Method for manufacturing joint disease treatment agents

Abstract

The present invention aims to provide a therapeutic agent for arthritis and a method for manufacturing the same, wherein the therapeutic agent comprises synovial-derived mesenchymal stem cells having molecules necessary for joint treatment. According to the present invention, a therapeutic agent for arthritis is provided, comprising synovial-derived mesenchymal stem cells having one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β.

Description

[Technical Field]

[0001] The present invention relates to a treatment agent for arthritis and a method for manufacturing the above-mentioned treatment agent for arthritis, wherein the treatment agent for arthritis comprises synovial-derived mesenchymal stem cells having molecules necessary for joint treatment. [Previous Technology]

[0002] In recent years, with the advancement of regenerative medicine and cell therapy technologies, the development of various cell therapies and research cell products utilizing autologous, allogeneic, or xenogeneic cells is being actively pursued. Among these, mesenchymal stem cells (MSCs) are expected to serve as a useful cell source for cell therapy. MSCs can be collected from various biological tissues, and it has been reported that they can be isolated from bone marrow tissue (Non-Patent Literature 1), adipose tissue (Non-Patent Literature 2), muscle tissue (Non-Patent Literature 3), synovial tissue (Non-Patent Literature 4), and periosteum (Non-Patent Literature 5). In particular, synovial-derived mesenchymal stem cells have been reported to have higher proliferative capacity and cartilage-forming capacity compared to mesenchymal stem cells derived from various mesenchymal tissues such as bone marrow (Non-Patent Literature 6). Furthermore, Patent Literature 1-3 discloses a method for treating articular cartilage injuries and meniscus injuries using synovial-derived mesenchymal stem cells. Patent document 4 describes a method for preparing and managing the quality of limb bud mesenchymal cell populations, cartilage precursor cell populations, and osteoprogenitor cell populations using molecules such as CD140b.

[0003] [Non-Patent Literature 1] Prockop, DJ, 1997, Science. 276:71-4 [Non-Patent Literature 2] Zuk, PA et al., 2002, Mol Biol Cell. 13:4279-95 [Non-Patent Literature 3] Cao et al., 2003, Nat Cell Biol. 5:640-6 [Non-Patent Literature 4] De Bari, C. et al., 2001, Arthritis Rheum. 44:1928-42 [Non-Patent Literature 5] Fukumoto, T. et al., 2003, OsteoarthritisCartilage. 11:55-64 [Non-Patent Literature 6] Sakaguchi, et al., 2005, Arthritis Rhum. 52:2521-9

[0004] [Patent Document 1] Japanese Patent No. 5928961 [Patent Document 2] Japanese Patent No. 5656183 [Patent Document 3] Japanese Patent No. 6864302 [Patent Document 4] International Publication No. WO2021 / 054449

[0005] Ensuring batch equivalence and identity is a key challenge in the quality management of cell products. However, since the cells themselves that constitute the product are not completely homogeneous structures and their characteristics are difficult to determine, ensuring batch equivalence and identity is usually difficult. Therefore, in order to manage product quality, not only are quality tests conducted on the final product, but the concept of QMS (Quality Management System), already applicable to medical devices, has been implemented to record and control manufacturing raw materials, material management, manufacturing process management, and process management testing, thereby managing the entire process. However, with the advancement of science and technology, the importance of identifying the characteristics of the cells themselves as the final product is increasing.

[0006] As a method for cell quality management, for example, cell type-specific surface markers are used to identify the cell type of the target final product (e.g., identifying it as mesenchymal stem cells). However, cells obtained by this quality management method have concerns about unstable therapeutic effects and are still not entirely satisfactory.

[0007] To date, methods for manufacturing joint therapeutic agents using synovial stem cells have been reported. However, the lack of adequate quality control to ensure the efficacy of these agents has led to inconsistent therapeutic effects. Cell products primarily rely on markers for cell type identification for quality control, rather than quality control related to potency (efficacy). [Summary of the Invention]

[0008] Since biomarkers representing potency (efficacy) are identified based on mechanisms of action, this invention aims to elucidate the mechanisms of action related to the effectiveness of cell therapy products, identify biomarkers based on these mechanisms of action, and thereby provide a method for manufacturing a therapeutic agent based on these biomarkers. Specifically, the object of this invention is to provide a therapeutic agent for arthritis and a method for manufacturing the aforementioned therapeutic agent, the therapeutic agent comprising synovial-derived mesenchymal stem cells containing molecules essential for joint treatment.

[0009] In order to solve the above-mentioned problems, the inventors conducted in-depth research and discovered that one or more of integrin β1 or platelet-derived growth factor receptor β are essential quality control markers for the efficacy of synovial stem cell therapy for joint diseases. This invention is based on the above findings.

[0010] That is, the following inventions are provided according to the present invention. <1> A treatment for arthritis comprising synovial-derived mesenchymal stem cells having surface antigens of either integrin β1 or platelet-derived growth factor receptor β. <2> The treatment for arthritis as described in <1>, wherein the synovial-derived mesenchymal stem cells have surface antigens of both integrin β1 and platelet-derived growth factor receptor β. <3> The treatment for arthritis as described in <1> or <2>, having a gene encoding a type II collagen α1 chain and producing a type II collagen α1 chain after transplantation. <4> The treatment for arthritis as described in any one of <1> to <3>, having a surface antigen of FGFR3. <5> The treatment for arthritis as described in any one of <1> to <4>, wherein the ratio of synovial-derived mesenchymal stem cells having surface antigens of either integrin β1 or platelet-derived growth factor receptor β to all cells contained in the treatment for arthritis is 30% or more. <6> A method for manufacturing an arthritis treatment agent according to any one of <1> to <5>, comprising: step A, treating synovial tissue with an enzyme; step B, washing the enzyme-treated mixture; step C, culturing synovial-derived mesenchymal stem cells contained in the washed mixture on a substrate; and step D, separating the cultured synovial-derived mesenchymal stem cells from the substrate. <7> The method according to <6>, wherein step B is a step of washing the enzyme-treated mixture until the residual enzyme concentration in the supernatant reaches less than 0.5 ng / mL. <8> The method according to <6> or <7>, wherein in step C, the culturing period of the synovial-derived mesenchymal stem cells is within 28 days. <9> The method according to any one of <6> to <8>, wherein in step D, the separation is performed by acting a cell-stripping solution on the mesenchymal stem cells for a period of no more than 120 minutes. <10> The method as described in any one of <6> to <9> further includes the step of sorting synovial-derived mesenchymal stem cells having any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β. [Effects of the Invention]

[0011] The arthritis treatment agent of the present invention exerts a therapeutic effect on arthritis by comprising synovial-derived mesenchymal stem cells containing one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β. According to the present invention, variations in the therapeutic effect of the manufactured arthritis treatment agent can be suppressed, and quality management of the product's therapeutic effect can be achieved.

Implementation Method

[0013] Hereinafter, the contents of the present invention will be described in detail. In this specification, "~" is used to mean that the values ​​recorded before and after it are included as lower and upper limits.

[0014] The arthropathy treatment agent of the present invention comprises synovial-derived mesenchymal stem cells having one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β (also referred to as PDGFRb in this specification). The synovial-derived mesenchymal stem cells may have only integrin β1 or platelet-derived growth factor receptor β, but preferably have both integrin β1 and platelet-derived growth factor receptor β.

[0015] Synovial-derived mesenchymal stem cells are preferably those that possess a gene encoding type II collagen α1 chains and produce type II collagen α1 chains after transplantation, thereby exerting a therapeutic effect. Synovial-derived mesenchymal stem cells are preferably those that possess the surface antigen FGFR3 (fibroblast growth factor receptor 3).

[0016] The ratio of synovial-derived mesenchymal stem cells containing any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β to all cells contained in the arthropathy treatment agent of the present invention is preferably 30% or more, and may also be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.

[0017] The arthritis treatment agent of the present invention can be manufactured by a method comprising: step A, treating synovial tissue with an enzyme; step B, washing the enzyme-treated mixture; step C, culturing synovial-derived mesenchymal stem cells contained in the washed mixture on a substrate; and step D, separating the cultured synovial-derived mesenchymal stem cells from the substrate.

[0018] <Step A of enzymatic treatment of synovial tissue> Synovial tissue can be collected from the non-load-bearing part of the joint under anesthesia. There are no particular limitations on the biological source of the synovial tissue; any organism can be used, preferably synovial tissue derived from mammals. For example, synovial tissue derived from primates (e.g., chimpanzees, Japanese macaques, humans) can be used, and synovial tissue derived from humans is particularly preferred.

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

[0020] When producing synovial-derived mesenchymal stem cells for the purpose of drug administration to humans, it is preferable to use synovial tissue collected from a donor whose tissue suitability antigen type is consistent with or similar to that of the recipient. More preferably, the recipient of the synovial tissue collection and the recipient of the synovial-derived mesenchymal stem cell transplantation are the same person. That is, it is preferable to use synovial tissue collected from the recipient itself (autologous transplantation). The amount of synovial tissue collected can be determined considering the type of donor or the necessary amount of synovial-derived mesenchymal stem cells. For example, synovial-derived mesenchymal stem cells can be obtained from 0.1g to 10g, preferably 0.1g to 2.0g, more preferably 0.1g to 1.5g, and even more preferably 0.1g to 1.0g of synovial tissue. The collected synovial tissue is chopped with scissors or the like as needed and then provided for the enzyme treatment described later.

[0021] 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 collagenases and one or more neutral proteases is preferred. A particularly preferred enzyme is Liberase (registered trademark). For example, Liberase MNP-S (manufactured by Roche Diagnostics KK) can be used as Liberase (registered trademark), which is an enzyme containing type I collagenase, type II collagenase, and thermolysin.

