was added per cell, and the cells were incubated on ice for 1 hour. After that, 2 × 10 cells per cryovial were frozen using the freezing solution CP-1 High Grade. ７As a control treatment without inhibition, purified Armenian hamster IgG isotype Ctrl was reacted on ice for 1 hour, and then frozen at a concentration of 2 x 10 cells per cryovial using freezing solution CP-1 High Grade. ７ The cells were cryopreserved at a concentration of 100 μL.

[0140] Female LEW / CrlCrlj rats were used to create a meniscus injury model to evaluate the meniscus regeneration effect. The meniscus injury and mesenchymal stem cell transplantation methods involved incising the skin over the knee joint under isoflurane anesthesia to expose the knee joint. The medial infrapatellar joint capsule was exposed and then incised longitudinally with a scalpel to expose the cartilage of the distal femur. The medial meniscus was detached from the synovial membrane, exposing the medial meniscus, and approximately two-thirds of the meniscus was resected. The patellar tendon and synovial membrane were sutured, followed by muscle sutures to create a meniscus injury model. The treated animals were then divided into three groups, and 1 x 10 CD140b-inhibited synovial stem cells (CD140b-ab-SMSCs) were transplanted. ７ 1 × 10 cells, and 1 × 10 control-treated synovial stem cells (IgG-SMSCs) ７ After the treatment, all rats were returned to their cages and allowed to move freely and eat and drink.

[0141] Three weeks after treatment, the animals were euthanized by exsanguination via aortic transection under isoflurane anesthesia. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial meniscus from both knee joints are shown in Figure 10. The regenerated area was identified based on differences in color and shape compared to the normal meniscus and is enclosed by a dashed line. In the negative control vehicle group, many subjects had menisci extending to the mid-segment. In the positive control IgG-SMSC group, many subjects had regenerated menisci extending to the anterior segment, revealing a large regenerated area. In contrast, the CD140b-ab-SMSC group, which contained molecularly inhibited cells, showed no significant difference compared to the positive control, demonstrating a similar level of meniscal regeneration.

[0142] To quantitatively evaluate the macroscopic findings of the regenerated meniscus in Figure 10, the area of ​​the regenerated meniscus (within the dashed line) was calculated using Image J (version 1.52) according to the following formula:

[0143] Meniscal regeneration area (mm ２ ) = Number of pixels in the meniscus regeneration area / 1 mm ２ Pixels per

[0144] The mean values, standard deviations, and statistical analyses of the regenerated areas of each group were all performed using Microsoft Excel 2007 (Microsoft Corp.). Statistical analyses were performed using Graph Pad Prism 5.04 software, with a round-robin test performed between all groups using Bonferroni's Multiple Comparison Test. The significance level was less than 5%, with a difference considered to exist, and the results are shown in Table 15.

[0145] Regarding the area of ​​the meniscus regeneration portion shown in Table 15, the Vehicle group had a 1.03 mm ２ is 0.74 mm in the CD140b-ab-SMSC group. ２ The CD140b-ab-SMSC group showed a significant increase in regeneration area compared to the IgG-SMSC group. On the other hand, the CD140b-ab-SMSC group showed a similar regeneration area to the IgG-SMSC group. This indicates that the CD140b molecule is not essential for meniscus regeneration by synovial stem cells.

[0146]

[0147] Reference Example 1 Confirmation of TSP2 Secretion from Rat Synovial Membrane-Derived Stem Cells and Treatment for Inhibition Thereof Frozen stocks of rat synovial membrane-derived stem cells prepared in Example 1 were put to sleep, and the same culture medium as in Example 1 was used to incubate the cells in CO ２The culture was carried out at a concentration of 5% at 37°C. On day 4 of culture, Silencer Select Pre-designed siRNA (Thermofisher Scientific Cat. No. 4390816 IDs146414) (hereafter referred to as Tsp2 siRNA) that inhibits TSP2 gene expression was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermofisher Scientific Cat. No. 13778150) and Opti-MEM I Reduced Serum Medium (Thermofisher Scientific Cat. No. 31985070) and added to the cells in culture. (siRNA final concentration 20 nmol / L). As a control treatment, Silencer Select Negative Control #1 siRNA (Thermofisher Scientific Cat. No. 4390844) (hereinafter referred to as NC siRNA), which does not inhibit the expression of any genes including TSP2, was similarly treated to the cells. Three days after Tsp2 or NC siRNA treatment, the medium in the flask was discarded and washed twice with PBS, after which TrypLE Express was added and the cells were left to stand in a 37 °C incubator for 5 minutes, and the cells were collected as synovium-derived mesenchymal stem cells. The supernatant was discarded by centrifugation and replaced with CP-1 High Grade, and frozen stocks of rat synovium-derived mesenchymal stem cells treated with Tsp2 or NC siRNA were prepared by freezing.

