CN117917960A - Therapeutic agent for arthropathy and method for producing therapeutic agent for arthropathy - Google Patents
Therapeutic agent for arthropathy and method for producing therapeutic agent for arthropathy Download PDFInfo
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- CN117917960A CN117917960A CN202280058697.0A CN202280058697A CN117917960A CN 117917960 A CN117917960 A CN 117917960A CN 202280058697 A CN202280058697 A CN 202280058697A CN 117917960 A CN117917960 A CN 117917960A
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- stem cells
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- meniscus
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Abstract
The present invention addresses the problem of providing an arthropathy therapeutic agent comprising synovial-derived mesenchymal stem cells having molecules necessary for joint treatment, and a method for producing the arthropathy therapeutic agent. According to the present invention, there is provided an arthropathy therapeutic agent comprising a synovial-derived mesenchymal stem cell having a surface antigen of at least one of integrin β1 and platelet-derived growth factor receptor β.
Description
Technical Field
The present invention relates to an arthropathy therapeutic agent comprising synovial-derived mesenchymal stem cells having molecules necessary for joint treatment, and a method for producing the arthropathy therapeutic agent.
Background
In recent years, with the progress of regenerative medicine and cytotherapy, development of various cytotherapeutic and research cell products using autologous, allogeneic or xenogeneic cells has been actively conducted. Among them, mesenchymal stem cells (MESENCHYMAL STEM CELL: MSC) are expected as a cell source for useful cell therapy. It has been reported that mesenchymal stem cells can be collected from various body tissues and 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), periosteal tissue (non-patent document 5) and the like. In particular, it has been reported that synovial-derived mesenchymal stem cells have higher proliferation and cartilage formation ability than mesenchymal stem cells derived from various mesenchymal tissues such as bone marrow (non-patent document 6). Patent documents 1 to 3 disclose methods for treating articular cartilage damage and meniscus damage using synovial membrane-derived mesenchymal stem cells. Patent document 4 describes a method for producing a limb bud mesenchymal cell group, a cartilage precursor cell group, and an osteoblast precursor cell group, each using a molecule such as CD140b, and a quality control method.
Prior art literature
Non-patent literature
Non-patent document 1: prockop, D.J.,1997, science.276:71-4
Non-patent document 2: zuk, P.A. et al 2002,Mol Biol Cell.13:4279-95
Non-patent document 3: cao et al 2003,Nat Cell Biol.5:640-6
Non-patent document 4: de Bari, c.et al, 2001,Arthritis Rheum.44:1928-42
Non-patent document 5: fukumoto, T.et al, 2003,Osteoarthritis Cartilage.11:55-64
Non-patent document 6: sakaguchi et al 2005,Arthritis Rhum.52:2521-9
Patent literature
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
Disclosure of Invention
Technical problem to be solved by the invention
In quality control of cell products, it is a problem to ensure equivalence and identity of each batch. However, since the cells constituting the product are not themselves completely uniform structures and it is difficult to determine the characteristics thereof, it is often difficult to ensure the equivalence and identity of each batch. Therefore, in order to manage the Quality of the product, not only Quality tests of the final product are performed, but also QMS (Quality MANAGEMENT SYSTEM: quality management system) concepts applied to medical equipment are introduced, and the manufacturing raw materials, material management, manufacturing process management, and process management tests are recorded and controlled, thereby performing management of the entire process. However, with advances in science and technology, the importance of identifying characteristics of cells themselves as end products is increasing.
As a method for quality control of cells, for example, identification of the cell type of a target final product (for example, identification as mesenchymal stem cells) is being performed using a cell type-specific surface marker as an index. However, the cells obtained by this quality control method have a concern that the therapeutic effect is unstable, and are still not completely satisfactory.
Methods for producing joint therapeutic agents using synovial stem cells have been reported. However, quality control for ensuring the effectiveness of the therapeutic agent is not sufficiently performed, and there is a problem in that the therapeutic effect is not stable. The cell product is mainly quality-managed with markers for determining cell type, but not quality-managed with respect to potency (validity).