[0022] The enzyme reaction can be carried out in an aqueous solution containing the enzyme, or an aqueous solution containing human serum can be used. The human serum can be autologous serum or allogeneic serum, but autologous serum is preferred. 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, further preferably 0.5 mg / ml to 10 mg / ml, further 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. The mass ratio of synovial tissue to enzyme is preferably 1000:1 to 10:1, more preferably 500:1 to 20:1, and further preferably 200:1 to 40:1.

[0023] The enzyme reaction can be carried out at a temperature preferably from 15°C to 40°C, more preferably from 20°C to 35°C, and even more preferably from 25°C to 35°C. The reaction time is 2 hours or more, more preferably 2.5 hours or more, and even more preferably 3 hours or more. There is no particular upper limit to the reaction time, which can be within 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, or 4 hours. The enzyme-treated mixture contains synovial-derived mesenchymal stem cells. The enzyme-treated mixture can be used to recover synovial-derived mesenchymal stem cells by transferring it to a centrifuge tube through a cell filter and centrifuging it.

[0024] <Step B of washing the enzyme-treated mixture> In step B, the enzyme-treated mixture is washed. In step B, it is preferable to wash until the residual enzyme concentration in the supernatant reaches 0.5 ng / mL or less. More preferably, the residual enzyme concentration in the supernatant is 0.3 ng / mL or less, further preferably 0.2 ng / mL or less, and most preferably 0.1 ng / mL or less.

[0025] Washing can be performed by resuspending the synovial-derived mesenchymal stem cells recovered by centrifugation in a culture medium and centrifuging again (e.g., at 400g for 5 minutes). As the culture medium, α-modified Eagle Minimum Essential Medium (αMEM) can be used, but there are no particular limitations. As described above, washing can be performed multiple times (more than twice) using the culture medium.

[0026] <Step C: Culturing Synovial-Derived Mesenchymal Stem Cells in a Washed Mixture on a Substrate> In step C, synovial-derived mesenchymal stem cells in a washed mixture are cultured on a substrate. Examples of substrates include planar plastic substrates such as culture plates, and three-dimensional substrates such as culture bags, microcarriers, or gels, but there are no particular limitations. The culture medium used in the culture can be prepared using a medium commonly used for animal cell culture as the basal medium. Examples of media commonly used for animal cell culture include αMEM, DMEM (Dulbecco Modified Eagle Medium), a mixture of DMEM and F12 (DMEM:F12=1:1), RPMI medium (GIBCO RPMI1640 medium, etc.), and a mixture of DMEM / F12 and RPMI (DMEM / F12:RPMI=1:1), but there are no particular limitations.

[0027] The culture medium can be either a serum-containing medium or a serum-free medium. When producing synovial-derived mesenchymal stem cells from autologous tissue for the purpose of administering medication to an organism, the culture medium can contain allogeneic serum. That is, when producing synovial-derived mesenchymal stem cells from human tissue for the purpose of administering medication to a human, a culture medium containing human serum can be used. When using serum, it can be autologous serum or allogeneic serum, but autologous serum is preferred. When using serum, the amount of serum added to the culture medium is, for example, 20% by volume or less, 10% by volume or less, or 5% by volume or less.

[0028] There are no particular limitations on the cell culture conditions, and common cell culture conditions can be used. For example, culture at a temperature of 30-40°C and 3-7% CO2 can be used, but there are no particular limitations. As an example, culture at a temperature of 37°C and 5% CO2 can be used.

[0029] In this invention, it is preferable to culture without changing the culture medium. Furthermore, in the above-described culture, it is preferable to produce synovial-derived mesenchymal stem cells without co-culturing with cells other than synovial-derived mesenchymal stem cells.

[0030] It is known that the longer the culture period, the more synovial-derived mesenchymal stem cells differentiate into chondrocytes. Therefore, if the culture period exceeds a certain length, the in-situ chondrogenic capacity of synovial-derived mesenchymal stem cells will decrease. Therefore, in this invention, in order to promote the proliferation of synovial-derived mesenchymal stem cells in an undifferentiated state and with good in-situ chondrogenic capacity, regulating the culture period is preferable. In step C, the culture period of synovial-derived mesenchymal stem cells is preferably within 28 days.

[0031] Furthermore, in this invention, it is necessary to consider the necessity of preparing a sufficient number of undifferentiated synovial stem cells to cover the damaged cartilage and promote regeneration of the affected area. Therefore, a culture period of 5 days or more, 7 days or more, or 10 days or more is preferred, 10 to 14 days, 10 to 21 days or 10 to 28 days is even more preferred, and 10 to 21 days is further preferred.

[0032] It is known that mesenchymal stem cells can be cultured in a chondrogenic medium supplemented with transforming growth factor β3 (TGF-β3), dexamethasone, and bone morphogenetic protein 2 (BMP-2) to differentiate into chondrocytes, and cartilage tissue can be produced in vitro. Therefore, in this invention, in order to prevent synovial-derived mesenchymal stem cells from differentiating into chondrocytes, it is preferable to culture isolated synovial-derived mesenchymal stem cells in the absence of TGF-β3, dexamethasone, or BMP-2.

[0033] It is also known that synovial-derived mesenchymal stem cells have an inversely proportional success rate to the number of passages of in vitro mesenchymal stem cells, while their in situ chondrogenic capacity decreases. Therefore, to prepare undifferentiated mesenchymal stem cells, it is preferable to manufacture primary or first-passage synovial-derived mesenchymal stem cells.

[0034] In this invention, the serum used in autologous therapy is derived from the patient's own body. Since the amount of serum that can be collected from a donor is limited in autologous therapy, and from the viewpoint of synovial-derived mesenchymal stem cell proliferation, a certain cell density is required. Therefore, it is preferable to seed and culture enzyme-treated synovial-derived mesenchymal stem cells at cell densities of 100 cells / cm² to 5000 cells / cm² or less, 200 cells / cm² to 5000 cells / cm² or less, 500 cells / cm² to 5000 cells / cm² or less, 500 cells / cm² to 2500 cells / cm² or less, or 500 cells / cm² to 2000 cells / cm² or less. Furthermore, to promote the proliferation of synovial-derived mesenchymal stem cells after enzyme treatment, culturing for 10 days or more is more preferable.

[0035] It is preferable that the number of cells obtained at the end of the culture is 1.0×10 7 or more, 2.0×10 7 or more, 2.5×10 7 or more, or 3.0×10 7 or more, 4.0×10 7 or more is even better, 5.0×10 7 or more is further better, and 6.0×10 7 or more is particularly good.

[0036] <Step D: Separation of Cultured Synovial-Derived Mesenchymal Stem Cells from the Substrate> In step D, the cultured synovial-derived mesenchymal stem cells are separated from the substrate. Preferably, separation in step D involves applying a cell-stripping solution to the mesenchymal stem cells for no more than 120 minutes. The cell-stripping solution is a solution containing a trypsin-like enzyme and EDTA. A particularly preferred enzyme is TrypLE. For example, TrypL Express (manufactured by Gibco) or TrypLE Select (manufactured by Gibco) can be used as TrypLE.

[0037] From the viewpoint of thoroughly exfoliating cells, it is preferable to apply the cell exfoliation solution to the mesenchymal stem cells for at least 10 minutes. It is preferable to apply the cell exfoliation solution to the mesenchymal stem cells for 10 to 120 minutes, and more preferably for 10 to 60 minutes. The application time can be 10 to 50 minutes, 10 to 40 minutes, 20 to 60 minutes, 20 to 50 minutes, or 20 to 40 minutes.

[0038] Mesenchymal stem cells are somatic stem cells derived from mesodermal tissue (mesenchyma). 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 fascia cells. Regarding the differentiation of mesenchymal stem cells into chondrocytes, it is known that adding BMP or TGF-β to the culture medium can promote the differentiation of undifferentiated mesenchymal stem cells into chondrocytes, and cartilage tissue can regenerate under in vitro conditions.

[0039] Mesenchymal stem cells can be identified by detecting characteristic molecules (e.g., enzymes, receptors, low-molecular-weight compounds) within them. Examples of characteristic molecules in mesenchymal stem cells include cell surface markers (positive markers) such as CD73, CD90, CD105, and CD166, but these are not limited to these. Examples of negative markers not expressed in mesenchymal stem cells include CD19, CD34, CD45, HLA-DR, CD11b, and CD14, but these are not limited to these. Furthermore, CD stands for Clusters of Differentiation, and HLA-DR stands for Human Leukocyte Antigen-D-related. Using these positive and negative markers, it is possible to identify them as mesenchymal stem cells. These markers can be detected using immunological methods, but they can also be detected by quantifying the mRNA levels of each molecule.

[0040] In this specification, synovial-derived mesenchymal stem cells are stem cells contained in the synovium. Synovial-derived mesenchymal stem cells are a type of mesenchymal stem cell. Synovial-derived mesenchymal stem cells can be detected, for example, by detecting CD90 positivity, CD45 negativity, and chondrogenic differentiation capacity, but the detection method is not particularly limited.

[0041] When synovial-derived mesenchymal stem cells produced by the above method are used to make a treatment for arthritis, the cells can be mixed with a pharmaceutically permissible carrier using conventional methods to prepare a formulation suitable for individual administration. Examples of carriers include isotonic distilled water for injection containing physiological saline, glucose, or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Furthermore, buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzyl ammonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc., can also be incorporated.

[0042] Preferably, the method for manufacturing the arthritis treatment agent of the present invention may further include a sorting step: sorting synovial-derived mesenchymal stem cells having any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β.

[0043] As a step in sorting synovial-derived mesenchymal stem cells that have one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β, a step in managing the expression level of integrin β1 or platelet-derived growth factor receptor β can be cited.

