Arthritis treatment agent, and method for manufacturing arthritis treatment agent

Abstract

The present invention aims to provide an arthritis treatment agent containing synovial membrane-derived mesenchymal stem cells having molecules essential for joint treatment, and a method for producing the arthritis treatment agent. [Solution] According to the present invention, an arthritis treatment agent is provided which comprises synovial mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β.

Description

Technical Field

[0001] The present invention relates to a therapeutic agent for arthritis containing synovium-derived mesenchymal stem cells having molecules essential for joint treatment, and a method for producing the above therapeutic agent for arthritis.

Background Art

[0002] In recent years, due to the progress of regenerative medicine technology and cell therapy technology, various cell therapies using autologous, allogeneic or xenogeneic cells and the development of research cell products have been actively carried out. Among them, mesenchymal stem cells (MSC) are expected as a cell source for useful 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 tissue (Non-Patent Document 1), adipose tissue (Non-Patent Document 2), muscle tissue (Non-Patent Document 3), synovial tissue (Non-Patent Document 4), and periosteal tissue (Non-Patent Document 5). In particular, it has been reported that synovium-derived mesenchymal stem cells have higher proliferative ability and chondrogenic ability than mesenchymal stem cells derived from various mesenchymal tissues such as bone marrow (Non-Patent Document 6). In addition, Patent Documents 1 to 3 disclose methods for treating articular cartilage injury and meniscus injury using synovium-derived mesenchymal stem cells. Patent Document 4 describes a method for preparing and quality controlling limb bud mesenchymal cell populations, chondrocyte progenitor cell populations, and osteoblast progenitor cell populations using molecules such as CD140b.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

[0004] [Patent Document 1] Patent No. 5928961 [Patent Document 2] Patent No. 5656183 [Patent Document 3] Patent No. 6864302 [Patent Document 4] International Publication No. WO2021 / 054449 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] In 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 perfectly uniform and it is difficult to identify their characteristics, ensuring the equivalence and identity of each lot is generally difficult. Therefore, in order to manage product quality, in addition to quality testing of the final product, the Quality Management System (QMS) concept, which has been applied in medical devices, has been adopted, and overall process management has been implemented by recording and controlling manufacturing raw materials, material management, manufacturing process management, and process control testing. However, with the advancement of science and technology, the importance of identifying the characteristics of the cells themselves, which constitute the final product, is increasing.

[0006] One method of cell quality control involves identifying the cell type of the target final product (for example, identifying it as a mesenchymal stem cell) using cell type-specific surface markers. However, there are concerns that cells obtained using this quality control method may not have consistent therapeutic effects, and the method is still not entirely satisfactory.

[0007] Methods for manufacturing joint treatment agents using synovial stem cells have been reported to date. However, there have been problems with inconsistent therapeutic effects due to insufficient quality control to ensure the efficacy of the treatment agents. Quality control of cell products has mainly been performed using markers to identify cell types, and quality control regarding potency (efficacy) has not been performed.

[0008] Since markers indicating potency (efficacy) are identified based on the mechanism of action, the present invention aims to elucidate the mechanism of action related to the efficacy of cell therapy products, identify markers based on the mechanism of action, and further provide a method for producing therapeutic agents based on those markers. In other words, the object of the present invention is to provide an arthropathy therapeutic agent containing synovial membrane-derived mesenchymal stem cells having molecules essential for joint treatment, and a method for producing the above arthropathy therapeutic agent. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the inventors have found that one or more of either integrin β1 or platelet-derived growth factor receptor β are essential quality control markers for the effectiveness of synovial stem cell-based treatment of joint diseases. The present invention was completed based on the above findings.

[0010] In other words, the present invention provides the following invention. <1> A therapeutic agent for arthritis containing synovial mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β. <2> Synovial mesenchymal stem cells possess both integrin β1 and platelet-derived growth factor receptor β surface antigens. <1> The arthritis treatment agent described above. <3> It possesses a gene encoding type II collagen α1 chain and produces type II collagen α1 chain after transplantation. <1> or <2> The arthritis treatment agent described above. <4> Having the surface antigen of FGFR3, <1> from <3> An arthritis treatment agent listed in any one of the following. <5> The proportion of synovial-derived mesenchymal stem cells possessing one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β, to the total number of cells contained in the arthritis treatment agent is 30% or more. <1> from <4> An arthritis treatment agent listed in any one of the following. <6> Step A, in which synovial tissue is treated with enzymes. Step B involves washing the mixture after enzyme treatment. Step C involves culturing synovial membrane-derived mesenchymal stem cells contained in the washed mixture on a substrate, and Step D: Separation of cultured synovial membrane-derived mesenchymal stem cells from the substrate. including, <1> from <5> A method for manufacturing an arthritis treatment agent as described in any one of the following. <7> Step B described above is a step in which the mixture after enzyme treatment is washed until the residual enzyme concentration in the supernatant is 0.5 ng / mL or less. <6> Methods used. <8> In step C described above, the period for culturing synovial membrane-derived mesenchymal stem cells is 28 days or less. <6> or <7> Methods used. <9> In step D described above, mesenchymal stem cells are separated by exposing them to a cell detachment solution for a period of 120 minutes or less. <6> from <8> The method described in any one of the following. <10> The process further includes selecting synovial mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β. <6> from <9> The method described in any one of the following. [Effects of the Invention]

[0011] The present invention provides a therapeutic agent for arthritis that contains synovial mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β, thereby exhibiting a therapeutic effect against arthritis. According to the present invention, fluctuations in the therapeutic effect of the manufactured arthritis therapeutic agent can be suppressed, and quality control of the product's therapeutic effect can be achieved. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the results of investigating the suppression of extracellular matrix adhesion ability in rat synovial membrane-derived mesenchymal stem cells by integrin β1 inhibition. [Figure 2] Figure 2 shows the results of investigating the suppression of cell proliferation in rat synovial membrane-derived mesenchymal stem cells by PDGFRb inhibition. [Figure 3] Figure 3 shows the Col2A1 nucleotide sequence (first half) of rat synovial stem cells with the Col2A1 gene wild type (Col2A1WT-rSMSC) and deletion type (Col2A1KO-rSMSC). [Figure 4] Figure 4 shows the Col2A1 nucleotide sequences (second half) of rat synovial stem cells with the wild-type (Col2A1WT-rSMSC) and deletion-type (Col2A1KO-rSMSC) Col2A1 gene. [Figure 5] Figure 5 shows the Col2A1 nucleotide sequence (first half) of rat synovial stem cells with the Col2A1 gene wild type (Col2A1WT-rSMSC) and deletion type (Col2A1KO-rSMSC). [Figure 6] Figure 6 shows the Col2A1 nucleotide sequences (second half) of rat synovial stem cells with the Col2A1 gene wild type (Col2A1WT-rSMSC) and deletion type (Col2A1KO-rSMSC). [Figure 7] Figure 7 shows the amino acid sequence translated based on the Col2A1 nucleotide sequence of rat synovial stem cells with the wild-type Col2A1 gene (Col2A1WT-rSMSC). [Figure 8] Figure 8 shows the amino acid sequence translated based on the Col2A1 nucleotide sequence of rat synovial stem cells lacking the Col2A1 gene (Col2A1KO-rSMSC). [Figure 9] Figure 9 shows the amino acid sequence translated based on the Col2A1 nucleotide sequence of rat synovial stem cells lacking the Col2A1 gene (Col2A1KO-rSMSC). [Figure 10] Figure 10 shows the CD120a nucleotide sequences of rat synovial stem cells with the wild-type (CD120aWT-rSMSC) and deletion-type (CD120aKO-rSMSC) CD120a gene. [Figure 11] Figure 11 shows the amino acid sequences translated based on the CD120a nucleotide sequence of rat synovial stem cells with the CD120a gene wild type (CD120aWT-rSMSC) and deletion type (CD120aKO-rSMSC). [Figure 12] Figure 12 shows the CD106 nucleotide sequences of rat synovial stem cells with the CD106 gene wild type (CD106WT-rSMSC) and deletion type (CD106KO-rSMSC). [Figure 13] Figure 13 shows the amino acid sequences translated based on the CD106 nucleotide sequences of rat synovial stem cells with the CD106 gene wild type (CD106WT-rSMSC) and deletion type (CD106KO-rSMSC). [Figure 14] Figure 14 shows the results of investigating the suppression of chondrogenic differentiation ability in rat synovial mesenchymal stem cells lacking Col2A1. [Figure 15] Figure 15 shows the results of investigating the suppression of chondrogenic differentiation ability in rat synovial mesenchymal stem cells lacking CD120a. [Figure 16] Figure 16 shows the results of investigating the suppression of chondrogenic differentiation ability in rat synovial mesenchymal stem cells lacking CD106. [Figure 17] Figure 17 shows the results of confirming the meniscus regeneration effect of rat synovial membrane-derived mesenchymal stem cells with integrin β1 inhibition. [Figure 18] Figure 18 shows the results of confirming the meniscus regeneration effect of rat synovial mesenchymal stem cells with PDGFRb inhibition. [Figure 19] Figure 19 shows the results of confirming the meniscus regeneration effect of rat synovial mesenchymal stem cells with CD44 inhibition. [Figure 20]Figure 20 shows the results of confirming the meniscus regeneration effect of rat synovial mesenchymal stem cells lacking Col2A1 (Col2A1KO-rSMSC). [Figure 21] Figure 21 shows the results of confirming the meniscus regeneration effect of rat synovial mesenchymal stem cells lacking CD120a (CD120aKO-rSMSC). [Figure 22] Figure 22 shows the results of confirming the meniscus regeneration effect of rat synovial mesenchymal stem cells lacking CD106 (CD106aKO-rSMSC). [Figure 23] Figure 23 shows the results of confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells with FGFR3 inhibition. [Modes for carrying out the invention]

[0013] The details of the present invention will be described below. In this specification, "~" is used to mean that the numbers before and after it are included as the lower limit and upper limit.