Since the label indicating the potency (effectiveness) is identified based on the mechanism of action, in the present invention, it is intended to clarify the mechanism of action related to the effectiveness of a cell therapy product, and to identify the label based on the mechanism of action, and further to provide a method for producing a therapeutic agent based on the label. That is, an object of the present invention is to provide an arthropathy therapeutic agent comprising synovial-derived mesenchymal stem cells having molecules necessary for joint treatment, and a method for producing the arthropathy therapeutic agent.
Means for solving the technical problems
As a result of intensive studies to solve the above problems, the present inventors have found that one or more of integrin β1 and platelet-derived growth factor receptor β is a quality control marker necessary for the therapeutic effect of treating joint diseases using synovial stem cells. The present invention has been completed based on the above findings.
That is, according to the present invention, the following invention can be provided.
< 1 > An arthropathy therapeutic agent comprising a synovial-derived mesenchymal stem cell having a surface antigen of at least one of integrin beta 1 or platelet-derived growth factor receptor beta.
< 2> The therapeutic agent for arthrosis according to < 1 >, wherein,
The synovial-derived mesenchymal stem cells have surface antigens of both integrin beta 1 and platelet-derived growth factor receptor beta.
< 3 > The therapeutic agent for arthrosis according to <1> or < 2 > which has a gene encoding type II collagen alpha 1 chain and produces type II collagen alpha 1 chain after transplantation.
< 4 > The therapeutic agent for arthrosis according to any one of < 1 > to < 3 > having a surface antigen of FGFR 3.
An arthrosis therapeutic agent according to any one of < 1 > to < 4 > wherein,
The ratio of synovial membrane-derived mesenchymal stem cells having a surface antigen of at least one of integrin beta 1 and platelet-derived growth factor receptor beta relative to all cells contained in the therapeutic agent for arthrosis is at least 30%.
A method for producing an arthropathy therapeutic agent according to any one of < 6 > 1 > to < 5 >, comprising:
Step A, treating synovial tissue with enzyme;
Step B, cleaning the enzyme-treated mixture;
Step C, culturing the mesenchymal stem cells of synovial origin contained in the cleaned mixture on the substrate; and
And step D, separating the cultured mesenchymal stem cells from the substrate.
The method of < 7 > according to < 6 >, wherein,
The step B is a step of washing the enzyme-treated mixture to a residual enzyme concentration of 0.5ng/mL or less in the supernatant.
The method according to < 8 > is < 6 > or < 7 >, wherein,
In the step C, the period of culturing the synovial-derived mesenchymal stem cells is 28 days or less.
The method according to any one of < 6 > to < 8 >, wherein,
In the step D, the mesenchymal stem cells are allowed to act on the cell-separating liquid for a period of time of 120 minutes or less.
The method of any one of < 10 > to < 6 > to < 9 >, further comprising a step of sorting the synovial-derived mesenchymal stem cells having the surface antigen of any one or more of integrin beta 1 or platelet-derived growth factor receptor beta.
Effects of the invention
The therapeutic agent for arthrosis of the present invention can exert therapeutic effects on arthrosis by comprising synovial-derived mesenchymal stem cells having a surface antigen of at least one of integrin beta 1 and platelet-derived growth factor receptor beta. According to the present invention, variation in the therapeutic effect of the produced therapeutic agent for arthropathy can be suppressed, and the therapeutic effect of the product can be quality-controlled.
Drawings
Fig. 1 shows the results of examining the inhibition of extracellular matrix adhesive capacity of rat synovial-derived mesenchymal stem cells based on the inhibition of integrin β1.
Fig. 2 shows the results of examining the inhibition of the cell proliferation ability of rat synovial-derived mesenchymal stem cells by PDGFRb inhibition.
FIG. 3 shows Col2A1 base sequences (first half) of Col2A1 gene wild type (Col 2A1 WT-rSMSC) and deletion type (Col 2A1 KO-rSMSC) rat synovial stem cells.