[0044] The expression level of integrin β1 or platelet-derived growth factor receptor β refers to the expression level of the gene or protein of integrin β1 or platelet-derived growth factor receptor β. The expression level of integrin β1 or platelet-derived growth factor receptor β can be calculated as an absolute value or a relative value (comparison to control or baseline expression level, etc.).

[0045] The expression level of integrin β1 or platelet-derived growth factor receptor β can be determined by any method known to those skilled in the art and can be performed according to conventional methods. As a method for determining the expression level, the amount of mRNA as a gene transcription product can be measured. There are no particular limitations on the method for measuring the desired amount of mRNA, and a suitable method can be selected from known methods. For example, the following methods can be used: gene amplification method using oligonucleotides that hybridize with the gene encoding integrin β1 or platelet-derived growth factor receptor β as primers, or hybridization method using oligonucleotides (polynucleotides) that hybridize with the gene encoding a specific protein molecule as probes. Specifically, examples include RT-PCR (reverse transcription polymerase chain reaction), real-time RT-PCR, DNA microarray, cell array, Northern blot, dot speckling, RNase protection detection, etc.

[0046] Regarding the primers or probes used in the above-described assay method, the amount of mRNA can be determined by labeling and detecting the signal intensity of the label. Real-time RT-PCR is preferable because it allows direct use of RNA as a sample, optical measurement of the gene amplification process, and gene quantification based on the number of cycles required for amplification. Furthermore, as a control, the expression levels of mRNAs such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin, which are housekeeping genes, can be used to standardize the expression levels of genes encoding integrin β1 or platelet-derived growth factor receptor β. Moreover, regarding the primers and probes used in the above-described assay method, those skilled in the art can appropriately design and prepare them based on information about the base sequence of genes encoding integrin β1 or platelet-derived growth factor receptor β.

[0047] The expression levels of integrin β1 or platelet-derived growth factor receptor β can be determined, for example, by using an immunological assay with an antibody or antibody fragment targeting integrin β1 or platelet-derived growth factor receptor β. Specifically, flow cytometer, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent antibody assay, and cell array assay can be used. These assay methods can also be performed using standard protocols or appropriately modified or altered standard protocols.

[0048] For example, when measuring the expression level of integrin β1 or platelet-derived growth factor receptor β in cells by flow cytometer, if the positive rate of integrin β1 or platelet-derived growth factor receptor β is preferably 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, then synovial-derived mesenchymal stem cells with any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β can be sorted.

[0049] When sorting synovial-derived mesenchymal stem cells containing any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β, sorting can be performed, for example, by comparing the expression levels of integrin β1 or platelet-derived growth factor receptor β in the cells measured using the above method with a predetermined baseline expression level. The baseline expression level can, for example, be the expression level of integrin β1 or platelet-derived growth factor receptor β in cells confirmed to have a certain quality (positive control), or it can be the expression level in cells confirmed to not have a certain quality (negative control).

[0050] By comparing the expression levels of integrin β1 or platelet-derived growth factor receptor β with the baseline expression levels, cells with expression levels of integrin β1 or platelet-derived growth factor receptor β that are equal to or higher than the expression levels of the positive control can be selected and used as a therapeutic agent for arthritis.

[0051] Furthermore, a cutoff value for the expression level of integrin β1 or platelet-derived growth factor receptor β can be preset, and the expression level of integrin β1 or platelet-derived growth factor receptor β in the measured cells can be compared with the cutoff value. For example, based on a regression line representing the correlation between the expression level of integrin β1 or platelet-derived growth factor receptor β and the therapeutic effect, the cutoff value can be used as the expression level of integrin β1 or platelet-derived growth factor receptor β that imparts the desired therapeutic effect. For example, cells in which the expression level of integrin β1 or platelet-derived growth factor receptor β is above the cutoff value can be sorted out and used as a treatment agent for arthritis.

[0052] The arthritis treatment agent of the present invention can be used for joint treatment. As a joint treatment, it can be used to treat diseases associated with joint injury, damage, or inflammation, and can also be used to treat joint diseases caused by degeneration of connective tissues such as cartilage and / or inflammation, or non-inflammatory joint diseases. Examples of joint treatments include, but are not limited to, diseases selected from the group consisting of meniscus injury, traumatic cartilage injury, osteochondritis dissecans, non-septic osteonecrosis, deforming arthritis (e.g., deforming knee arthritis), rheumatoid arthritis (e.g., chronic rheumatoid arthritis), gout, reactive arthritis, atrophic arthritis, angiomyopathic arthritis, inflammatory arthritis, and articular cartilage defects.

[0053] The treatment method for joints using the arthritis treatment agent of the present invention includes the following steps: transplanting the arthritis treatment agent of the present invention by covering the cartilage injury site or the meniscus injury site with synovial-derived mesenchymal stem cells; and regenerating cartilage tissue in situ at the cartilage injury site or the meniscus injury site by differentiating the synovial-derived mesenchymal stem cells contained in the arthritis treatment agent into chondrocytes.

[0054] When the arthritis treatment agent of the present invention is transplanted to a patient, in order to effectively treat the cartilage injury or meniscus injury, it is preferable to apply 2.0×10 7 to 1.0×10 11, 2.5×10 7 to 1.0×10 11, 3.0×10 7 to 1.0×10 11, 4.0×10 7 to 1.0×10 11, 2.5×10 7 to 1.0×10 10, 2.5×10 7 to 1.0×10 9, or 2.5×10 7 to 1.0×10 8 synovial-derived mesenchymal stem cells, or 2.0×10 7 to 1.0×10 8 synovial-derived mesenchymal stem cells to each cartilage injury or meniscus injury.

[0055] By transplanting synovial-derived mesenchymal stem cells to the site of cartilage or meniscus injury, the site of cartilage or meniscus injury is covered by synovial-derived mesenchymal stem cells. Transplantation of synovial-derived mesenchymal stem cells can be performed via open surgery or arthroscopic surgery. To minimize invasiveness, arthroscopic transplantation of synovial-derived mesenchymal stem cells is preferred.

[0056] The cartilage injury site or the meniscus injury site can be covered by a suspension of synovial-derived mesenchymal stem cells, or by cell sheets of synovial-derived mesenchymal stem cells. For example, bioabsorbable gels such as gelatin or collagen can be used as gel-like substances. The synovial-derived mesenchymal stem cells have a high adhesion ability to the cartilage injury site or the meniscus injury site.

[0057] In treating cartilage damage, the low-invasive surgical procedure of the present invention is characterized by covering the cartilage injury site with synovial-derived mesenchymal stem cells, and includes the following steps: maintaining a position so that the cartilage injury site faces upward; placing a cell sheet of synovial-derived mesenchymal stem cells, a suspension of synovial-derived mesenchymal stem cells, or a gel containing synovial-derived mesenchymal stem cells on the surface of the cartilage injury site; and maintaining the position for a specific time, thereby allowing the synovial-derived mesenchymal stem cells to adhere to the surface of the cartilage injury site.

[0058] In treating meniscus injuries, the low-invasive surgical procedure of the present invention is characterized by covering the injured meniscus with synovial-derived mesenchymal stem cells, and includes the following steps: maintaining a position so that the injured meniscus faces downward; injecting a suspension of synovial-derived mesenchymal stem cells into the knee joint; and maintaining the position for a specific time to allow the synovial-derived mesenchymal stem cells to adhere to the injured meniscus.

[0059] To ensure reliable adhesion of synovial-derived mesenchymal stem cells to the surface of the cartilage injury site or meniscus injury site, the transplanted synovial-derived mesenchymal stem cells should be held on the surface of the cartilage injury site or meniscus injury site for at least 10 minutes, preferably 15 minutes. To achieve this, the position should be maintained with the cartilage injury site or meniscus injury site facing upwards and the synovial-derived mesenchymal stem cells held in the upward-facing cartilage injury site or meniscus injury site for at least 10 minutes, preferably 15 minutes.

[0060] To ensure a stronger attachment between the synovial-derived mesenchymal stem cells and the cartilage or meniscus injury, the cartilage or meniscus injury site accompanied by the synovial-derived mesenchymal stem cells can be further covered with periosteum. The procedure is completed after the synovial-derived mesenchymal stem cells are held on the surface of the cartilage or meniscus injury for at least 10 minutes.

[0061] In this invention, the transplanted synovial-derived mesenchymal stem cells are divided into chondrocytes at the site of cartilage injury or meniscus injury, and then the cartilage tissue is regenerated in situ at the site of cartilage injury or meniscus injury.

[0062] During the in situ chondrogenesis of synovial-derived mesenchymal stem cells, cartilage tissue regenerates according to the local microenvironment (nutrient supply and cytokine environment, etc.), thus eliminating the need for external manipulation. Regarding the results of in situ chondrogenesis of synovial-derived mesenchymal stem cells, cartilage tissue regenerates at the site of cartilage injury or meniscus injury, repairing the damage. Moreover, in the case of cartilage injury, the bone region, the boundary between cartilage and bone, the central part of the cartilage, the surface region, and the region adjacent to the original cartilage are formed in the form of the original cartilage tissue, or in the case of meniscus injury, meniscal cartilage is formed.