[0014] The present invention provides a therapeutic agent for arthritis, comprising synovial mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β (also referred to herein as PDGFRb). Synovial mesenchymal stem cells may have either integrin β1 or platelet-derived growth factor receptor β, but preferably they have both integrin β1 and platelet-derived growth factor receptor β.

[0015] Synovial mesenchymal stem cells preferably possess a gene encoding type II collagen α1 chain, and can exert a therapeutic effect by producing type II collagen α1 chain after transplantation. Synovial mesenchymal stem cells preferably have the surface antigen FGFR3 (fibroblast growth factor receptor 3).

[0016] The ratio of synovial-derived mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β, to the total number of cells in the arthropathy treatment agent of the present invention is preferably 30% or more, and may 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 present invention provides a therapeutic agent for arthritis, Step A, in which synovial tissue is treated with enzymes. Step B involves washing the mixture after enzyme treatment. Step C involves culturing synovial membrane-derived mesenchymal stem cells contained in the washed mixture on a substrate, and Step D: Separation of cultured synovial membrane-derived mesenchymal stem cells from the substrate. It can be manufactured by a method that includes [a specific component].

[0018] <Process A: Enzymatic treatment of synovial tissue> Synovial tissue can be harvested from the non-weight-bearing portion of the joint under anesthesia. The biological origin of the synovial tissue is not particularly limited, and synovial tissue from any organism, preferably mammals, can be used. For example, synovial tissue from primates (e.g., chimpanzees, Japanese macaques, 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 it is preferably derived from a single donor.

[0020] When producing synovial-derived mesenchymal stem cells for administration to humans, it is preferable to use synovial tissue collected from a donor whose histocompatibility antigen type matches or is similar to that of the recipient. More preferably, the subject from which the synovial tissue is collected and the subject to which the synovial-derived mesenchymal stem cells are transplanted are the same subject. That is, it is preferable to use synovial tissue collected from the recipient itself (autologous transplantation). The amount of synovial tissue to be collected can be determined considering the type of donor or the required 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 then shredded with scissors or the like as needed and subjected to the enzymatic treatment described later.

[0021] The synovial tissue is treated with enzymes. The enzyme is not particularly limited as long as it contains a protease, but preferably it is a mixed enzyme containing one or more collagenases and one or more neutral proteases. A particularly preferred enzyme is Liberase®. As Liberase®, 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).

[0022] The enzymatic reaction can be carried out in an aqueous solution containing the enzyme, and an aqueous solution containing human serum may be used. The human serum may be the patient's own serum or an allogeneic, differently typed serum, but the patient's own 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, even more preferably 0.5 mg / ml to 10 mg / ml, even 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. The mass ratio of synovial tissue to enzyme is preferably 1000:1 to 10:1, more preferably 500:1 to 20:1, and even more preferably 200:1 to 40:1.

[0023] The enzymatic reaction can preferably be carried out at a temperature of 15°C to 40°C, more preferably 20°C to 35°C, and even more preferably 25°C to 35°C. The reaction time should be at least 2 hours, 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, but it may be within 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, or 4 hours. The enzymatically treated mixture contains synovial membrane-derived mesenchymal stem cells. The enzyme-treated mixture can be transferred to a centrifuge tube through a cell strainer and centrifuged to recover synovial-derived mesenchymal stem cells.

[0024] <Step B: Washing the mixture after enzyme treatment> In step B, the mixture after enzyme treatment is washed. In step B, the supernatant can preferably be washed until the residual enzyme concentration is 0.5 ng / mL or less. More preferably, the residual enzyme concentration in the supernatant is 0.3 ng / mL or less, even more preferably 0.2 ng / mL or less, and particularly preferably 0.1 ng / mL or less.

[0025] Washing can be performed by resuspending the synovial-derived mesenchymal stem cells recovered by the centrifugation process described above in a culture medium and centrifugating again (e.g., at 400g for 5 minutes). While α-modified Eagle's Minimum Essential Medium (αMEM) can be used as the culture medium, it is not particularly limited. Washing may be performed multiple times (two or more times) using the culture medium as described above.

[0026] <Step C: Culturing synovial-derived mesenchymal stem cells contained in the washed mixture on a substrate> In step C, synovial membrane-derived mesenchymal stem cells contained in the washed mixture are cultured on a substrate. Examples of substrates include flat plastic substrates such as culture plates, and three-dimensional substrates such as culture bags, microcarriers, or gels, but are not particularly limited. The culture medium used in cell culture can be prepared using the same medium as that used for normal animal cell culture as the base medium. Examples of media commonly used for normal animal cell culture include αMEM, DMEM (Dulbecco Modified Eagle Medium), a mixed medium of DMEM and F12 (DMEM:F12=1:1), RPMI medium (such as GIBCO® RPMI1640 medium), and a mixed medium of DMEM / F12 and RPMI (DMEM / F12:RPMI=1:1), but are not particularly limited.

[0027] The culture medium may contain serum or not. When producing synovial mesenchymal stem cells from the patient's own tissue for the purpose of administration to a living organism, the culture medium may contain allogeneic serum. That is, when producing synovial mesenchymal stem cells from human tissue for the purpose of administration to a human, a culture medium containing human serum may be used. When serum is used, it may be the patient's own serum or allogeneic serum, but the patient's own serum is preferred. When serum is used, the amount of serum added to the culture medium should be, for example, 20% by volume or less, 10% by volume or less, or 5% by volume or less.

[0028] The cell culture conditions are not particularly limited, and standard cell culture conditions can be used. For example, culture at a temperature of 30-40°C and 3-7% CO2 is possible, but it is not particularly limited. As an example, culture at a temperature of 37°C and 5% CO2 is possible.

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

[0030] The differentiation of synovial mesenchymal stem cells into chondrocytes progresses with increasing culture duration, and it is known that the in situ chondrogenic ability of synovial mesenchymal stem cells decreases when the culture period exceeds a certain length. Therefore, in the present invention, it is preferable to adjust the culture period in order to proliferate synovial mesenchymal stem cells in an undifferentiated state and in a state with good in situ chondrogenic ability. In step C, it is preferable that the culture period of synovial mesenchymal stem cells is 28 days or less.

[0031] Furthermore, in the present invention, it is necessary to consider the need to prepare a sufficient number of undifferentiated synovial stem cells to cover the cartilage damage and regenerate the affected area. Therefore, the culture period is preferably 5 days or more, 7 days or more, or 10 days or more, more preferably 10 to 14 days, 10 to 21 days, or 10 to 28 days, and even more preferably 10 to 21 days.

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

[0033] It is known that synovial-derived mesenchymal stem cells exhibit a decrease in in situ chondrogenesis ability that is inversely proportional to the passage number of mesenchymal stem cells in vitro. Therefore, in order to prepare undifferentiated mesenchymal stem cells, it is preferable to produce synovial-derived mesenchymal stem cells at the primary or first passage level.

[0034] In the present invention, the serum used in self-treatment is self-derived, and the amount of serum that can be collected from a donor in self-treatment is limited. Also, since a certain cell density or higher is required from the perspective of the proliferation of synovium-derived mesenchymal stem cells, the synovium-derived mesenchymal stem cells after enzyme treatment are seeded at a cell density of 100 cells / cm

[0036] , , or more and 5000 cells / cm 2 or less, 200 cells / cm 2 or more and 5000 cells / cm 2 or less, 500 cells / cm 2 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​In step D, cultured synovial-derived mesenchymal stem cells are separated from the substrate. Preferably, in step D, the mesenchymal stem cells can be separated by allowing a cell detachment solution to act on them for a period of 120 minutes or less. The cell detachment solution is a solution containing a trypsin-like enzyme and EDTA. A particularly preferred enzyme is TrypLE. As TrypLE, for example, TrypL Express (Gibco) or TrypLE Select (Gibco) can be used.