FIG. 4 shows Col2A1 base sequences (second half) of Col2A1 gene wild type (Col 2A1 WT-rSMSC) and deletion type (Col 2A1 KO-rSMSC) rat synovial stem cells.
FIG. 5 shows Col2A1 base sequences (first half) of Col2A1 gene wild type (Col 2A1 WT-rSMSC) and deletion type (Col 2A1 KO-rSMSC) rat synovial stem cells.
FIG. 6 shows Col2A1 base sequences (latter half) of Col2A1 gene wild type (Col 2A1 WT-rSMSC) and deletion type (Col 2A1 KO-rSMSC) rat synovial stem cells.
FIG. 7 shows the amino acid sequence translated based on the Col2A1 base sequence of Col2A1 gene wild type (Col 2A1 WT-rSMSC) rat synovial stem cells.
FIG. 8 shows the amino acid sequence translated based on the Col2A1 base sequence of Col2A1 gene-deleted (Col 2A1 KO-rSMSC) rat synovial stem cells.
FIG. 9 shows the amino acid sequence translated based on the Col2A1 base sequence of Col2A1 gene-deleted (Col 2A1 KO-rSMSC) rat synovial stem cells.
FIG. 10 shows the nucleotide sequences of CD120a of the wild-type (CD 120 aWT-rSMSC) and deleted (CD 120 aKO-rSMSC) rat synovial stem cells of the CD120a gene.
FIG. 11 shows the amino acid sequences translated based on the CD120a base sequences of the CD120a gene wild type (CD 120 aWT-rSMSC) and deletion type (CD 120 aKO-rSMSC) rat synovial stem cells.
FIG. 12 shows the nucleotide sequences of CD106 gene wild type (CD 106 WT-rSMSC) and deletion type (CD 106 KO-rSMSC) rat synovial stem cells.
FIG. 13 shows the amino acid sequences translated based on the CD106 base sequences of the CD106 gene wild type (CD 106 WT-rSMSC) and deletion type (CD 106 KO-rSMSC) rat synovial stem cells.
FIG. 14 shows the results of examining the inhibition of the ability of the rat mouse to differentiate into cartilage in the Col2A 1-deficient mesenchymal stem cells.
FIG. 15 shows the results of examining the inhibition of the cartilage differentiation ability in the rat synovial-derived mesenchymal stem cells in which CD120a had been deleted.
Fig. 16 shows the results of examining the inhibition of the cartilage differentiation ability in the rat synovial-derived mesenchymal stem cells in which CD106 had been deleted.
FIG. 17 shows the results of confirming the effect of meniscus regeneration of rat synovial-derived mesenchymal stem cells in which integrin beta 1 was inhibited.
Fig. 18 shows the result of confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells in which PDGFRb was inhibited.
Fig. 19 shows the result of confirming the meniscus regeneration effect of the rat synovial-derived mesenchymal stem cells with inhibited CD 44.
FIG. 20 shows the results of confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells (Col 2A1 KO-rSMSC) lacking Col2A 1.
FIG. 21 shows the results of confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells (CD 120 aKO-rSMSC) lacking CD120 a.
FIG. 22 shows the results of confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells (CD 106 aKO-rSMSC) lacking CD 106.
Fig. 23 shows the result of confirming the meniscus regeneration effect of rat synovial-derived mesenchymal stem cells in which FGFR3 was inhibited.
Detailed Description
Hereinafter, the present invention will be described in detail. In the present specification, "to" means that the numerical values described before and after "are included as a lower limit value and an upper limit value.
The therapeutic agent for arthrosis of the present invention comprises a synovial-derived mesenchymal stem cell having a surface antigen of at least one of integrin beta 1 and platelet-derived growth factor receptor beta (also referred to as PDGFRb in the present specification).
The synovial-derived mesenchymal stem cells may have only either integrin β1 or platelet-derived growth factor receptor β, but preferably have both integrin β1 and platelet-derived growth factor receptor β.