[0063] The present invention will be further described in detail through the following embodiments, but the present invention is not limited to the embodiments. [Embodiments]

[0064] <Example 1> Preparation of Rat Synovial Derivative Mesenchymal Stem Cells LEW / CrlCrlj rats were used to establish rat synovial derivative mesenchymal stem cells. Collagenase V (Sigma Cat. No. C9263) was added to αMEM no nucleosides (Gibco Cat. No. 12561056) medium to achieve a concentration of 2 or 3 mg / mL in synovial tissue collected under isoflurane anesthesia, and the reaction was carried out at 37°C for 2 hours. Cooled medium was added to stop the reaction, and residual tissue was removed through a 40 μm cell filter. The recovered cells were seeded into cell culture flasks and cultured with αMEM no nucleosides at 37°C with a CO2 concentration of 5%. The αMEM no nucleosides contained Fetal Bovine Serum (Gibco Cat. No. 10270106) to achieve a final concentration of 20%, L-glutamine 200 mmol / L (Gibco Cat. #25030081) to achieve a final concentration of 1%, and Antibiotic-Antimycotic (100X) (Gibco Cat. No. 15240062) to achieve a final concentration of 1%. After 8 days of culture, the culture medium in the flask was discarded, and the cells were washed twice with PBS (phosphate-buffered saline). TrypLE Express (Gibco Cat. No. 12604-013) was added, and the cells were incubated at 37°C for 5 minutes. The cells were then recovered as synovial-derived mesenchymal stem cells. The supernatant was discarded by centrifugation and replaced with COS-banker (COSMO BIO Cat. No. COS-CFM01) to prepare cryopreservation medium for rat synovial-derived mesenchymal stem cells.

[0065] <Example 2> The extracellular matrix adhesion ability of rat synovial-derived mesenchymal stem cells was inhibited by inhibiting integrin β1. As a preparation of rat synovial-derived mesenchymal stem cells with inhibited integrin β1, the cryopreservation solution of rat synovial-derived mesenchymal stem cells prepared in Example 1 was activated and thawed. After culturing in αMEM no nucleosides at 37°C with a CO2 concentration of 5% for 1 week, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. The αMEM no nucleosides contained Fetal Bovine Serum to achieve a final concentration of 20%, L-glutamine 200 mmol / L to achieve a final concentration of 1%, and Antibiotic-Antimycotic (100X) to achieve a final concentration of 1%. Cells (integrin β1-rSMSCs) were recovered after reacting with 12 μg of Purified anti-mouse / rat CD29 Antibody (BioLegend Cat. No. 102202) at 5 × 10⁶ cells per cell number under ice for 1 hour. As a control without integrin β1 inhibition, Purified Armenian Hamster IgG Isotype Ctrl (BioLegend Cat. No. 400902) cells were recovered after reacting with ice for 1 hour. Untreated rSMSCs (non-treated rSMSCs) were also prepared using only the reaction solvent and then used for subsequent treatments.

[0066] To confirm the inhibition of integrin β1, the adhesion function of the extracellular matrix, one of the functions of integrin β1, was investigated. For the extracellular matrix adhesion reaction, cells were washed with 10 mmol / L PBS containing MgCl₂·6H₂O (hereinafter PBS(+)), and then resuspended in PBS(+) to a density of 1×10⁵ cells / 10 μL / well. These cells were then seeded into Collagen Type I Cellware 8-Well Culture Slides (Corning Cat. No. 354630). After standing at room temperature for 10 minutes, the cells were washed with PBS(+). Subsequently, the cells were observed under a microscope (OLIMPUS Cat. No. IX71) at 10x objective. An image of the field of view showing the most adhered cells was obtained, and the number of adhered cells was calculated.

[0067] The results are shown in Figure 1. Regarding the number of cells adhering to the membrane, the number of non-treated rSMSCs was 1637 cells, IgG-rSMSCs was 1214 cells, and integrin β1-rSMSCs was 194 cells. It was observed that the number of cells adhering to the membrane was significantly reduced by inhibiting integrin β1. This confirms that treatment with Purified anti-mouse / rat CD29 Antibody can inhibit integrin β1 in rat synovial stem cells.

[0068] <Example 3> The ability of rat synovial-derived mesenchymal stem cells to inhibit cell proliferation by inhibiting PDGFRb was used to prepare rat synovial-derived mesenchymal stem cells with inhibited PDGFRb. The cryopreservation solution prepared in Example 1 was activated and thawed, and after being cultured in αMEM no nucleosides at 37°C with a CO2 concentration of 5% for 1 week, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. Fetal Bovine Serum was added to the αMEM no nucleosides to achieve a final concentration of 20%, L-glutamine 200 mmol / L was added to achieve a final concentration of 1%, and Antibiotic-Antimycotic (100X) was added to achieve a final concentration of 1%.

[0069] For each cell number of 1 × 10⁶ cells, 40 and 120 μg of Anti-PDGF Receptorβ Human Goat-Poly (R&D Systems Cat. No. AF385) were reacted at ice temperature for 1 hour, and then seeded at 1000 cells / well in 96-well plates (Corning Cat No. 353072) and cultured at 37°C with a CO₂ concentration of 5% (PDGFRb-rSMSC). As a control treatment without PDGFRb inhibition, Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was reacted at ice temperature for 1 hour and then seeded at 1000 cells / well in 96-well plates (IgG-rSMSC). Additionally, Non-treated rSMSCs were also established, where cells were seeded at 1000 cells / well in 96-well plates without any reaction.

[0070] To confirm that PDGFRb was inhibited, the cell proliferation capacity, which is a function of PDGFRb, was examined. Cells were cultured at 37°C with a CO2 concentration of 5% and on day 6, cell proliferation was quantitatively evaluated using the ATP assay of CellTiter-Glo (Promega Cat. No. G7571).

[0071] The results are shown in Figure 2a. The ATP concentration of 40 μg / mL PDGFRb-rSMSC was 3.78 ± 0.84 μmol / L, the ATP concentration of 120 μg / mL PDGFRb-rSMSC was 4.12 ± 1.29 μmol / L, the ATP concentration of IgG-rSMSC was 6.08 ± 0.63 μmol / L, and the ATP concentration of Non-treated-rSMSC was 6.81 ± 0.82 μmol / L. It was observed that cell proliferation was significantly inhibited by PDGFRb inhibition. This confirms that treatment with Anti-PDGF Receptorβ Human Goat-Poly can inhibit the proliferation of rat synovial stem cells.

[0072] To confirm the ligand specificity of PDGFRb, as a preparation of PDGFRb-inhibited rat synovial-derived mesenchymal stem cells, the cryopreservation solution prepared in Example 1 was activated and thawed. αMEM no nucleosides were added to achieve a final concentration of 20% Fetal Bovine Serum, 1% L-glutamine (200 mmol / L), and 1% Antibiotic-Antimycotic (100X). After culturing at 37°C with a CO2 concentration of 5% for 1 week, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent.

[0073] At a cell number of 1×10⁶ cells, 10, 20, and 40 μg of Anti-PDGF Receptorβ Human Goat-Poly (R&D Systems Cat. No. AF385) were reacted at ice-cold for 1 hour, and then seeded at 1000 cells / well in 96-well plates (Corning Cat No. 353072) and cultured at 37°C with a CO₂ concentration of 5% (PDGFRb-rSMSC). As a control treatment without PDGFRb inhibition, Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was reacted at ice-cold for 1 hour and then seeded at 1000 cells / well in 96-well plates (IgG-rSMSC). Additionally, Non-treated-rSMSCs were also established, with cells seeded at only 1000 cells / well in 96-well plates without any reaction.

[0074] On the second day after cell seeding, the culture supernatant was discarded, and the culture medium was replaced with αMEM no nucleosides (Gibco Cat.No.10270106). This αMEM no nucleosides contained Fetal Bovine Serum to achieve a final concentration of 0.5%, L-glutamine 200 mmol / L to achieve a final concentration of 1%, Antibiotic-Antimycotic (100X) to achieve a final concentration of 1%, and PDGF-BB,Rat,Recombinant (R&D Systems Cat.No.520-BB-050) to achieve a final concentration of 4 ng / mL. For experimental levels, Anti-PDGF Receptorβ Human Goat-Poly (R&D Systems Cat.No.AF385) was added to achieve final concentrations of 10, 20, and 40 μg / mL, respectively, bringing the total culture medium volume to 100 μL. For the positive control, Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was added to bring the total culture medium volume to 100 μL. For the negative control, no antibody was added; only 100 μL of culture medium was added. On day 6 of culture, cell proliferation was quantitatively evaluated using the ATP assay with CellTiter-Glo (Promega Cat. No. G7571).

[0075] The results are shown in Figure 2b. The ATP concentration of 10 μg / mL PDGFRb-rSMSC was 0.62 ± 0.12 μmol / L, 20 μg / mL PDGFRb-rSMSC was 0.65 ± 0.05 μmol / L, 40 μg / mL PDGFRb-rSMSC was 0.24 ± 0.05 μmol / L, IgG-rSMSC was 0.49 ± 0.17 μmol / L, and Non-treated-rSMSC was 0.24 ± 0.11 μmol / L. It was observed that 40 μg / mL PDGFRb-rSMSC significantly reduced the proliferation of IgG-rSMSC cells. This confirms that treatment with Anti-PDGF Receptorβ Human Goat-Poly can inhibit PDGFRb in rat synovial stem cells in a ligand-specific manner.