[0037] The time for which the cell detachment solution is applied to mesenchymal stem cells is preferably 10 minutes or more, in order to sufficiently detach the cells. The time for which the cell detachment solution is applied to mesenchymal stem cells is preferably 10 to 120 minutes, and more preferably 10 to 60 minutes. It may also 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). They are known to be present in bone marrow, synovial membrane, periosteum, adipose tissue, and muscle tissue, and are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and muscle cells. Regarding the differentiation of mesenchymal stem cells into chondrocytes, it is known that adding BMP or TGF-β to the culture medium promotes the differentiation of undifferentiated mesenchymal stem cells into chondrocytes, enabling the regeneration of cartilage tissue under in vitro conditions.

[0039] Mesenchymal stem cells can be identified by detecting molecules characteristic of mesenchymal stem cells, such as enzymes, receptors, and small molecule compounds. Examples of molecules characteristic of mesenchymal stem cells include, but are not limited to, cell surface markers (positive markers) such as CD73, CD90, CD105, and CD166. Examples of 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 confirm the presence of mesenchymal stem cells. Immunological methods can be used to detect these markers, but detection may also be performed by quantifying the mRNA levels of each molecule.

[0040] In this specification, synovial-derived mesenchymal stem cells are stem cells contained in the synovial membrane. 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 potential, but the detection method is not particularly limited.

[0041] When synovial-derived mesenchymal stem cells produced by the above method are to be used as a treatment for arthritis, the cells may be mixed with a pharmaceutically acceptable carrier by conventional methods to form a formulation suitable for administration to an individual. Examples of carriers include saline solution, glucose, and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.) to make it isotonic for injection. Furthermore, buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. may also be added.

[0042] The method for producing the arthropathy treatment agent of the present invention may further include a step of selecting synovial mesenchymal stem cells having one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β.

[0043] One step in selecting synovial mesenchymal stem cells that have one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β, is to control the expression level of integrin β1 or platelet-derived growth factor receptor β.

[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 (such as a ratio or difference from a comparison control or reference expression level).

[0045] The expression level of integrin β1 or platelet-derived growth factor receptor β can be measured by any method known to those skilled in the art and can be carried out according to conventional methods. The expression level may be measured by measuring the amount of mRNA, which is the transcript of the gene. The method for measuring mRNA is not particularly limited as long as it can measure the desired amount of mRNA, and can be appropriately selected from known methods. For example, gene amplification methods using oligonucleotides that hybridize to the gene encoding integrin β1 or platelet-derived growth factor receptor β as primers, or hybridization methods using oligo(poly)nucleotides that hybridize to the gene encoding a specific protein molecule as probes can be used. Specifically, examples include RT-PCR (reverse transcription polymerase chain reaction), real-time RT-PCR, DNA microarray, cell array, Northern blotting, dot blotting, and RNase protection assays.

[0046] The primers and probes used in the above measurement method 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 the direct use of RNA in the sample and enables gene quantification from the number of temperature cycles required for amplification by optically measuring the gene amplification process. Furthermore, as a control, the expression levels of housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin mRNA can be used to standardize the expression levels of genes encoding integrin β1 or platelet-derived growth factor receptor β. Note that the primers and probes used in the above measurement method can be appropriately designed and prepared by those skilled in the art based on the nucleotide sequence information of the 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 measured immunologically, for example, using antibodies or antibody fragments against integrin β1 or platelet-derived growth factor receptor β. Specifically, these methods include flow cytometry, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunofluorescence assay, and cell array assay. These measurement methods can also be performed using standard protocols or protocols that have been appropriately modified or changed from standard protocols.

[0048] For example, when measuring the expression level of integrin β1 or platelet-derived growth factor receptor β in cells by flow cytometry, synovial mesenchymal stem cells having one or more surface antigens of either integrin β1 or platelet-derived growth factor receptor β can be selected 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.

[0049] When selecting synovial-derived mesenchymal stem cells that possess one or more surface antigens, either integrin β1 or platelet-derived growth factor receptor β, this can be done, for example, by comparing the expression level of integrin β1 or platelet-derived growth factor receptor β in cells measured by the method described above with a predetermined reference expression level. The reference expression level may be, for example, the expression level of integrin β1 or platelet-derived growth factor receptor β in cells that have already been confirmed to have a certain quality (positive control), or it may be the expression level of cells that have already been confirmed not to have a certain quality (negative control).

[0050] By comparing the expression level of integrin β1 or platelet-derived growth factor receptor β with the reference expression level, cells in which the expression level of integrin β1 or platelet-derived growth factor receptor β is equal to or higher than the expression level of the positive control can be selected and used as a treatment for arthritis.

[0051] Alternatively, a cutoff value for the expression level of integrin β1 or platelet-derived growth factor receptor β may be set in advance, and the expression level of integrin β1 or platelet-derived growth factor receptor β measured for cells may be compared with the cutoff value. The cutoff value can be, for example, determined based on a regression line showing the correlation between the expression level of integrin β1 or platelet-derived growth factor receptor β and the therapeutic effect, and set to the expression level of integrin β1 or platelet-derived growth factor receptor β that gives the desired therapeutic effect. For example, cells in which the expression level of integrin β1 or platelet-derived growth factor receptor β is equal to or greater than the cutoff value can be selected and used as a treatment for arthritis.

[0052] The arthropathy treatment agent of the present invention can be used for the treatment of joints. Examples of joint treatment include the treatment of diseases involving joint injury, damage, or inflammation, and include the treatment of joint diseases resulting from degeneration and / or inflammation of connective tissue such as cartilage, or non-inflammatory joint diseases. Examples of joint treatment include, but are not limited to, the treatment of diseases selected from the group consisting of meniscal tears, traumatic cartilage injuries, osteochondritis dissecans, avascular osteonecrosis, osteoarthritis (e.g., osteoarthritis of the knee), rheumatoid arthritis (e.g., chronic rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defects.

[0053] The method of treating joints using the arthropathy treatment agent of the present invention is: A step of implanting the arthropathy treatment agent of the present invention so as to cover the cartilage injury site or meniscus injury site with synovial mesenchymal stem cells; and A process of regenerating cartilage tissue in situ at the site of cartilage damage or meniscus damage by differentiating synovial membrane-derived mesenchymal stem cells contained in arthritis treatment agents into chondrocytes; Includes.

[0054] When implanting the arthropathy treatment agent of the present invention into a patient, in order to efficiently treat the cartilage damage or meniscus damage, 2.0 × 10⁻¹⁰ units should be used per cartilage damage or meniscus damage area. 7 ~1.0×10 11 1, or 2.5 × 10 7 ~1.0×10 11 1, or 3.0 × 10 7 ~1.0×10 11 pieces, 4.0×10 7 ~1.0×10 11 1, or 2.5 × 10 7 ~1.0×10 10 1, or 2.5 × 10 7 ~1.0×10 9 1, or 2.5 × 10 7 ~1.0×10 8 Individual synovial-derived mesenchymal stem cells, or 2.0 × 10⁶ cells. 7 ~1.0×10 8It is preferable to use synovial membrane-derived mesenchymal stem cells.

[0055] By transplanting synovial-derived mesenchymal stem cells (MSCs) into the cartilage or meniscus injury site, the site becomes covered with MSCs. MSCs can be transplanted by open surgery or arthroscopic surgery. To minimize invasiveness, arthroscopic transplantation is preferred.

[0056] Cartilage or meniscus injuries may be covered with a suspension of synovial-derived mesenchymal stem cells or with a cell sheet of synovial-derived mesenchymal stem cells. For example, bioabsorbable gels such as gelatin or collagen can be used as the gel-like substance. Synovial-derived mesenchymal stem cells have a high ability to adhere to cartilage or meniscus injuries.

[0057] In the treatment of cartilage damage, the minimally invasive procedure of the present invention is characterized by covering the cartilage damage area with synovial-derived mesenchymal stem cells, and involves the following steps: Maintain a position that faces upwards towards the cartilage injury; A cell sheet of synovial-derived mesenchymal stem cells, a suspension of synovial-derived mesenchymal stem cells, or a gel-like substance containing synovial-derived mesenchymal stem cells is placed on the surface of the cartilage injury; and Maintaining a specific body position for a certain period of time to allow synovial-derived mesenchymal stem cells to adhere to the surface of the cartilage injury site; Includes.

[0058] In the treatment of meniscus injuries, the minimally invasive procedure of the present invention is characterized by covering the meniscus injury site with synovial-derived mesenchymal stem cells, and involves the following steps: Maintain a position where the injured meniscus is facing downwards; Injecting a suspension of synovial membrane-derived mesenchymal stem cells into the knee joint; and Maintaining a specific body position for a certain period of time to allow synovial-derived mesenchymal stem cells to adhere to the meniscus injury site; Includes.