Preferably, the synovial-derived mesenchymal stem cells can exert therapeutic effects by having a gene encoding type II collagen α1 chain and producing type II collagen α1 chain after transplantation.
Preferably, the synovial-derived mesenchymal stem cells have a surface antigen of FGFR3 (fibroblast growth factor receptor3: fibroblast growth factor receptor 3).
The ratio of the synovial membrane-derived mesenchymal stem cells having the surface antigen of at least one of integrin β1 and platelet-derived growth factor receptor β to all cells contained in the therapeutic agent for arthrosis of the present invention is preferably 30% or more, but may be 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
The arthropathy therapeutic agent of the present invention can be produced by a method comprising:
Step A, treating synovial tissue with enzyme;
Step B, cleaning the enzyme-treated mixture;
Step C, culturing the mesenchymal stem cells of synovial origin contained in the cleaned mixture on the substrate; and
And step D, separating the cultured mesenchymal stem cells from the substrate.
Process A of enzymatic treatment of synovial tissue
Synovial tissue can be harvested from the non-weight bearing portion of the joint under anesthesia.
The biological source of the synovial tissue is not particularly limited, and synovial tissue derived from any organism (preferably mammalian) can be used. For example, synovial tissue derived from primates (e.g., chimpanzees, macaque, human) can be used, and it is particularly preferable that synovial tissue derived from human can be used.
The synovial tissue may be synovial tissue from a single donor or may be synovial tissue from multiple donors, but is preferably synovial tissue from a single donor.
For the purpose of administration to humans, in the case of producing mesenchymal stem cells derived from synovial membrane, it is preferable to use tissue derived from a donor having a type of histocompatibility antigen identical or similar to that of the recipient. More preferably, the object for collecting synovial membrane is the same as the object for transplanting synovial membrane-derived mesenchymal stem cells. That is, it is preferable to use synovial tissue collected from the recipient itself (autograft).
The amount of synovial tissue collected can be determined in consideration of the kind of donor or the amount of necessary synovial-derived mesenchymal stem cells. For example, the mesenchymal stem cells derived from the synovial membrane can be obtained from 0.1g to 10g (preferably 0.1g to 2.0g, more preferably 0.1g to 1.5g, still more preferably 0.1g to 1.0 g) of the synovial membrane-derived tissue. The collected synovial tissue is minced with scissors or the like as needed, and then subjected to enzyme treatment described later.
Synovial tissue is treated with enzymes.
The enzyme is not particularly limited as long as it is an enzyme containing protease, but a mixed enzyme containing 1 or more collagenases and 1 or more neutral proteases is preferable. A particularly preferred enzyme is Liberase (registered trademark). As Liberase (registered trademark), for example, liberase MNP-S (manufactured by Roche Diagnostics K.K.) which is an enzyme comprising a type I collagenase, a type II collagenase and a neutral protease (thermolysin: thermolysin) can be used.
The enzyme reaction can be carried out in an aqueous solution containing an enzyme, or an aqueous solution containing human serum can be used. The human serum may be autologous serum or allogeneic serum, but is preferably autologous serum.
The enzyme concentration in the enzyme treatment is preferably 0.01mg/ml to 10mg/ml, more preferably 0.1mg/ml to 10mg/ml, still more preferably 0.5mg/ml to 5.0mg/ml, particularly preferably 0.5mg/ml to 2.0mg/ml, and most preferably 0.7mg/ml to 2.0mg/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.
The enzyme reaction can be carried out at a temperature of preferably 15℃to 40℃and more preferably 20℃to 35℃and still more preferably 25℃to 35 ℃.
The reaction time is not less than 2 hours, more preferably not less than 2.5 hours, still more preferably not less than 3 hours. The upper limit of the reaction time is not particularly limited, and may be 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, or 4 hours or less.
The enzyme-treated mixture contains synovial-derived mesenchymal stem cells.