[0076] <Example 4> Production of Col2A1-deleted rat synovial-derived mesenchymal stem cells The rat synovial-derived mesenchymal stem cells produced in Example 1 underwent a Col2A1 gene deletion operation, and the deletion of the Col2A1 gene was confirmed by Sanger sequencing analysis. The Col2A1 base sequences of wild-type (Col2A1WT-rSMSC) and deleted-type (Col2A1KO-rSMSC) rat synovial stem cells are shown in Figures 3, 4, 5, and 6, and the amino acid sequences translated based on these sequences are shown in Figures 7, 8, and 9. The Col2A1 base sequence of wild-type (Col2A1WT-rSMSC) rat synovial stem cells is shown in sequence number 1; the Col2A1 base sequence of one chromosome of Col2A1-deleted (Col2A1KO-rSMSC) rat synovial stem cells is shown in sequence number 2; and the Col2A1 base sequence of the other chromosome of Col2A1-deleted (Col2A1KO-rSMSC) rat synovial stem cells is shown in sequence number 3. The amino acid sequence of wild-type (Col2A1WT-rSMSC) is shown in sequence number 4; and the amino acid sequence of Col2A1-deleted (Col2A1KO-rSMSC) is shown in sequences number 5 and 6. The results revealed that in Col2A1KO-rSMSCs, starting from the amino acid translation start codon ATG, there were DNA sequences with deletions of bases 55 through 62 and heteroframeshift mutants with an insertion at base 59. One allele mutated from the 19th amino acid sequence due to base deletion and inserted a stop codon at 29, resulting in a mutant sequence that should have contained 1419 amino acids but was translated into 28 amino acid residues. Another allele mutated from the 20th amino acid sequence due to base insertion and inserted a stop codon at 50, resulting in a mutant sequence that should have contained 1419 amino acids but was translated into 49 amino acid residues. The mutant's base sequence was determined to be capable of producing synovial stem cells (Col2A1KO-rSMSCs) with a gene sequence that lacks Col2A1 function because the triple helix structure, which is an important functional domain of Col2A1, was not translated.

[0077] <Comparative Example 1> Production of CD120a-deleted rat synovial-derived mesenchymal stem cells: The rat synovial-derived mesenchymal stem cells produced in Example 1 were subjected to CD120a gene deletion, and the deletion of the CD120a gene was confirmed by Sanger sequencing analysis. The CD120a base sequences of wild-type (CD120aWT-rSMSC) and deleted (CD120aKO-rSMSC) rat synovial stem cells, and the amino acid sequences translated based on these sequences, are shown in Figures 10 and 11. The CD120a base sequences of rat synovial stem cells of wild-type (CD120aWT-rSMSC) and deletion type (CD120aKO-rSMSC) are shown in sequence number 7, the amino acid sequences of wild-type (CD120aWT-rSMSC) are shown in sequence number 8, and the amino acid sequences of deletion type (CD120aKO-rSMSC) are shown in sequence number 9. The results showed that in CD120aKO-rSMSC, starting from the amino acid translation start codon ATG, it is a frameshift mutant with DNA having the 16th base deleted. Due to the base deletion, the sequence mutates starting from the 6th amino acid and inserts the 19th start codon, thus revealing a mutant sequence that should have 461 amino acids but is now translated into 19 amino acid residues. The mutant's base sequence was determined to be a synovial stem cell (CD120aKO-rSMSC) with a gene sequence that has lost CD120a function because the sequence constituting the protein region of CD120a was not translated.

[0078] <Comparative Example 2> Production of CD106-deleted rat synovial-derived mesenchymal stem cells: The rat synovial-derived mesenchymal stem cells produced in Example 1 were subjected to CD106 gene deletion, and the deletion of the CD106 gene was confirmed by Sanger sequencing analysis. The CD106 base sequences of wild-type (CD106WT-rSMSC) and deleted (CD106KO-rSMSC) rat synovial stem cells, and the amino acid sequences translated based on these sequences, are shown in Figures 12 and 13. The CD106 base sequences of rat synovial stem cells of wild-type (CD106WT-rSMSC) and deletion type (CD106KO-rSMSC) are shown in sequence number 10, the amino acid sequence of wild-type (CD106WT-rSMSC) is shown in sequence number 11, and the amino acid sequence of deletion type (CD106KO-rSMSC) is shown in sequence number 12. The results showed that in CD106KO-rSMSC, starting from the amino acid translation start codon ATG, it is a frameshift mutant with DNA containing the deletion of bases 1059 to 1076. Due to the base deletion, the sequence mutated starting from amino acid 354, and a terminal codon was inserted at 356. Therefore, the mutant sequence, which should have contained 739 amino acids, was now translated into 355 amino acid residues. The mutant's base sequence was determined to be capable of producing synovial stem cells (CD106KO-rSMSCs) with a gene sequence that lacks CD106 function because the sequence of amino acids 699 to 720, which is the transmembrane region of CD106, was not translated.

[0079] <Example 5> Inhibition of chondrogenic differentiation capacity in rat synovial-derived mesenchymal stem cells lacking Col2A1. Col2A1 is one of the components of cartilage. To confirm Col2A1 deficiency at the cellular functional level, the chondrogenic differentiation capacity of Col2A1KO-rSMSO was tested. 2.5 × 10⁵ Col₂A₁KO-rSMSCs prepared in Example 4 were suspended in DMEM high glucose (Thermo Cat. No. 11965092) and centrifuged at 450g for 10 minutes. The cells were then cultured at 37°C with 5% CO₂ to induce chondrocyte differentiation. The DMEM high glucose solution contained TGF-β3 (R&DSystems Cat. No. 243-B3-002) to achieve a final concentration of 10 ng / mL, Dexamethasone (Wako Cat. No. 041-18861) to achieve a final concentration of 3.92 μg / mL, L-Ascorbic Acid 2-phosphate (Cayman Chemical Cat. No. 16457) to achieve a final concentration of 50 μg / mL, and L-proline (MP Biomedicals) to achieve a final concentration of 50 μg / mL. The final concentration of cell blocks was 40 μg / mL with the addition of sodium pyruvate (Invitrogen Cat. No. 11360070) to achieve a final concentration of 1 μg / mL, ITS-X supplement (x100) (Wako Cat. No. 094-06761) to achieve a final concentration of 1%, and BMP-2 (R&D Systems Cat. No. 355-BM-010) to achieve a final concentration of 0.5 μg / mL. As a control cell, chondrogenic differentiation was induced in the same manner as with Col2A1WT-rSMSCs. After 3 weeks of culture, the diameter and weight of cell blocks were measured, and the chondrogenic capacity was evaluated based on histological staining of the cell blocks. The results are shown in Figure 14. In Col2A1WT-rSMSCs, the short diameter was 1.55±0.14 mm, the long diameter was 2.04±0.25 mm, and the weight was 1.9±0.26 mg. In Col2A1KO-rSMSCs, the short diameter was 0.54±0.04 mm, the long diameter was 0.76±0.18 mm, and the weight was 0.85±0.4 mg, indicating a significant reduction in cartilage size and weight. Col2A1 is a component of cartilage, thus the reduction in cartilage size and weight can be seen due to the absence of Col2A1.Furthermore, it was found that safranin O-fast green staining and type II collagen immunostaining were disappearing in Col2A1KO-rSMSCs. This indicates that Col2A1 deficiency not only eliminates the ability to produce type II collagen but also affects the production of mucopolysaccharide-rich cartilage matrix. This suggests that type II collagen not only contributes to the formation of cartilage skeletal structure but also plays a role in inducing cartilage differentiation / matrix production.

[0080] <Comparative Example 3> Inhibition of chondrogenic differentiation capacity in rat synovial-derived mesenchymal stem cells lacking CD120a was investigated. The chondrogenic differentiation capacity of CD120aKO-rSMSO prepared in Comparative Example 1 was examined. Chondrogenic differentiation induction was performed under the same differentiation medium and culture conditions as in Example 5. As a control cell, chondrogenic differentiation was induced in the same manner as CD120aWT-rSMSC. After 3 weeks of culture, the diameter and weight of cell blocks were measured, and the chondrogenic differentiation capacity was evaluated based on histological staining of the cell blocks. The results are shown in Figure 15. In CD120aWT-rSMSCs, the short diameter was 1.55±0.14 mm, the long diameter was 2.04±0.25 mm, and the weight was 1.9±0.26 mg. In CD120aKO-rSMSCs, the short diameter was 1.19±0.17 mm, the long diameter was 1.53±0.04 mm, and the weight was 1.55±1.20 mg. No significant reduction in cartilage size or weight was observed. Furthermore, no difference in the staining properties of Safranin O-Fast Green and type II collagen immunostaining caused by CD120a deficiency was observed. Therefore, it is considered that CD120a is not a molecule that contributes to the cartilage differentiation capacity of cells.

[0081] <Comparative Example 4> Inhibition of chondrogenic differentiation capacity in rat synovial-derived mesenchymal stem cells lacking CD106 was investigated. The chondrogenic differentiation capacity of CD106KO-rSMSO prepared in Comparative Example 2 was examined. Chondrogenic differentiation induction of 2.5 × 10⁵ cells of CD106KO-rSMSC was performed under the same differentiation medium and culture conditions as in Example 5. As a control cell, chondrogenic differentiation was induced in the same manner as CD106WT-rSMSC. After 3 weeks of culture, the diameter and weight of cell blocks were measured, and the chondrogenic differentiation capacity was evaluated based on histological staining of the cell blocks. The results are shown in Figure 16. In CD106WT-rSMSCs, the short diameter was 1.55±0.14 mm, the long diameter was 2.04±0.25 mm, and the weight was 1.9±0.26 mg. In CD106KO-rSMSOs, the short diameter was 1.58±0.56 mm, the long diameter was 1.75±0.52 mm, and the weight was 2.38±1.54 mg. No significant reduction in cartilage size or weight was observed. Furthermore, no difference in the staining properties of Safranin O-Fast Green and type II collagen immunostaining caused by CD106 deficiency was observed. Therefore, CD106 is not considered a molecule that contributes to the cartilage differentiation capacity of cells.

[0082] <Example 6> Confirmation of the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with inhibited integrin β1. As described in Example 2, rat synovial-derived mesenchymal stem cells with inhibited integrin β1 were prepared. After activating and thawing cryopreservation medium and culturing for 1 week, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. For a cell count of 5 x 10⁶ cells, 12 μg of Purified anti-mouse / rat integrin β1 Antibody was added, and the cells were reacted at ice temperature for 1 hour before being recovered for transplantation (integrin β1-rSMSC). As a control without inhibition, Purified Armenian Hamster IgG Isotype Ctrl was reacted at ice temperature for 1 hour before being recovered for transplantation (IgG-rSMSC).