[0059] To ensure that synovial-derived mesenchymal stem cells adhere securely to the surface of the cartilage or meniscus injury, it is preferable to hold the transplanted synovial-derived mesenchymal stem cells on the surface of the cartilage or meniscus injury for at least 10 minutes, preferably 15 minutes. To achieve this, the body position is maintained for at least 10 minutes, preferably 15 minutes, with the aim of orienting the cartilage or meniscus injury upward and holding the synovial-derived mesenchymal stem cells to the upward-facing cartilage or meniscus injury.

[0060] The cartilage or meniscus injury site, which contains synovial-derived mesenchymal stem cells, can be further covered with periosteum to strengthen the adhesion of the synovial-derived mesenchymal stem cells to the cartilage or meniscus injury site. The surgery is completed after the synovial-derived mesenchymal stem cells are retained on the surface of the cartilage or meniscus injury site for at least 10 minutes.

[0061] In this invention, transplanted synovial-derived mesenchymal stem cells differentiate into chondrocytes at the site of cartilage injury or meniscal injury, and then regenerate cartilage tissue in situ at the site of cartilage injury or meniscal injury.

[0062] During the in situ chondrogenesis process of synovial-derived mesenchymal stem cells, cartilage tissue regenerates according to the local microenvironment (nutrient supply and cytokine environment, etc.), so no external intervention is required. As a result of in situ chondrogenesis by synovial-derived mesenchymal stem cells, cartilage tissue is regenerated at the site of cartilage damage or meniscal damage, repairing the damage. In the case of cartilage damage, 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 the original cartilage tissue, or in the case of meniscal damage, meniscal cartilage is formed.

[0063] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0064] <Example 1> Preparation of rat synovial membrane-derived mesenchymal stem cells LEW / CrlCrlj rats were used to establish mesenchymal stem cells derived from rat synovial membrane. Synovial tissue collected under isoflurane anesthesia was mixed with αMEM no nucleosides (Gibco Cat. No. 12561056) medium to a concentration of 2 or 3 mg / mL of collagenase V (Sigma Cat. No. C9263) and reacted at 37°C for 2 hours. The reaction was stopped by adding cooled medium, and the residue was removed by passing the mixture through a 40 μm cell strainer. The harvested cells were seeded in a cell culture flask and cultured in αMEM no nucleosides at 5% CO2 concentration and 37°C, with Fetal Bovine Serum (Gibco Cat. No. 10270106) added to a final concentration of 20%, L-glutamie 200 mmol / L (Gibco Cat. #25030081) added to a final concentration of 1%, and Antibiotic-Antimycotic (100X) (Gibco Cat. No. 15240062) added to a final concentration of 1%. After 8 days of culture, the culture medium in the flask was discarded, 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 to harvest the cells as synovial-derived mesenchymal stem cells. The supernatant was discarded by centrifugation and replaced with COS-banker (COSMO BIO Cat.No.COS-CFM01) to create a frozen stock of rat synovial-derived mesenchymal stem cells.

[0065] <Example 2> Inhibition of extracellular matrix adhesion ability of rat synovial mesenchymal stem cells by integrin β1 inhibition To prepare rat synovial mesenchymal stem cells with inhibited integrin β1, the frozen stock of rat synovial mesenchymal stem cells prepared in Example 1 was awakened, and the cells were cultured for 1 week at 37°C with αMEMno nucleosides to which Fetal Bovine Serum was added to a final concentration of 20%, L-glutamie 200 mmol / L to a final concentration of 1%, and Antibiotic-Antimycotic (100X) to a final concentration of 1%. The harvested cells were suspended in PBS containing 2% FBS as the reaction solvent. Cell count: 5 × 10⁶ 6 12 μg of Purified anti-mouse / ratCD29 Antibody (BioLegend Cat.No.102202) was added per cell, and the cells were collected after reacting on ice for 1 hour (integrin β1-rSMSC). As a control treatment without integrin β1 inhibition, Purified Armenian Hamster IgG Isotype Ctrl (BioLegend Cat.No.400902) was reacted on ice for 1 hour, and the cells were collected (IgG-rSMSC). In addition, untreated cells (Non-treated-rSMSC) were prepared by performing the same reaction with only the reaction solvent, and subjected to the following adhesion treatment.

[0066] To confirm that integrin β1 is inhibited, we investigated one of the functions of integrin β1: adhesion to the extracellular matrix. For the extracellular matrix adhesion reaction, we washed the cells with PBS containing 10 mmol / L MgCl2·6H2O (hereinafter referred to as PBS(+)), resuspended the cells in PBS(+), and performed a 1 × 10⁶ test. 5 The cells were prepared to a concentration of 10 μL / well and seeded onto Collagen Type I Cellware 8-Well Culture Slides (Corning Cat. No. 354630). After standing at room temperature for 10 minutes, the slides were washed with PBS(+). Subsequently, the slides were observed under a microscope (OLIMPUS Cat. No. IX71) with a 10x objective lens. An image of one field of view was also acquired from the area where the most adherent cells were observed, and the number of adherent cells was counted.

[0067] The results are shown in Figure 1. The number of adherent cells was 1637 cells for non-treated-rSMSCs, 1214 cells for IgG-rSMSCs, and 194 cells for integrin β1-rSMSCs. A significant decrease in the number of adherent cells was observed upon inhibition of 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> Inhibition of cell proliferation of rat synovial membrane-derived mesenchymal stem cells by PDGFRb inhibition To prepare rat synovial mesenchymal stem cells with inhibited PDGFRb, the frozen stock prepared in Example 1 was awakened, and cultured in αMEM no nucleosides containing Fetal Bovine Serum to a final concentration of 20%, L-glutamie 200 mmol / L to a final concentration of 1%, and Antibiotic-Antimycotic (100X) to a final concentration of 1% for 1 week at 37°C with a CO2 concentration of 5%. The harvested cells were then suspended in PBS containing 2% FBS as the reaction solvent.

[0069] Number of cells 1×10 6 Anti-PDGF Receptorβ Human Goat-Poly (R&D Systems Cat.No.AF385) was reacted at 40 or 120 μg per cell under ice conditions for 1 hour, and then the cells were seeded at 1000 cells / well in a 96-well plate (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 under ice conditions for 1 hour and then seeded at 1000 cells / well in a 96-well plate (IgG-rSMSC). Non-treated-rSMSC cells were also prepared, in which only 1000 cells / well were seeded in a 96-well plate without any reaction.

[0070] To confirm that PDGFRb was inhibited, we examined the cell proliferation function, which is a key function of PDGFRb. Cells were cultured at 5% CO2 concentration and 37°C, and their proliferation was quantitatively evaluated on day 6 using ATPassay with Cell Titer 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, 120 μg / mL PDGFRb-rSMSC was 4.12 ± 1.29 μmol / L, IgG-rSMSC was 6.08 ± 0.63 μmol / L, and non-treated-rSMSC was 6.81 ± 0.82 μmol / L, indicating a significant suppression of cell proliferation by PDGFRb inhibition. This confirms that treatment with Anti-PDGF Receptorβ Human Goat-Poly can suppress the proliferation of rat synovial stem cells.

[0072] To confirm the ligand specificity of PDGFRb, rat synovial mesenchymal stem cells with inhibited PDGFRb were prepared. The frozen stock prepared in Example 1 was awakened, and αMEMno nucleosides were cultured for 1 week at 37°C with a CO2 concentration of 5% using Fetal Bovine Serum to a final concentration of 20%, L-glutamie 200 mmol / L to a final concentration of 1%, and Antibiotic-Antimycotic (100X) to a final concentration of 1%. The harvested cells were suspended in PBS containing 2% FBS as the reaction solvent.

[0073] Number of cells 1×10 6Cells were reacted with 10, 20, or 40 μg of Anti-PDGF Receptorβ Human Goat-Poly (R&D Systems Cat. No. AF385) per cell under ice conditions for 1 hour, and then seeded at 1000 cells / well in 96-well plates (Corning Cat. No. 353072). Culture was started 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 under ice conditions for 1 hour and seeded at 1000 cells / well in 96-well plates (IgG-rSMSC). Non-treated-rSMSC cells were also prepared, in which cells were seeded at 1000 cells / well in 96-well plates without any reaction.

[0074] The day after cell seeding, the culture supernatant was discarded, and the medium was replaced with αMEMno nucleosides (Gibco Cat. No. 10270106) containing Fetal Bovine Serum to a final concentration of 0.5%, L-glutamie 200 mmol / L to a final concentration of 1%, Antibiotic-Antimycotic (100X) to a final concentration of 1%, and PDGF-BB, Rat, Recombinant (R&D Systems Cat. No. 520-BB-050) to 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 final concentrations of 10, 20, and 40 μg / mL, bringing the total volume of medium to 100 μL. For the positive control, Normal Goat IgG Control (R&D Systems Cat. No. AB-108-C) was added, bringing the total volume of medium 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 by an ATP assay using Cell Titer 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. 40 μg / mL PDGFRb-rSMSC showed a significant decrease in cell proliferation compared to IgG-rSMSC. This confirms that treatment with Anti-PDGF Receptorβ Human Goat-Poly can ligand-specifically inhibit PDGFRb in rat synovial stem cells.