[0148] Frozen stocks of rat synovium-derived mesenchymal stem cells treated with Tsp2 or NC siRNA were awakened and cultured in culture medium. Portions of the culture supernatant were collected on days 1, 3, and 7 after the start of culture. The culture medium was not replaced during this time. For evaluation, the collected culture supernatant was diluted 300-fold with PBS using a rat Thbs2 ELISA kit (CLOUD-CLONE CORP. Cat. No. SED822RA), and the TSP2 concentration was measured. The concentration obtained after measurement was multiplied by 300 and converted to the original concentration calculated. The results are shown in Table 16.

[0149]

[0150] For untreated synovial stem cells (labeled SMSC (no treated) in the table), TSP2 secretion was observed in the culture supernatant at 1358, 6102, and 7203 ng / mL on Days 1, 3, and 7. For rat synovial-derived mesenchymal cells treated with control NC siRNA (labeled NC-SMSC in the table), TSP2 secretion was also observed in the culture supernatant at 1848, 5817, and 9432 ng / mL on Days 1, 3, and 7. On the other hand, for rat synovial-derived mesenchymal cells treated with Tsp2 siRNA (labeled TSP2kd-SMSC in the table), TSP2 secretion was observed at 133 and 436 ng / mL on Days 1 and 3, respectively, which was less than one-tenth of that of NC-SMSC, but at 1630 ng / mL on Day 7, a level equivalent to that of Day 1 for other treatments. TSP2 was not detected in the culture medium (GM in the table). This indicates that rat synovium-derived mesenchymal cells secrete TSP2, and that rat synovium-derived mesenchymal cells in which TSP2 secretion was inhibited for at least 3 days by treatment with Tsp2 siRNA were prepared.

[0151] Reference Example 2: Meniscus regeneration effect of rat synovium-derived stem cells with inhibited TSP2 secretion Rat synovium-derived stem cells with inhibited TSP2 secretion were prepared as described in Comparative Example 1. A meniscus injury model for evaluating meniscus regeneration effect was created in the same manner as in Example 4. Treated animals were divided into three groups, and TSP2kd-SMSCs were injected at 1 x 10 ７ 1 × 10 cells, NC-SMSCs ７ Cells, and the freezing solvent CP1 (Vehicle) alone were each administered into the joint capsule.

[0152] Three weeks after treatment, the animals were euthanized by exsanguination via infra-aortic section under isoflurane anesthesia. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial meniscus from both knee joints are shown in Figure 11. The regenerated area was identified based on differences in color and shape from the normal meniscus and is enclosed by a dashed line. In the positive control NC-SMSC group, the regenerated meniscus area appeared larger than in the negative control Vehicle group, but no clear difference was observed compared to the TSP2kd-SMSC group.

[0153] To quantitatively evaluate the macroscopic findings of the regenerated meniscus in Figure 11, the area was calculated as described in Example 4 and evaluated by statistical analysis. Regarding the area of ​​the regenerated meniscus shown in Table 17, the TSP2kd-SMSC group had a 0.83 mm ２ is 1.06 mm of IgG-rSMSCs ２ Although there was a difference, it was not statistically significant. ２ Although there was a difference in the TSP2 activity against the TSP2-dependent fibroblast growth factor receptor 2 (TFGR), the difference was not statistically significant. This suggests that the TSP2 molecule in synovial stem cells is not an important molecule that contributes to meniscus regeneration.

[0154]

Claims

1. Synovial membrane-derived mesenchymal stem cells that are positive for 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 of claim 1, which are positive for hepatocyte growth factor, transforming growth factor β-induced protein, and tumor necrosis factor superfamily 15.

3. The synovium-derived mesenchymal stem cells according to claim 1, which are positive for integrin β1.

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

5. Synovial membrane-derived mesenchymal stem cells according to claim 1, obtained by a method comprising: step A of treating synovial tissue with an enzyme; step B of culturing the synovial membrane-derived mesenchymal stem cells contained in the mixture obtained after washing the mixture after the enzyme treatment in a culture medium; and step C of cryopreserving the cultured synovial membrane-derived mesenchymal stem cells.

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

7. A therapeutic agent for arthropathy, comprising the synovium-derived mesenchymal stem cells or cell population described in any one of claims 1 to 6.

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

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

10. The method according to claim 8 or 9, wherein step B is carried out using a medium containing an ascorbic acid derivative.

11. In the step C, the cell concentration when the cells are frozen is 1 x 10 ６ The method of claim 8 or 9, wherein the concentration is 10 ...

12. The method according to claim 8 or 9, wherein in step C, after the cells have been subjected to the freezing treatment, they are stored in a frozen state for two days or more.

13. The method according to claim 8 or 9, wherein step C comprises freezing the cells at -70°C to -90°C, followed by freezing at -140°C to -160°C.

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

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