The enzyme-treated mixture can be transferred to a centrifuge tube through a cell filter and subjected to centrifugation to recover mesenchymal stem cells derived from synovium.
Process B > < step of washing enzyme-treated mixture
In step B, the enzyme-treated mixture is washed.
In step B, the supernatant is preferably washed to a residual enzyme concentration of 0.5ng/mL or less. The residual enzyme concentration in the supernatant is more preferably 0.3ng/mL or less, still more preferably 0.2ng/mL or less, and particularly preferably 0.1ng/mL or less.
The cleaning can be performed by the following method: the mesenchymal stem cells derived from the synovial membrane, which were collected by centrifugation, were resuspended in the medium and centrifuged again (for 5 minutes with 400 g). As the medium, an alpha-modified Eagle minimal basal medium (alpha MEM) can be used, but is not particularly limited. As described above, the washing may be performed a plurality of times (2 times or more) using the medium.
Step C > < culturing synovial-derived mesenchymal stem cells contained in the mixture after washing on a substrate)
In step C, mesenchymal stem cells derived from synovial membrane contained in the mixture after washing are cultured on a substrate.
The substrate may be a planar plastic substrate such as a culture plate, a three-dimensional substrate such as a culture bag, a microcarrier (microcarrier), or a gel, but is not particularly limited.
The medium used for the culture can be prepared by using a medium commonly used for the culture of animal cells as a basal medium. Examples of the medium usually used for culturing animal cells include αmem, DMEM (Dulbecco Modified Eagle Medium), a mixed medium of DMEM and F12 (DMEM: f12=1:1), RPMI medium (GIBCO (registered trademark) RPMI1640 medium, etc.), a mixed medium of DMEM/F12 and RPMI (DMEM/f12:rpmi=1:1), and the like, but are not particularly limited thereto.
The medium may be a serum-containing medium or a serum-free medium. For the purpose of administration to a living body, when mesenchymal stem cells derived from synovial membrane are produced from autologous tissue, the culture medium may contain the same serum. That is, for the purpose of administration to a human, when mesenchymal stem cells derived from synovial membrane are produced from human tissues, a medium containing human serum may be used. When serum is used, it may be autologous serum or allogeneic serum, but is preferably autologous serum. When serum is used, the amount of serum added to the medium is, for example, 20 vol% or less, 10 vol% or less, or 5 vol% or less.
The culture conditions of the cells are not particularly limited, and ordinary cell culture conditions can be employed. For example, the culture at a temperature of 30 to 40℃and 3 to 7% CO 2 can be mentioned, but the culture is not particularly limited. As an example, cultivation at 37℃and 5% CO 2 is given.
In the present invention, it is preferable to perform the culture without changing the medium. In the above-described culture, it is preferable that the synovial-derived mesenchymal stem cells are produced without co-culturing with other cells than the synovial-derived mesenchymal stem cells.
It is known that differentiation of synovial-derived mesenchymal stem cells into chondrocytes proceeds with the extension of the culture period, and therefore if the culture period exceeds a specific length, the chondrogenic capacity of synovial-derived mesenchymal stem cells in situ (in situ) is decreased. Therefore, in the present invention, in order to proliferate synovial-derived mesenchymal stem cells in an undifferentiated state and in a state having good in situ (in situ) cartilage formation ability, it is preferable to regulate the culture period. In the step C, the period of culturing the synovial-derived mesenchymal stem cells is preferably 28 days or less.
In the present invention, it is necessary to prepare a sufficient number of undifferentiated synovial stem cells to cover the cartilage damaged portion and regenerate the affected portion. Therefore, the cultivation 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 still more preferably 10 to 21 days.
It is known that cartilage tissue can be produced in vitro (in vitro) by culturing mesenchymal stem cells in a cartilage formation medium supplemented with transforming growth factor β3 (TGF- β3), dexamethasone (Dexamethasone), and bone morphogenic protein (bone morphogenetic protein) 2 (BMP-2) to differentiate into chondrocytes. Therefore, in the present invention, in order to prevent differentiation of the synovial-derived mesenchymal stem cells into chondrocytes, it is preferable to culture the isolated synovial-derived mesenchymal stem cells in the absence of TGF- β3, dexamethasone or BMP-2.