[0076] <Example 4> Preparation of Col2A1-deficient rat synovial membrane-derived mesenchymal stem cells The rat synovial mesenchymal stem cells prepared in Example 1 were subjected to a deletion of the Col2A1 gene, and the deletion of the Col2A1 gene was confirmed by Sanger sequencing analysis. The Col2A1 nucleotide sequences of rat synovial stem cells with wild-type Col2A1 (Col2A1WT-rSMSC) and deleted Col2A1 (Col2A1KO-rSMSC) genes 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 nucleotide sequence of rat synovial stem cells with wild-type Col2A1 (Col2A1WT-rSMSC) genes is shown as Sequence ID No. 1, the Col2A1 nucleotide sequence of one chromosome of rat synovial stem cells with Col2A1 gene deletion (Col2A1KO-rSMSC) genes is shown as Sequence ID No. 2, and the Col2A1 nucleotide sequence of the other chromosome of rat synovial stem cells with Col2A1 gene deletion (Col2A1KO-rSMSC) genes is shown as Sequence ID No. 3. The amino acid sequence of the wild-type Col2A1 gene (Col2A1WT-rSMSC) is shown in SEQ ID NO: 4, and the amino acid sequences of the deleted Col2A1 gene (Col2A1KO-rSMSC) are shown in SEQ ID NOs: 5 and 6. As a result, it was found that Col2A1KO-rSMSC is a heterozygous frameshift mutant with DNA that has a deletion of bases from position 55 to 62 starting from the amino acid translation start codon ATG, and an insertion at base 59. In one allele, the deletion of a base causes a mutation in the sequence starting from the 19th amino acid, and a stop codon is inserted at position 29, resulting in a nucleotide sequence that translates as a mutant of 28 amino acid residues instead of the original 1419 amino acids. In the other allele, the insertion of a base causes a mutation in the sequence starting from the 20th amino acid, and a stop codon is inserted at position 50, resulting in a nucleotide sequence that translates as a mutant of 49 amino acid residues instead of the original 1419 amino acids. In this mutant, the sequence from amino acid 133 to 1146, which is the triple helix structure domain—a crucial functional domain of Col2A1—is not translated. Therefore, we determined that we were able to obtain synovial stem cells (Col2A1KO-rSMSC) with a gene sequence lacking Col2A1 function.

[0077] <Comparative Example 1> Preparation of mesenchymal stem cells derived from the synovial membrane of CD120a-deficient rats The CD120a gene was deleted from rat synovial mesenchymal stem cells prepared in Example 1, and the deletion of the CD120a gene was confirmed by Sanger sequencing analysis. The CD120a nucleotide sequences and the amino acid sequences translated based on those sequences of rat synovial stem cells with wild-type CD120a (CD120aWT-rSMSC) and deletion type CD120a (CD120aKO-rSMSC) are shown in Figures 10 and 11. The CD120a nucleotide sequences of rat synovial stem cells with wild-type CD120a (CD120aWT-rSMSC) and deletion type CD120a (CD120aKO-rSMSC) are shown in Sequence ID No. 7, the amino acid sequence of wild-type CD120a (CD120aWT-rSMSC) is shown in Sequence ID No. 8, and the amino acid sequence of CD120a deletion type (CD120aKO-rSMSC) is shown in Sequence ID No. 9. As a result, it was found that CD120aKO-rSMSCs are frameshift mutants with DNA that has a deletion at the 16th base, starting from the amino acid translation start codon ATG. Due to the deletion of the base, a mutation occurs in the sequence from the 6th amino acid, and a stop codon is inserted at the 19th position. Therefore, it was found that the translated base sequence is a mutant of 19 amino acid residues, instead of the original 461 amino acids. Since the protein region sequence that makes up CD120a is not translated in this mutant base sequence, it was determined that synovial stem cells (CD120aKO-rSMSCs) with a gene sequence lacking CD120a function were obtained.

[0078] <Comparative Example 2> Production of CD106-deficient rat synovial membrane-derived mesenchymal stem cells The CD106 gene was deleted from the rat synovial mesenchymal stem cells prepared in Example 1, and the deletion of the CD106 gene was confirmed by Sanger sequencing analysis. The CD106 nucleotide sequences and the amino acid sequences translated based on those sequences of rat synovial stem cells with wild-type CD106 (CD106WT-rSMSC) and deleted CD106 (CD106KO-rSMSC) are shown in Figures 12 and 13. The CD106 nucleotide sequences of rat synovial stem cells with wild-type CD106 (CD106WT-rSMSC) and deleted CD106 (CD106KO-rSMSC) are shown in Sequence ID No. 10, the amino acid sequence of wild-type CD106 (CD106WT-rSMSC) is shown in Sequence ID No. 11, and the amino acid sequence of deleted CD106 (CD106KO-rSMSC) is shown in Sequence ID No. 12. As a result, it was found that CD106KO-rSMSCs are frameshift mutants with DNA that has deleted bases from position 1059 to 1076, starting from the amino acid translation start codon ATG. Due to the deletion of bases, a mutation occurs in the sequence starting from amino acid 354, and a stop codon is inserted at position 356. Therefore, it was found that the translated base sequence is a mutant of 355 amino acid residues, instead of the original 739 amino acids. Since the sequence exhibiting this mutant does not translate the transmembrane region of CD106 from amino acid 699 to 720, it was determined that synovial stem cells (CD106KO-rSMSCs) with a gene sequence lacking CD106 function were obtained.

[0079] <Example 5> Suppression of chondrogenic differentiation ability in rat synovial mesenchymal stem cells lacking Col2A1 Col2A1 is one of the components of cartilage. To confirm Col2A1 deletion at the cellular function level, the cartilage differentiation ability of Col2A1KO-rSMSO was investigated. Col2A1KO-rSMSC prepared in Example 4 was 2.5 × 10⁶. 5Cells were treated with TGF-β3 (R&D Systems Cat. No. 243-B3-002) to a final concentration of 10 ng / mL, Dexamethasone (Wako 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, Sodium Pyruvate (Invitrogen Cat. No. 11360070) to a final concentration of 1 μg / mL, ITS-X supplement (x100) (Wako Cat. No. 094-06761) to a final concentration of 1%, and BMP-2 (R&D Systems Cat. The cells were suspended in DMEM high glucose (Thermo Cat. No. 11965092) with No. 355-BM-010 added, centrifuged at 450g for 10 minutes, and then cultured at 5% CO2 concentration and 37°C to induce chondrogenic differentiation. As a control, Col2A1WT-rSMSC cells were similarly induced to differentiate into cartilage. After 3 weeks of culture, the diameter and weight of the cell aggregates were measured, and the chondrogenic potential was evaluated from histological staining of the cell aggregates. The results are shown in Figure 14. Col2A1WT-rSMSC cells had a short diameter of 1.55±0.14 mm, a long diameter of 2.04±0.25 mm, and a weight of 1.9±0.26 mg, while Col2A1KO-rSMSC cells had a short diameter of 0.54±0.04 mm, a long diameter of 0.76±0.18 mm, and a weight of 0.85±0.4 mg, showing a significant decrease in cartilage size and weight. Since Col2A1 is a component of cartilage, it can be inferred that Col2A1 deletion leads to a decrease in cartilage size and weight. In addition, Col2A1KO-rSMSCs were found to have lost their staining ability in safranin O-fast green staining and type II collagen immunohistochemistry. This indicates that Col2A1 deletion not only leads to a loss of type II collagen production ability but also affects the production of mucopolysaccharides in the cartilage matrix. This suggests that type II collagen contributes not only to the formation of the cartilage tissue skeleton but also to the induction of cartilage differentiation and matrix production.

[0080] <Comparative Example 3> Suppression of chondrogenic differentiation ability in rat synovial mesenchymal stem cells lacking CD120a The chondrogenic potential of CD120aKO-rSMSO prepared in Comparative Example 1 was investigated. Chondrogenic differentiation induction was carried out using the same differentiation medium and culture conditions as in Example 5. CD120aWT-rSMSC was also similarly induced as a control cell. After 3 weeks of culture, the diameter and weight of the cell aggregates were measured, and the chondrogenic potential was evaluated from histological staining of the cell aggregates. The results are shown in Figure 15. CD120aWT-rSMSC had a short diameter of 1.55±0.14 mm, a long diameter of 2.04±0.25 mm, and a weight of 1.9±0.26 mg, while CD120aKO-rSMSC had a short diameter of 1.19±0.17 mm, a long diameter of 1.53±0.04 mm, and a weight of 1.55±1.20 mg. No significant decrease in cartilage size or weight was observed. Furthermore, since no difference in staining properties was observed in safranin O-fast green and type II collagen immunostaining due to CD120a deletion, CD120a is thought to be a molecule that does not contribute to the chondrogenic differentiation ability of cells.