[0083] LEW / CrlCrlj rats were used in the creation of a meniscus injury model to evaluate the regeneration effect of the meniscus. In the method of meniscus injury and transplantation of mesenchymal stem cells, the skin of the knee joint was incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule below the knee was exposed, and a longitudinal incision was made with a scalpel to expose the cartilage at the distal end of the femur. The medial meniscus was detached from the synovium to expose the medial meniscus, and about 2 / 3 of the entire meniscus was removed. The patellar tendon and synovium were sutured, followed by suturing of the muscles to create a meniscus injury model. Subsequently, the treated animals were divided into 3 groups, and 5 x 10⁶ cells of integrin β1-inhibited synovial stem cells (integrin β1-rSMSC), 5 x 10⁶ cells of uninhibited control synovial stem cells (IgG-rSMSC), and solvent alone were injected into the joint capsule. The day after cell injection, 12 μg of Purified anti-mouse / rat integrin β1 Antibody, Armenian Hamster IgG Isotype Ctrl, or solvent was injected into the joint capsule per knee. After treatment, all rats were returned to their cages and allowed to move and eat freely.

[0084] Three weeks after treatment, the animals were euthanized under isoflurane anesthesia by exsanguination of the inferior aorta. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial menisci from both knee joints are shown in Figure 17. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled in dashed. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. In the IgG-rSMSC group (positive control), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion was larger. On the other hand, in the integrin β1-rSMSC group (cells with suppressed or missing molecules), the observed regenerated portion of the meniscus was smaller compared to the positive control.

[0085] In order to quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 17, the area of ​​the meniscus regeneration portion (within the dashed line) was calculated using ImageJ (version 1.52) by the following formula.

[0086] Meniscus regeneration area (mm²) = Number of pixels in the meniscus regeneration area / Number of pixels per 1 mm²

[0087] The mean, standard deviation, and statistical analysis of the regenerated area of ​​each group were performed using Microsoft Excel 2007 (Microsoft Corp.). For statistical analysis, the Student's T-Test was performed twice for the positive control group and the molecular inhibition group, and for the molecular inhibition group and the solvent group, and the p-values ​​were calculated. Since the test was repeated, Bonferroni correction was applied and the calculated p-values ​​were multiplied by the number of tests (2). For the significance level, 5% (α=0.05) was set as the presence of a difference, and the results are shown in Table 1.

[0088] Regarding the average area of ​​the regenerated meniscus recorded in Table 1, the integrin β1-rSMSC group (2.1 mm²) was significantly reduced compared to the IgG-rSMSC group (3.4 mm²). On the other hand, the integrin β1-rSMSC group had a similar regeneration area compared to the solvent group (1.7 mm²). This confirms that integrin β1 molecules in synovial stem cells are important molecules that contribute to meniscus regeneration.

[0089] [Table 1] Table 1: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) Integrin β1-rSMSC 2.1±0.5* IgG-rSMSC 3.4±1.0 solvent 1.7±0.9 *P<0.05 vs IgG-rSMSC group

[0090] <Example 7> Confirmation of the meniscus regeneration effect of PDGFRb-inhibited rat synovial-derived mesenchymal stem cells As described in Example 3, the preparation of PDGFRb-inhibited rat synovial-derived mesenchymal stem cells was performed. After activating and thawing cryopreservation medium and culturing for 1 week, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. At a cell count of 5 x 10⁶ cells, 12 μg of Anti-PDGF Receptor β Human Goat-Poly (R&D Systems Cat. No. AF385) was reacted at ice temperature for 1 hour before being recovered for transplantation (PDGFRb-rSMSC). As a control treatment without inhibition, Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was reacted at ice temperature for 1 hour and then recovered for transplantation (IgG-rSMSC).

[0091] A meniscus injury model for evaluating meniscus regeneration was created as described in Example 6. The treated animals were then divided into three groups, with 5 x 10⁶ cells of PDGFRb-inhibited synovial stem cells (PDGFRb-rSMSC), 5 x 10⁶ cells of uninhibited control synovial stem cells (IgG-rSMSC), and solvent alone injected into the joint capsule, respectively. The day after cell injection, 12 μg of Anti-PDGF Receptorβ Human Goat-Poly, Normal Goat IgG Control, or solvent was injected into the joint capsule per knee. After treatment, all rats were returned to their cages and allowed free movement and feeding.

[0092] Three weeks after treatment, the animals were euthanized under isoflurane anesthesia by severing the inferior aorta and bleeding them out. The meniscus was then exposed from the knee joint, the medial meniscus was removed, and photographs were taken. Images of the removed medial menisci from both knee joints are shown in Figure 18. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled with a dashed line. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. In the IgG-rSMSC group (positive control), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion was larger. On the other hand, in the PDGFRb-rSMSC group (cells with suppressed or missing molecules), the observed regenerated portion of the meniscus was smaller compared to the positive control.

[0093] To quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 18, the area of ​​the meniscus regeneration portion (within the dashed line) was measured as described in Example 6. The results are shown in Table 2. Regarding the average area value of the meniscus regeneration portion, the PDGFRb-rSMSC group (2.2 mm²) was significantly reduced compared to the IgG-rSMSC group (3.2 mm²). On the other hand, the PDGFRb-rSMSC group was significantly increased compared to the solvent group (1.3 mm²). This confirms that the PDGFRb molecule in synovial stem cells is a molecule that contributes to meniscus regeneration.

[0094] [Table 2] Table 2: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) PDGFRb-rSMSC 2.2±0.9*† IgG-rSMSC 3.2±0.7 solvent 1.3±1.2 *P<0.05 vs IgG-rSMSC group†P<0.05 vs solvent group

[0095] <Comparative Example 5> Confirmation of the meniscus regeneration effect of CD44-inhibited rat synovial-derived mesenchymal stem cells: As a preparation of CD44-inhibited rat synovial-derived mesenchymal stem cells, after activation, thawing, and cryopreservation in a 1-week culture, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. At a cell count of 5 x 10⁶ cells, 12 μg of Anti-CD44 Rabbit IgG clone Hermes-1 (Absolute Antibody Cat. No. Ab00628-23.0) was reacted at ice temperature for 30 minutes before being recovered for transplantation (CD44-rSMSC). As a control without inhibition, Rabbit IgG Isotype Control (invitrogen Cat. No. 10500C) was reacted at ice temperature for 1 hour and then recovered for transplantation (IgG-rSMSC).

[0096] A meniscus injury model for evaluating meniscus regeneration was created as described in Example 6. The treated animals were then divided into three groups, with 5 x 10⁶ cells of CD44-inhibited synovial stem cells (CD44-rSMSC), 5 x 10⁶ cells of uninhibited control synovial stem cells (IgG-rSMSC), and solvent alone injected into the joint capsule, respectively. The day after cell injection, 12 μg of Anti-CD44 Rabbit IgG clone Hermes-1, Rabbit IgG Isotype Control, or solvent was injected into the joint capsule per knee. After treatment, all rats were returned to their cages and allowed free movement and feeding.

[0097] Four weeks after treatment, the animals were euthanized under isoflurane anesthesia by exsanguination of the inferior aorta. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial menisci from both knee joints are shown in Figure 19. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled with a dashed line. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. In the IgG-rSMSC and CD44-rSMSC groups (positive controls), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion was larger.

[0098] To quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 19, the area of ​​the meniscus regeneration portion (within the dashed line) was measured as described in Example 6. The results are shown in Table 3. Regarding the average area value of the meniscus regeneration portion, there was no significant difference between the IgG-rSMSC group's 4.0 mm² and the CD44-rSMSC group's 3.4 mm², indicating they were of similar size. On the other hand, the CD44-rSMSC group showed a significant increase compared to the solvent group's 2.7 mm². Therefore, it is concluded that CD44 in synovial stem cells is not a molecule that contributes to meniscus regeneration.

[0099] [Table 3] Table 3: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) CD44-rSMSC 3.4±0.9† IgG-rSMSC 4.0±1.0 solvent 2.7±0.7 †P < 0.05 vs solvent group

[0100] <Example 8> Confirmation of the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells lacking Col2A1 (Col2A1KO-rSMSC): As described in Example 4, rat synovial-derived mesenchymal stem cells lacking Col2A1 were adjusted by expansion culture after Col2A1 deficiency was confirmed and used for transplantation (Col2A1KO-rSMSC). After confirming the wild-type Col2A1 sequence as a positive control, they were adjusted by expansion culture and used for transplantation (Col2A1WT-rSMSC).

[0101] A meniscus injury model for evaluating the regeneration effect of meniscus was prepared as described in Example 6. The treated animals were then divided into 3 groups, and Col2A1KO-rSMSC, Col2A1WT-rSMSC 5x10 6 cells, and solvent alone were injected into the joint capsule, respectively.

[0102] Three weeks after treatment, the animals were euthanized under isoflurane anesthesia by exsanguination of the inferior aorta. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial menisci from both knee joints are shown in Figure 20. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled in dashed. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. In the Col2A1WT-rSMSC group (positive control), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion was larger. On the other hand, in the Col2A1KO-rSMSC group, the observed regenerated portion was smaller compared to the positive control.

[0103] To quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 20, the area of ​​the meniscus regeneration portion (within the dashed line) was measured as described in Example 6. The results are shown in Table 4. Regarding the average area value of the meniscus regeneration portion, the Col2A1WTrSMSC group significantly increased to 3.8 mm² compared to 2.9 mm² in the Col2A1KO-rSMSC group. On the other hand, the Col2A1KO-rSMSC group also showed a significant increase compared to 2.1 mm² in the solvent group. This confirms that the Col2A1 molecule in synovial stem cells is a molecule that contributes to meniscus regeneration.