It is also known that synovial-derived mesenchymal stem cells are inversely proportional to the number of passages of in vitro (in vitro) mesenchymal stem cells and have a reduced in situ (in situ) cartilage forming ability. Therefore, in order to prepare undifferentiated mesenchymal stem cells, it is preferable to manufacture synovial-derived mesenchymal stem cells in the first generation or passage 1.
In the present invention, the serum used in the autologous treatment is derived from autologous, the amount of serum that can be collected from a donor in the autologous treatment is limited, and a cell density of not less than a certain level is required in view of proliferation of the synovial-derived mesenchymal stem cells, so that it is preferable to inoculate and culture the synovial-derived mesenchymal stem cells after the enzyme treatment at a cell density of not less than 100 cells/cm 2 and not more than 5000 cells/cm 2, not less than 200 cells/cm 2 and not more than 5000 cells/cm 2, not less than 500 cells/cm 2 and not more than 5000 cells/cm 2, not less than 500 cells/cm 2 and not more than 2500 cells/cm 2, or not less than 500 cells/cm 2 and not more than 2000 cells/cm 2. In order to proliferate synovial-derived mesenchymal stem cells after the enzyme treatment, the cells are preferably cultured for 10 days or more.
The number of cells obtained at the end of the culture is preferably 1.0X10 7 cells or more, 2.0X10 7 cells or more, 2.5X10 7 cells or more, or 3.0X10 7 cells or more, more preferably 4.0X10 7 cells or more, still more preferably 5.0X10 7 cells or more, and particularly preferably 6.0X10 7 cells or more.
Step D > -separating the cultured synovial-derived mesenchymal stem cells from the substrate
In step D, the cultured synovial-derived mesenchymal stem cells are isolated from the substrate. In step D, it is preferable that the separation is performed by allowing the cell-releasing liquid to act on the mesenchymal stem cells for a period of time of 120 minutes or less. The cell-removing liquid is a solution containing trypsin-like enzyme and EDTA. A particularly preferred enzyme is TrypLE. As the TrypLE, trypL Express (manufactured by Gibco), TRYPLE SELECT (manufactured by Gibco), or the like can be used, for example.
In view of sufficiently separating cells, the time for allowing the cell separation liquid to act on the mesenchymal stem cells is preferably 10 minutes or longer. The time for allowing the cell-releasing liquid to act on the mesenchymal stem cells is preferably 10 minutes to 120 minutes, more preferably 10 minutes to 60 minutes or less. It may be 10 to 50 minutes, 10 to 40 minutes, 20 to 60 minutes, 20 to 50 minutes or 20 to 40 minutes.
Mesenchymal stem cells are adult stem cells derived from mesodermal tissue (stroma). Mesenchymal stem cells are known to exist in bone marrow, synovium, periosteum, adipose tissue, muscle tissue, and are known to have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, and fascia cells. Regarding differentiation of mesenchymal stem cells into chondrocytes, it is known that differentiation of undifferentiated mesenchymal stem cells into chondrocytes can be promoted by adding BMP or TGF- β to a culture medium, and that cartilage tissue can be regenerated under in vitro (in vitro) conditions.
Mesenchymal stem cells can be confirmed by detecting characteristic molecules (e.g., enzymes, receptors, low molecular compounds, etc.) in mesenchymal stem cells. The characteristic molecules in the mesenchymal stem cells include, but are not limited to, cell surface markers (positive markers) CD73, CD90, CD105, CD166, and the like. The negative markers not expressed in the mesenchymal stem cells include, but are not limited to, CD19, CD34, CD45, HLA-DR, CD11b, and CD 14. In addition, CD is an abbreviation for Clusters of differentiation (cluster of differentiation), HLA-DR is an abbreviation for human leukocyte antigen-D-related (human leukocyte antigen-D). These positive and negative markers can be used to confirm that they are mesenchymal stem cells. Although immunological methods can be used for detection of these markers, detection can also be performed by quantifying the amount of mRNA of each molecule.