[0081] <Comparative Example 4> Suppression of chondrogenic differentiation ability in rat synovial mesenchymal stem cells lacking CD106 The cartilage differentiation potential of CD106KO-rSMSO prepared in Comparative Example 2 was investigated. CD106KO-rSMSC 2.5×10 5 Chondrogenic differentiation of cells was induced using the same differentiation medium and culture conditions as in Example 5. CD106WT-rSMSC cells were also induced similarly as control cells. After 3 weeks of culture, the diameter and weight of the cell aggregates were measured, and the chondrogenic differentiation potential was evaluated from histological staining of the cell aggregates. The results are shown in Figure 16. CD106WT-rSMSC cells measured 1.55±0.14 mm in short diameter, 2.04±0.25 mm in long diameter, and weighed 1.9±0.26 mg, while CD106KO-rSMSC cells measured 1.58±0.56 mm in short diameter, 1.75±0.52 mm in long diameter, and weighed 2.38±1.54 mg. No significant decrease in cartilage size or weight was observed. Furthermore, no difference was observed in the staining properties of safranin O-fast green and type II collagen immunostaining due to CD106 deletion, suggesting that CD106 is a molecule that does not contribute to the chondrogenic differentiation potential of cells.

[0082] <Example 6> Confirmation of the meniscus regeneration effect of rat synovial membrane-derived mesenchymal stem cells with integrin β1 inhibition. The preparation of rat synovial mesenchymal stem cells with inhibited integrin β1 was carried out as described in Example 2. Frozen stocks were awakened, cultured for one week, and the harvested cells were suspended in PBS containing 2% FBS as the reaction solvent. Cell count: 5 x 10⁶ 6 Each cell was treated with 12 μg of Purified anti-mouse / rat integrin β1 Antibody and incubated on ice for 1 hour before being collected for transplantation (integrin β1-rSMSC). As a control without inhibition, Purified Armenian Hamster IgG Isotype Ctrl was incubated on ice for 1 hour before being collected for transplantation (IgG-rSMSC).

[0083] 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 of the knee joint under isoflurane anesthesia to expose the knee joint. The medial joint capsule below the patella was exposed, and a longitudinal incision was made with a scalpel to expose the cartilage of the distal femur. The medial meniscus was separated from the synovial membrane to expose the medial meniscus, and approximately two-thirds of it was resected. The patellar tendon and synovial membrane were sutured, followed by the muscle, to create a meniscus injury model. The treated animals were then divided into three groups, and integrin β1-inhibited synovial stem cells (integrin β1-rSMSC) were administered in 5x10⁶ doses. 6 Cells, synovial stem cells (IgG-rSMSC) treated with a control without inhibition, 5x10 6 Cells and / or the solvent alone were administered intra-articularly. The day after cell administration, 12 μg of Purified anti-mouse / rat integrin β1 Antibody, Armenian Hamster IgG Isotype Ctrl, or the solvent was administered intra-articularly per knee. After the procedure, all rats were returned to their cages and allowed to exercise and eat freely.

[0084] Three weeks after the procedure, the animals were euthanized by dissection of the inferior aorta under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 17 shows images of the excised medial meniscus from both knee joints. The regenerated area was identified and enclosed by a dashed line based on differences in color and shape compared to the normal meniscus. In the solvent group (negative control), many cases showed meniscus regeneration extending to the middle segment. In the IgG-rSMSC group (positive control), many cases showed meniscus regeneration extending to the anterior segment, and the regenerated area was larger. On the other hand, in the integrin β1-rSMSC group (cells with molecular inhibition or deletion), the regenerated area of ​​the meniscus appeared smaller compared to the positive control.

[0085] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 17, the area of ​​the meniscus regeneration area (within the dashed line) was calculated using ImageJ (version 1.52) with the following formula.

[0086] Meniscal regeneration area (mm 2 ) = Number of pixels in the meniscus regeneration area / 1mm 2 Number of pixels per unit

[0087] The mean, standard deviation, and statistical analysis of the regenerated area for each group were all performed using Microsoft Excel 2007 (Microsoft Corp.). Statistical analysis involved performing Student's T-Test twice: once against the positive control group and once against the molecular inhibition group, and again against the molecular inhibition group and once against the solvent group. The p-values ​​for each test were calculated. Due to the repeated testing, Bonferroni correction was applied, and the calculated p-value was multiplied by the number of tests (2). A significance level of 5% (α=0.05) was used to indicate a difference, and the results are shown in Table 1.

[0088] Regarding the average area of ​​the meniscus regeneration portion listed in Table 1, the integrin β1-rSMSC group was 2.1 mm². 2 This is IgG-rSMSC 3.4mm 2 It was significantly reduced compared to the solvent group. On the other hand, the integrin β1-rSMSC group was 1.7 mm in the solvent group. 2The regeneration area was comparable to that of the other group. This confirms that the integrin β1 molecule in synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0089] [Table 1] *P<0.05 vs IgG-rSMSC group

[0090] <Example 7> Confirmation of the meniscus regeneration effect of rat synovial membrane-derived mesenchymal stem cells with PDGFRb inhibition. The preparation of rat synovial mesenchymal stem cells with inhibited PDGFRb was carried out as described in Example 3. Frozen stocks were awakened, cultured for one week, and the harvested cells were suspended in PBS containing 2% FBS as the reaction solvent. Cell count: 5 x 10⁶ 6 Cells were treated with 12 μg of Anti-PDGF ReceptorβHuman Goat-Poly (R&D Systems Cat.No.AF385) under ice cooling for 1 hour, and then collected for transplantation (PDGFRb-rSMSC). As a control treatment without inhibition, Normal Goat IgG Control (R&D Systems Cat.No.AB-108-C) was treated under ice cooling for 1 hour, and then collected for transplantation (IgG-rSMSC).

[0091] A meniscus injury model to evaluate the meniscus regeneration effect was prepared as described in Example 6. Subsequently, the treated animals were divided into three groups, and PDGFRb-inhibited synovial stem cells (PDGFRb-rSMSCs) were divided into 5x10⁻¹⁰ groups. 6 Cells, synovial stem cells (IgG-rSMSC) treated with a control without inhibition, 5x10 6 Cells and solvents alone were administered intra-articularly. The day after cell administration, 12 μg of Anti-PDGF Receptorβ Human Goat-Poly, Normal Goat IgG Control, or the solvent was administered intra-articularly per knee. After the procedure, all rats were returned to their cages and allowed to exercise and eat freely.

[0092] Three weeks after the procedure, the animals were euthanized by dissection of the inferior aorta under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 18 shows images of the excised medial meniscus from both knee joints. The regenerated area was identified and enclosed by a dashed line based on differences in color and shape compared to the normal meniscus. In the solvent group (negative control), many cases showed meniscus regeneration extending to the middle segment. In the IgG-rSMSC group (positive control), many cases showed meniscus regeneration extending to the anterior segment, and the regenerated area was larger. On the other hand, in the PDGFRb-rSMSC group (cells with molecular inhibition or deletion), the regenerated area of ​​the meniscus appeared smaller compared to the positive control.

[0093] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 18, the area of ​​the meniscus regeneration area (within the dashed line) was measured as described in Example 6. The results are shown in Table 2. The average area value of the meniscus regeneration area was 2.2 mm for the PDGFRb-rSMSC group. 2 This is IgG-rSMSC 3.2mm 2 It was significantly reduced compared to the solvent group. On the other hand, the PDGFRb-rSMSC group was 1.3 mm 2 The levels were significantly increased. This confirmed that PDGFRb molecules in synovial stem cells are molecules that contribute to meniscus regeneration.

[0094] [Table 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 rat synovial membrane-derived mesenchymal stem cells with CD44 inhibition. To prepare rat synovial mesenchymal stem cells with inhibited CD44, frozen stock cells were awakened, cultured for one week, and the harvested cells were suspended in PBS containing 2% FBS as the reaction solvent. (Cell count: 5 x 10⁶) 6Cells were treated with 12 μg of Anti-CD44 Rabbit IgGclone Hermes-1 (Absolute Antibody Cat.No. Ab00628-23.0) per cell for 30 minutes under ice cooling, and then collected for transplantation (CD44-rSMSC). As a control treatment without inhibition, Rabbit IgG Isotype Control (invitrogen Cat.No. 10500C) was treated with ice cooling for 1 hour, and then collected for transplantation (IgG-rSMSC).