[0104] [Table 4] Table 4: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) Col2A1KO-rSMSC 2.9±0.8*† Col2A1WT-rSMSC 3.8±0.6 solvent 2.1±1.0 *P<0.05 vs IgG-rSMSC group†P<0.05 vs solvent group

[0105] <Comparative Example 6> Confirmation of the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with CD120a deletion (CD120aKO-rSMSC): As described in Comparative Example 1, rat synovial-derived mesenchymal stem cells with CD120a deletion were adjusted by expansion culture after confirming CD120a deletion and used for transplantation (CD120aKO-rSMSC). After confirming the CD120a wild-type sequence as a positive control, they were adjusted by expansion culture and used for transplantation (CD120aWT-rSMSC).

[0106] A meniscus injury model for evaluating the regeneration effect of meniscus was prepared as described in Example 6. Subsequently, the treated animals were divided into 3 groups, and CD120aKO-rSMSC, CD120aWT-rSMSC 5x10 6 cells, and solvent alone were injected into the joint capsule respectively.

[0107] Three weeks after treatment, the animals were euthanized under isoflurane anesthesia by exsanguination of the inferior aorta. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial menisci from both knee joints are shown in Figure 21. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled with a dashed line. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. On the other hand, in the CD120aWT-rSMSC and CD120aKO-rSMSC groups (positive controls), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion was larger compared to the solvent group.

[0108] To quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 21, the area of ​​the meniscus regeneration portion (within the dashed line) was measured as described in Example 6. The results are shown in Table 5. Regarding the average area value of the meniscus regeneration portion, no significant difference was observed between the CD120aKO-rSMSC group (3.1 mm²) and the CD120aWT-rSMSC group (3.3 mm²). On the other hand, the CD120aKO-rSMSC group showed a significant increase compared to the solvent group (1.8 mm²). Therefore, it is concluded that CD120a in synovial stem cells is not a molecule that contributes to meniscus regeneration.

[0109] [Table 5] Table 5: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) CD120aKO-rSMSC 3.1±0.7† CD120aWT-rSMSC 3.3±1.6 solvent 1.8±0.5 †P < 0.05 vs solvent group

[0110] <Comparative Example 7> Confirmation of the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells lacking CD106 (CD106aKO-rSMSC): As described in Comparative Example 1, rat synovial-derived mesenchymal stem cells lacking CD106 were adjusted by expansion culture after confirming CD106 deficiency and then used for transplantation (CD106KO-rSMSC). As a positive control, after confirming the CD106 wild-type sequence, they were adjusted by expansion culture and then used for transplantation (CD106WT-rSMSC).

[0111] A meniscus injury model for evaluating the regeneration effect of meniscus was prepared as described in Example 6. The treated animals were then divided into 3 groups, and CD106KO-rSMSC, CD106WT-rSMSC 5x10 6 cells, and solvent alone were injected into the joint capsule respectively.

[0112] Three weeks after treatment, the animals were euthanized under isoflurane anesthesia by severing the inferior aorta and bleeding them. The meniscus was then exposed from the knee joint, the medial meniscus was removed, and photographs were taken. Images of the removed medial menisci from both knee joints are shown in Figure 22. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled with a dashed line. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. On the other hand, in the CD106WT-rSMSC and CD106KO-rSMSC groups (positive controls), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion of the meniscus was larger compared to the solvent group.

[0113] To quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 22, the area of ​​the meniscus regeneration portion (within the dashed line) was measured as described in Example 6. The results are shown in Table 6. Regarding the average area value of the meniscus regeneration portion, no significant difference was observed between the CD106WT-rSMSC group (3.3 mm²) and the CD106KO-rSMSC group (3.7 mm²). On the other hand, the CD106KO-rSMSC group showed a significant increase compared to the solvent group (1.7 mm²). Therefore, it is believed that CD106 in synovial stem cells is not a molecule that contributes to meniscus regeneration.

[0114] [Table 6] Table 6: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) CD106KO-rSMSC 3.7±0.7† CD106WT-rSMSC 3.3±0.7 solvent 1.7±0.7 †P < 0.05 vs solvent group

[0115] <Example 9> The steps for establishing rat synovial-derived mesenchymal stem cells, the differences in processing time during cell recovery, and the expression rates of integrin β1 and PDGFRb were the same as in Example 1. Rat synovial-derived mesenchymal stem cells were obtained by isolating cells from rat synovium and culturing them for 8 days. The culture medium in the flask was discarded, and after washing twice with PBS, TrypLE Express (Gibco Cat. No. 12604-013) was added. The cells were then incubated at 37°C for 5, 30, 60, and 120 minutes, respectively, before being peeled off and recovered.

[0116] Ten⁶ cells recovered under various conditions were resuspended in 500 μL of PBS. For dead cell staining, LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen Cat. No. L34957) was added to 0.5 μL of cell suspension and incubated at room temperature for 30 min. After centrifugation, the supernatant was discarded, and 1 mL of FACS buffer (final concentration 2 mmol / L EDTA·2Na, PBS containing 1% bovine serum albumin) was added to resuspend the cells. To determine the expression rate of integrin β1, 5 μL of PE anti-mouse / rat integrin β1 Antibody (Biolegend Cat. No. 102207) or PE Armenian Hamster IgG Isotype Ctrl Antibody (Biolegend Cat. No. 400907) was added and reacted at 4 °C for 30 min. Afterwards, the supernatant was discarded by centrifugation, and the mixture was resuspended in 1 mL of FACS buffer. This was repeated by centrifugation, and the supernatant was discarded again. The mixture was then resuspended in 500 μL of FACS buffer for assay. To determine the expression rate of PDGFRb, 5 μL of Anti-PDGF Receptorβ, 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 the mixture was incubated at 4°C for 30 minutes. Afterwards, the supernatant was discarded by centrifugation, and the mixture was resuspended in 1 mL of FACS buffer. Then, 1 μL of Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC (invitrogen Cat. No. A16006), was added, and the mixture was incubated at 4°C for 30 minutes. Afterwards, the supernatant was discarded by centrifugation, and the suspension was resuspended in 1 mL of FACS buffer. The suspension was then centrifuged again to discard the supernatant, and the suspension was resuspended in 500 μL of FACS buffer. The suspension was then used for flow cytometry (Attune NxT, AutoForcusing Cytometer model: AFC2, invitrogen) to measure the expression rates of integrin β1 and PDGFRb.

[0117] The expression rates of integrin β1 and PDGFRb based on the differences in the dissection treatment time of synovial-derived mesenchymal stem cells are recorded in Table 7. Even when the dissection treatment time was extended from the usual 5 minutes to 120 minutes, the expression rate of integrin β1 remained above 90%. On the other hand, the expression rate of PDGFRb decreased in a time-dependent manner, being 91.7% at 5 minutes, 76.9% at 30 minutes, 63.3% at 60 minutes, and 32.8% at 120 minutes. As shown in Example 7, PDGFRb is a molecule required for meniscus regeneration in synovial stem cells. Since meniscus regeneration can be further expected when the PDGFRb expression rate exceeds half during the cell dissection treatment time, a treatment time of 60 minutes or less is preferred, and the required molecule expression rate can be set to 60% or more.

[0118] [Table 7] Table 7: Dissection processing time and expression rates of integrin β1 and PDGFRb during cell recovery expression rate Processing time 5 minutes 30 minutes 60 minutes 120 minutes Integrin β1 98.7% 99.7% 96.1% 97.8% PDGFRb 91.6% 76.9% 63.3% 32.8%

[0119] <Example 10> Culture days and integrin β1 expression rate during cell recovery for establishing rat synovial-derived mesenchymal stem cells were obtained in the same manner as in Example 1. Cells were isolated from rat synovium and seeded at a density of 7.5 x 10⁴ cells in a 75 cm² flask. The cells were cultured for 8, 21, and 28 days, respectively. The culture medium in the flask was discarded, and the cells were washed twice with PBS. TrypLE Express (Gibco Cat. No. 12604-013) was added, and the cells were incubated at 37°C for 120 minutes. The cells were then peeled off and recovered. Subsequently, dead cell and integrin β1 staining procedures were performed in the same manner as in Example 9 for analysis.

[0120] The expression rate of integrin β1 based on the difference in culture days among synovial-derived mesenchymal stem cells is recorded in Table 8. The expression rate of integrin β1 in synovial-derived mesenchymal stem cells decreased in a culture day-dependent manner, reaching 97.8% at 8 days, 62.1% at 21 days, and 56.1% at 28 days. As shown in Example 6, integrin β1 is a molecule required for meniscus regeneration in synovial stem cells, therefore, a cell positivity rate of more than half of the cells is preferred. Furthermore, as shown in Example 7, the expression rate of the molecule required for meniscus regeneration can be set to 60% or more, therefore, a culture period of 21 days or less is preferred.

[0121] [Table 8] Table 8: Culture time and integrin β1 expression rate after cell isolation expression rate Training days 8 days 21 days 28 days Integrin β1 97.8% 62.1% 56.1%

[0122] <Example 11> For human synovial-derived stem cells (Cryopreserved Synoviocytes, Normal, P1, Model: CDD-H-2910-N, Batch: ST1414, ST1420, ST1434, ST1462) purchased from Articular Engineering, the positivity rates of integrin β1 and PDGFRb, which are essential protein molecules for drug efficacy, were also determined by flow cytometer. The assay equipment used was Attune NxT, AutoForcusing Cytometer (Model: AFC2, Invitrogen). Integrin β1 (APC Mouse Anti-Human CD29 Cat: 559883) and PDGFRb (Anti-PDGF Receptorβ, Human, Goat-Poly Cat: AF385) were used as antibodies, respectively.