[0096] A meniscus injury model to evaluate the meniscus regeneration effect was prepared as described in Example 6. Subsequently, the treated animals were divided into three groups, and 5 x 10⁻¹ synovial stem cells (CD44-rSMSC) with inhibited CD44 were introduced. 6 Cells, synovial stem cells (IgG-rSMSC) treated with a control without inhibition, 5x10 6 Cells and solvents were administered intra-articularly, respectively. The day after cell administration, 12 μg of Anti-CD44 Rabbit IgG clone Hermes-1, Rabbit IgG Isotype Control, or the solvent was administered intra-articularly per knee. After the procedure, all rats were returned to their cages and allowed to exercise and eat freely.

[0097] Four weeks after the procedure, the animals were euthanized by dissection of the inferior aorta under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 19 shows images of the excised medial meniscus from both knee joints. The regenerated portion was identified and enclosed by a dashed line based on differences in color and shape compared to the normal meniscus. In the solvent group (negative control), many cases showed meniscus regeneration extending to the middle segment. In the IgG-rSMSC and CD44-rSMSC groups (positive controls), many cases showed meniscus regeneration extending to the anterior segment, resulting in a larger regenerated area.

[0098] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 19, the area of ​​the meniscus regeneration area (within the dashed line) was measured as described in Example 6. The results are shown in Table 3. The average area value of the meniscus regeneration area was 3.4 mm for the CD44-rSMSC group. 2 This is IgG-rSMSC 4.0 mm 2 There was no significant difference compared to the other group, and they were similar. On the other hand, the CD44-rSMSC group was 2.7 mm in size compared to the solvent group. 2 It was significantly increased in comparison. From this, it is thought that CD44 in synovial stem cells is a molecule that does not contribute to meniscus regeneration.

[0099] [Table 3] †P<0.05 vs solvent group

[0100] <Example 8> Confirmation of the meniscus regeneration effect of rat synovial membrane-derived mesenchymal stem cells lacking Col2A1 (Col2A1KO-rSMSC) As described in Example 4, rat synovial mesenchymal stem cells lacking Col2A1 were prepared for transplantation by expanding culture after confirming Col2A1 deletion (Col2A1KO-rSMSC). As a positive control, Col2A1 wild-type sequences were confirmed and then prepared for transplantation by expanding culture (Col2A1WT-rSMSC).

[0101] A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in Example 6. The treated animals were then divided into three groups, with Col2A1KO-rSMSC and Col2A1WT-rSMSC being divided into 5x10⁻¹⁰⁻¹ 6 Only the cells and the solvent were administered intra-articularly.

[0102] Three weeks after the procedure, the animals were euthanized by dissection of the inferior aorta under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 20 shows images of the excised medial meniscuses from both knee joints. The regenerated portion was identified and enclosed by a dashed line based on differences in color and shape compared to the normal meniscus. In the solvent group (negative control), many cases showed meniscus regeneration extending to the middle segment. In the Col2A1WT-rSMSC group (positive control), many cases showed meniscus regeneration extending to the anterior segment, resulting in a larger regenerated area. Conversely, in the Col2A1KO-rSMSC group, the regenerated area appeared smaller compared to the positive control.

[0103] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 20, the area of ​​the meniscus regeneration area (within the dashed line) was measured as described in Example 6. The results are shown in Table 4. The average area value of the meniscus regeneration area was 2.9 mm for the Col2A1KO-rSMSC group. 2 In comparison, Col2A1WTrSMSC has a 3.8mm 2 This was significantly increased. On the other hand, the Col2A1KO-rSMSC group was 2.1 mm higher than the solvent group. 2 The levels were significantly increased compared to the previous level. This confirmed that the Col2A1 molecule in synovial stem cells is a molecule that contributes to meniscus regeneration.

[0104] [Table 4] *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 membrane-derived mesenchymal stem cells lacking CD120a (CD120aKO-rSMSC) As described in Comparative Example 1, rat synovial mesenchymal stem cells lacking CD120a were prepared for transplantation by expanding culture after confirmation of CD120a deletion (CD120aKO-rSMSC). As a positive control, CD120a wild-type sequence confirmation was performed, and then prepared for transplantation by expanding culture (CD120aWT-rSMSC).

[0106] A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in Example 6. The treated animals were then divided into three groups, and 5x10⁻¹ CD120aKO-rSMSC and CD120aWT-rSMSC were applied. 6 Only the cells and the solvent were administered intra-articularly.

[0107] Three weeks after the procedure, the animals were euthanized by dissection of the inferior aorta under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 21 shows images of the excised medial meniscus from both knee joints. The regenerated portion was identified and enclosed by a dashed line based on differences in color and shape compared to the normal meniscus. In the solvent group (negative control), many cases showed meniscus regeneration extending to the middle segment. On the other hand, in the CD120aWT-rSMSC and CD120aKO-rSMSC groups (positive controls), many cases showed meniscus regeneration extending to the anterior segment, and the regenerated portion of the meniscus was larger compared to the solvent group.

[0108] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 21, the area of ​​the meniscus regeneration area (within the dashed line) was measured as described in Example 6. The results are shown in Table 5. The average area value of the meniscus regeneration area was 3.1 mm for the CD120aKO-rSMSC group. 2 In comparison, the CD120aWT-rSMSC has a 3.3mm diameter. 2 No significant difference was observed. On the other hand, the CD120aKO-rSMSC group was 1.8 mm in size compared to the solvent group. 2 It was significantly increased compared to [previous level]. From this, it is thought that CD120a in synovial stem cells is a molecule that does not contribute to meniscus regeneration.

[0109] [Table 5] †P<0.05 vs solvent group

[0110] <Comparative Example 7> Confirmation of the meniscus regeneration effect of rat synovial membrane-derived mesenchymal stem cells lacking CD106 (CD106aKO-rSMSC) As described in Comparative Example 1, rat synovial mesenchymal stem cells lacking CD106 were prepared for transplantation by expanding culture after confirmation of CD106 deletion (CD106KO-rSMSC). As a positive control, CD106 wild-type sequences were confirmed and then prepared for transplantation by expanding culture (CD106WT-rSMSC).

[0111] A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in Example 6. The treated animals were then divided into three groups, and 5x10⁻¹ CD106KO-rSMSC and CD106WT-rSMSC were applied. 6 Only the cells and the solvent were administered intra-articularly.

[0112] Three weeks after the procedure, the animals were euthanized by dissection of the inferior aorta under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 22 shows images of the excised medial meniscus from both knee joints. The regenerated portion was identified and enclosed by a dashed line based on differences in color and shape compared to the normal meniscus. In the solvent group (negative control), many cases showed meniscus regeneration extending to the middle segment. On the other hand, in the CD106WT-rSMSC and CD106KO-rSMSC groups (positive controls), many cases showed meniscus regeneration extending to the anterior segment, and the regenerated portion of the meniscus was larger compared to the solvent group.

[0113] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 22, the area of ​​the meniscus regeneration area (within the dashed line) was measured as described in Example 6. The results are shown in Table 6. The average area value of the meniscus regeneration area was 3.7 mm for the CD106KO-rSMSC group. 2 In comparison, the CD106WT-rSMSC has a 3.3mm diameter. 2 No significant difference was observed. On the other hand, the CD106KO-rSMSC group showed a 1.7 mm difference compared to the solvent group. 2 It was significantly increased compared to [previous level]. From this, it is thought that CD106 in synovial stem cells is a molecule that does not contribute to meniscus regeneration.

[0114] [Table 6] †P<0.05 vs solvent group

[0115] <Example 9> Rat synovial membrane-derived mesenchymal stem cell establishment process, differences in processing time during cell harvesting, and integrin β1 and PDGFRb expression rates Similar to Example 1, cells were isolated from rat synovial membrane and cultured for 8 days to obtain rat synovial membrane-derived mesenchymal stem cells. The culture medium in the flask was discarded, the flask was washed twice with PBS, and TrypLE Express (Gibco Cat. No. 12604-013) was added. The flask was then incubated at 37°C for 5, 30, 60, and 120 minutes, after which the cells were detached and collected.

[0116] Cells collected under each condition 10 6Cells 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 mmol / L EDTA·2Na and 1% bovine serum albumin) was added to suspend the cells. To measure the expression rate of integrin β1, 5 μL of PE anti-mouse / rat integrin β1 Antibody (Biolegen Cat. No. 102207) or PE Armenian Hamster IgG Isotype Ctrl Antibody (Biolegen Cat. No. 400907) was added and incubated at 4°C for 30 minutes. The reagent was then centrifuged, the supernatant discarded, and resuspended in 1 mL of FACS buffer. It was then centrifuged again, the supernatant discarded, and resuspended in 500 μL of FACS buffer for measurement. To measure the expression rate of PDGFRb, 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 the mixture was reacted at 4°C for 30 minutes. The reagent was then centrifuged, the supernatant discarded, and resuspended in 1 mL of FACS buffer. Furthermore, 1 μL of Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, FITC (invitrogen Cat. No. A16006) was added, and the mixture was reacted at 4°C for 30 minutes. Subsequently, the sample was centrifuged, the supernatant discarded, and resuspended in 1 mL of FACS buffer. After centrifugation again, the supernatant was discarded, and the sample was resuspended in 500 μL of FACS buffer. The integrin β1 and PDGFRb expression rates were then measured using a flow cytometer (Attune NxT, Autofocusing Cytometer model: AFC2, invitrogen).

[0117] Table 7 shows the expression rates of integrin β1 and PDGFRb in synovial mesenchymal stem cells at different exfoliation treatment times. Even when the exfoliation 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, reaching 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 necessary for meniscus regeneration in synovial stem cells, and a greater meniscus regeneration effect can be expected when the PDGFRb expression rate accounts for more than half of the cells during the cell exfoliation treatment time. Therefore, a treatment time of 60 minutes or less is desirable, and the required molecular expression rate can be set at 60% or higher.

[0118] [Table 7]

[0119] <Example 10> Culture period and integrin β1 expression rate at the time of cell harvesting when rat synovial membrane-derived mesenchymal stem cell establishment Similar to Example 1, cells were isolated from rat synovial membrane and 75 cm³ of the sample. 2 A flask with an area of ​​7.5 x 10 4 Cells were seeded and cultured for 8, 21, and 28 days to obtain rat synovial mesenchymal stem cells. The culture medium in the flask was discarded, the cells were washed twice with PBS, and TrypLE Express (Gibco Cat. No. 12604-013) was added. The cells were incubated at 37°C for 120 minutes, and then detached and collected. Subsequently, dead cell and integrin β1 staining procedures were performed as in Example 9, and the cells were subjected to measurement.

[0120] Table 8 shows the integrin β1 expression rates of synovial-derived mesenchymal stem cells at different culture durations. The integrin β1 expression rate of synovial-derived mesenchymal stem cells decreased in a culture duration-dependent manner, reaching 97.8% after 8 days, 62.1% after 21 days, and 56.1% after 28 days. As shown in Example 6, integrin β1 is a molecule necessary for meniscus regeneration in synovial stem cells, so a majority of cells being integrin β1 positive is desirable. Also, as shown in Example 7, since the expression rate of molecules necessary for meniscus regeneration can be set at 60% or higher, a culture duration of 21 days or less is desirable.

[0121] [Table 8]

[0122] <Example 11> For human synovial stem cells (Cryopreserved Synoviocytes, Normal, P1, Model No.: CDD-H-2910-N, Lots: ST1414, ST1420, ST1434, ST1462) purchased from Articular Engineering, the positive rates for integrin β1 and PDGFRb, protein molecules essential for drug efficacy, were measured by flow cytometry. The measurement instruments used were Attune NxT and Autofocusing Cytometer (model: AFC2, invitrogen). The antibodies used were integrin β1 (APC Mouse Anti-Human CD29 Cat: 559883) and PDGFRb (Anti-PDGF Receptor β, Human, Goat-Poly Cat: AF385), respectively.

[0123] As a result, the surface antigen positivity rates for integrin β1 were 99.4%, 99.6%, 99.6%, and 99.5% for each lot. Furthermore, the surface antigen positivity rates for PDGFRb were found to be 92.2%, 90.7%, 95.2%, and 85.9% for each lot. Therefore, if these human synovial-derived stem cells have therapeutic efficacy as a treatment for arthritis, it is appropriate to manage them with specification values ​​of 90% or higher for integrin β1 and 80% or higher for PDGFRb.

[0124] Thus, the expression of integrin β1 and PDGFRb was confirmed not only in rat synovial stem cells in Examples 9 and 10, but also in human synovial stem cells, demonstrating that these can be established as quality control items for cell efficacy.

[0125] <Example 12> Confirmation of the meniscus regeneration effect of rat synovial stem cells with inhibited FGFR3 To prepare rat synovial stem cells with inhibited FGFR3, the frozen stock of rat synovial stem cells prepared in Example 1 was awakened and cultured for 1 week at 37°C with αMEM no nucleosides to which Fetal Bovine Serum was added to a final concentration of 20%, L-glutamie 200 mmol / L to a final concentration of 1%, and Antibiotic-Antimycotic (100×) to a final concentration of 1%. The harvested cells were suspended in PBS containing 2% FBS as the reaction solvent. Cell count: 5 × 10⁶ 6 100 μg of FGFR3 Polyclonal Antibody (Invitrogen Cat. No. PA5-34574) was added per cell, and the cells were reacted under ice conditions for 1 hour before being collected (FGFR3-rSMSC). As a control treatment without FGFR3 inhibition, Rabbit IgG Isotype Control (Thermo Fisher Scientific Cat. No. 10500C) was reacted under ice conditions for 1 hour before being collected (IgG-rSMSC).

[0126] 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 of the knee joint under isoflurane anesthesia to expose the knee joint. The medial joint capsule below the patella was exposed, and a longitudinal incision was made with a scalpel to expose the cartilage of the distal femur. The medial meniscus was separated from the synovial membrane to expose it, and approximately two-thirds of it was resected from the anterior end. The patellar tendon and synovial membrane were sutured, followed by suture of the muscles to create a meniscus injury model. The treated animals were then divided into three groups, and 5 × 10⁶ synovial stem cells (FGFR3-rSMSC) inhibited from FGFR3 were transplanted. 6 Synovial stem cells (IgG-rSMSCs) treated with a control without inhibition were divided into 5 × 10 6 Only the cells and the solvent were administered intraarticularly. After the procedure, all rats were returned to their cages and allowed to exercise and eat freely.

[0127] Three weeks after the procedure, the animals were euthanized by discharging blood through inferior aortic transection under isoflurane anesthesia. Subsequently, the meniscus was exposed through the knee joint, and the medial meniscus was excised and photographed. Figure 23 shows images of the excised medial meniscus from both knee joints. The regenerated area was identified and enclosed by a dashed line based on differences in color and shape compared to a normal meniscus. In the solvent group (negative control), many animals had meniscus regeneration extending to the middle segment. In the IgG-rSMSC group (positive control), many cases showed meniscus regeneration extending to the anterior segment, resulting in a larger regenerated area. Conversely, in the FGFR3-rSMSC group (cells with molecular inhibition or deletion), the regenerated area appeared smaller compared to the positive control.

[0128] To quantitatively evaluate the macroscopic findings of the meniscus regeneration area in Figure 23, the area of ​​the meniscus regeneration area (within the dashed line) was calculated using Image J (version 1.52) with the following formula.

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

[0130] The mean, standard deviation, and statistical analysis of the regenerated area for each group were all performed using Microsoft Excel 2007 (Microsoft Corp.). Statistical analysis involved performing Student's T-Test twice: once against the positive control group and once against the molecular inhibition group, and again against the molecular inhibition group and once against the solvent group. The p-values ​​were calculated for each test. While the significance level was 5% (α=0.05), a difference level of 2.5% (α=0.025) was achieved by applying Bonferroni correction (dividing by the number of tests, 2). The results are shown in Table 9.

[0131] Regarding the average area of ​​the meniscus regeneration portion listed in Table 9, the FGFR3-rSMSC group was 1.2 mm². 2 This is IgG-rSMSC 1.9mm 2 It was significantly reduced compared to the solvent group. On the other hand, the FGFR3-rSMSC group showed a 1.0 mm decrease compared to the solvent group. 2 The regeneration area was comparable to that of the other group. This confirms that the FGFR3 molecule in synovial stem cells is an important molecule that contributes to meniscus regeneration.

[0132] [Table 9] *P< 0.025 vs IgG-rSMSC group †P<0.025 vs solvent group

Claims

1. A method for quality control of cells for treating arthritis, comprising: step A of treating synovial tissue with an enzyme; step B of washing the mixture after enzyme treatment; step C of culturing synovial-derived mesenchymal stem cells contained in the washed mixture on a substrate; and step D of separating the cultured synovial-derived mesenchymal stem cells from the substrate, wherein the cell population has a positive rate of 80% or more for integrin β1 or platelet-derived growth factor receptor β.

2. The cell quality control method according to claim 1, wherein the cell population has a gene encoding type II collagen α1 chain and produces type II collagen α1 chain after transplantation.

3. The cell quality control method according to claim 1 or 2, wherein the cell population has the surface antigen of FFFR3.

4. The cell quality control method according to claim 1, wherein step B is a step of washing the mixture after enzyme treatment until the residual enzyme concentration in the supernatant is 0.5 ng / mL or less.

5. The cell quality control method according to claim 1 or 4, wherein in step C, the period for culturing synovial membrane-derived mesenchymal stem cells is 28 days or less.

6. The cell quality control method according to claim 1 or 4, wherein in step D, mesenchymal stem cells are separated by allowing a cell detachment solution to act on them for a period of 120 minutes or less.

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