[0123] As a result, the positive rates of integrin β1 surface antigen in each batch were 99.4%, 99.6%, 99.6%, and 99.5%, respectively. Furthermore, the positive rates of PDGFRb surface antigen in each batch were found to be 92.2%, 90.7%, 95.2%, and 85.9%, respectively. Therefore, it was found that when this human synovial-derived stem cells have a therapeutic effect as a treatment for arthritis, it is appropriate to manage integrin β1 at ≥90% and PDGFRb at ≥80% as standard values.

[0124] Thus, the expression of integrin β1 and PDGFRb can be confirmed not only in rat synovial-derived stem cells in Examples 9 and 10, but also in human synovial-derived stem cells, which suggests that they can be set as a quality management item for cell efficacy.

[0125] <Example 12> Confirmation of the meniscus regeneration effect of FGFR3-inhibited rat synovial-derived stem cells. To prepare FGFR3-inhibited rat synovial-derived stem cells, the cryopreservation solution of the rat synovial-derived stem cells prepared in Example 1 was activated and thawed. αMEM no nucleosides were added to achieve a final concentration of 20% Fetal Bovine Serum, 1% L-glutamine (200 mmol / L), and 1% Antibiotic-Antimycotic (100×). After culturing at 37°C with 5% CO2 for 1 week, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. For each cell number of 5 × 10⁶ cells, 100 μg of FGFR3 Polyclonal Antibody (Invitrogen Cat.No. PA5-34574) was added, and the cells (FGFR3-rSMSCs) were recovered after reacting at ice-cold for 1 hour. As a control treatment without FGFR3 inhibition, the cells (IgG-rSMSCs) were recovered after reacting at ice-cold for 1 hour in Rabbit IgG Isotype Control (Thermo Fisher Scientific Cat. No. 10500C).

[0126] LEW / CrlCrlj rats were used in the creation of a meniscus injury model to evaluate the regeneration effect of the meniscus. In the method of meniscus injury and transplantation of mesenchymal stem cells, the skin of the knee joint was incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule below the knee was exposed, and a longitudinal incision was made with a scalpel to expose the cartilage at the distal end of the femur. The medial meniscus was detached from the synovium to expose the medial meniscus, and about 2 / 3 was removed from the front. The patellar tendon and synovium were sutured, followed by suturing of the muscles to create a meniscus injury model. Subsequently, the treated animals were divided into 3 groups, and 5 × 10⁶ cells of FGFR3-inhibited synovial stem cells (FGFR3-rSMSC), 5 × 10⁶ cells of uninhibited control synovial stem cells (IgG-rSMSC), and solvent alone were injected into the joint capsule. After treatment, all rats were returned to their cages and allowed to move and eat freely.

[0127] Three weeks after treatment, the animals were euthanized under isoflurane anesthesia by exsanguination of the inferior aorta. The meniscus was then exposed from the knee joint, and the medial meniscus was removed and photographed. Images of the removed medial menisci from both knee joints are shown in Figure 23. The regenerated portion was identified based on the difference in hue and shape compared to the normal meniscus and circled with a dashed line. In the solvent group (negative control), the majority of menisci showed a shape extending to the middle segment. In the IgG-rSMSC group (positive control), there were more cases of meniscus regeneration extending to the anterior segment, and the observed regenerated portion was larger. On the other hand, in the FGFR3-rSMSC group (cells with suppressed or missing molecules), the observed regenerated portion of the meniscus was smaller compared to the positive control.

[0128] In order to quantitatively evaluate the visual appearance of the meniscus regeneration portion in Figure 23, the area of ​​the meniscus regeneration portion (within the dashed line) was calculated using Image J (version 1.52) by the following formula.

[0129] Meniscus regeneration area (mm²) = Number of pixels in the meniscus regeneration area / Number of pixels per 1 mm²

[0130] The mean, standard deviation, and statistical analysis of the regenerated area of ​​each group were performed using Microsoft Excel 2007 (Microsoft Corp.). For statistical analysis, two Student's T-Tests were performed on the positive control group and the molecular inhibition group, as well as on the molecular inhibition group and the solvent group, and the p-values ​​were calculated. Furthermore, a significance level of 5% (α=0.05) was defined as 2.5% (α=0.025) after Bonferroni correction and division by the number of tests (2), and the results are recorded in Table 9.

[0131] Regarding the average area of ​​the regenerated meniscus recorded in Table 9, the 1.2 mm² of the FGFR3-rSMSC group was significantly reduced compared to the 1.9 mm² of the IgG-rSMSC group. On the other hand, the FGFR3-rSMSC group had the same regeneration area as the solvent group (1.0 mm²). This confirms that the FGFR3 molecule in synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0132] [Table 9] Table 9: Area of ​​regenerated meniscus (mean ± standard deviation) Group Area (mm) 2 ) FGFR3-rSMSC 1.2±0.5*† IgG-rSMSC 1.9±0.6 solvent 1.0±0.5 *P < 0.025 vs IgG-rSMSC group†P < 0.025 vs solvent group [Simplified Explanation of the Diagram]

[0012] Figure 1 shows the results of detecting the extracellular matrix adhesion ability of rat synovial-derived mesenchymal stem cells by inhibiting integrin β1. Figures 2a and 2b show the results of detecting the cell proliferation ability of rat synovial-derived mesenchymal stem cells by inhibiting PDGFRb. Figure 3 shows the Col2A1 base sequence (first half) of wild-type (Col2A1WT-rSMSC) and deletion-type (Col2A1KO-rSMSC) rat synovial stem cells. Figure 4 shows the Col2A1 base sequence (second half) of wild-type (Col2A1WT-rSMSC) and deletion-type (Col2A1KO-rSMSC) rat synovial stem cells. Figure 5 shows the Col2A1 base sequence (first half) of wild-type (Col2A1WT-rSMSC) and deletion-type (Col2A1KO-rSMSC) rat synovial stem cells. Figure 6 shows the Col2A1 base sequences (second half) of wild-type (Col2A1WT-rSMSC) and Col2A1-deleted (Col2A1KO-rSMSC) rat synovial stem cells. Figure 7 shows the amino acid sequences translated from the Col2A1 base sequences of wild-type (Col2A1WT-rSMSC) rat synovial stem cells. Figure 8 shows the amino acid sequences translated from the Col2A1 base sequences of Col2A1-deleted (Col2A1KO-rSMSC) rat synovial stem cells. Figure 9 shows the amino acid sequences translated from the Col2A1 base sequences of Col2A1-deleted (Col2A1KO-rSMSC) rat synovial stem cells. Figure 10 shows the CD120a base sequences of CD120a wild-type (CD120aWT-rSMSC) and CD120a deletion (CD120aKO-rSMSC) rat synovial stem cells. Figure 11 shows the amino acid sequences translated from the CD120a base sequences of CD120a wild-type (CD120aWT-rSMSC) and CD120a deletion (CD120aKO-rSMSC) rat synovial stem cells. Figure 12 shows the CD106 base sequences of CD106 wild-type (CD106WT-rSMSC) and CD106 deletion (CD106KO-rSMSC) rat synovial stem cells. Figure 13 shows the amino acid sequences translated from the CD106 base sequences of CD106 wild-type (CD106WT-rSMSC) and CD106 deletion (CD106KO-rSMSC) rat synovial stem cells. Figure 14 shows the results of the examination on the ability of rat synovial-derived mesenchymal stem cells lacking Col2A1 to inhibit chondrogenesis.Figure 15 shows the results of the examination on the inhibitory capacity for chondrogenesis in rat synovial-derived mesenchymal stem cells lacking CD120a. Figure 16 shows the results of the examination on the inhibitory capacity for chondrogenesis in rat synovial-derived mesenchymal stem cells lacking CD106. Figure 17 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with inhibited integrin β1. Figure 18 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with inhibited PDGFRb. Figure 19 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with inhibited CD44. Figure 20 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells lacking Col2A1 (Col2A1KO-rSMSC). Figure 21 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells lacking CD120a (CD120aKO-rSMSC). Figure 22 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells (CD106aKO-rSMSC) lacking CD106. Figure 23 shows the results confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with inhibited FGFR3.

Claims

1. A treatment for arthritis comprising synovial-derived mesenchymal stem cells having one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β.

2. The arthropathy treatment agent as claimed in claim 1, wherein the synovial-derived mesenchymal stem cells have surface antigens of both integrin β1 and platelet-derived growth factor receptor β.

3. The arthropathy treatment agent as described in claim 1 or 2, which has a gene encoding type II collagen α1 chain and produces type II collagen α1 chain after transplantation.

4. The arthropathy treatment agent as described in claim 1 or 2, which has the surface antigen FGFR3.

5. The arthropathy treatment agent as claimed in claim 1 or 2, wherein the ratio of synovial-derived mesenchymal stem cells having any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β to all cells contained in the arthropathy treatment agent is 30% or more.

6. A method for manufacturing the arthritis treatment agent according to claim 1, comprising: Step A: Treat synovial tissue with an enzyme; Step B: Wash the enzyme-treated mixture; Step C: Culture the synovial-derived mesenchymal stem cells contained in the washed mixture on a substrate; and Step D: Separate the cultured synovial-derived mesenchymal stem cells from the substrate.

7. The method as described in claim 6, wherein step B is a step of washing the enzyme-treated mixture until the residual enzyme concentration in the supernatant reaches below 0.5 ng / mL.

8. The method as described in claim 6 or 7, wherein in step C above, the period for culturing synovial-derived mesenchymal stem cells is within 28 days.

9. The method as described in claim 6 or 7, wherein in the aforementioned step D, separation is performed by subjecting the mesenchymal stem cells to a cell stripping solution for a period of no more than 120 minutes.

10. The method as described in claim 6 or 7, further comprising the step of sorting synovial-derived mesenchymal stem cells having any one or more surface antigens of integrin β1 or platelet-derived growth factor receptor β.