In the present specification, the synovial-derived mesenchymal stem cells are stem cells contained in a synovial source. The synovial-derived mesenchymal stem cell is one of mesenchymal stem cells. The synovial-derived mesenchymal stem cells can be detected by detecting, for example, CD 90-positive, CD 45-negative, and cartilage differentiation ability, but the detection method is not particularly limited.
When the synovial membrane-derived mesenchymal stem cells produced by the above method are used as an arthropathy therapeutic agent, the cells may be mixed with a pharmaceutically acceptable carrier or the like by a conventional method to prepare a preparation in a form suitable for administration to an individual. Examples of the carrier include distilled water for injection, which is isotonic by adding physiological saline, glucose or other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Further, a buffer (e.g., phosphate buffer, sodium acetate buffer), a soothing agent (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc. may be mixed.
Preferably, the method for producing an arthropathy therapeutic agent of the present invention may further comprise a step of sorting synovial mesenchymal stem cells having a surface antigen of at least one of integrin β1 and platelet-derived growth factor receptor β.
The step of sorting the synovial membrane-derived mesenchymal stem cells having any one or more surface antigens selected from integrin β1 and platelet-derived growth factor receptor β includes a step of controlling the expression level of integrin β1 or platelet-derived growth factor receptor β.
The expression level of integrin β1 or platelet-derived growth factor receptor β refers to the expression amount of the gene or protein of integrin β1 or platelet-derived growth factor receptor β. The expression level of integrin beta 1 or platelet-derived growth factor receptor beta can be calculated as an absolute value or a relative value (comparison of a control or a ratio or difference with a reference expression level, etc.).
The level of integrin beta 1 or platelet-derived growth factor receptor beta expression can be determined by any method known to those skilled in the art and can be carried out according to conventional methods. As the measurement of the expression level, the amount of mRNA as a gene transcript can be measured. The method for measuring the amount of mRNA is not particularly limited as long as it is a method capable of measuring the desired amount of mRNA, and can be appropriately selected from known methods. For example, the following method can be utilized: a gene amplification method using an oligonucleotide hybridizing to a gene encoding integrin beta 1 or platelet-derived growth factor receptor beta as a primer, or a hybridization method using an oligonucleotide (poly) hybridizing to a gene encoding a specific protein molecule as a probe. Specifically, the method includes RT-PCR (reverse transcription polymerase chain reaction), real-time RT-PCR, DNA microarray, cell array, northern blot, spot blot, and RNase protection assay (RNase protection assay).
The primer or probe used in the above measurement method can be labeled and the signal intensity of the label can be detected to measure the amount of mRNA. The real-time RT-PCR method is preferable because it can directly use RNA as a sample, and can perform gene quantification based on the number of temperature cycles required for amplification by optically measuring a gene amplification process. In addition, as a control, the expression level of the gene encoding integrin β1 or platelet-derived growth factor receptor β can be normalized using the expression level of mRNA such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or Actin (β -Actin) as a housekeeping gene. In addition, regarding the primers and probes used in the above-described measurement method, those skilled in the art can appropriately design and prepare based on information on the base sequence of the gene encoding integrin β1 or platelet-derived growth factor receptor β.
Regarding the determination of the expression level of integrin β1 or platelet-derived growth factor receptor β, for example, the following method can be used: immunological assays are performed using antibodies or antibody fragments directed against integrin beta 1 or platelet-derived growth factor receptor beta. Specifically, flow cytometry, western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent antibody labeling, cell array, and the like can be cited. These measurement methods can also be implemented by a conventional protocol or a conventional protocol modified or changed as appropriate.
For example, when the expression level of integrin β1 or platelet-derived growth factor receptor β in a cell is measured by flow cytometry, 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, it is possible to sort into synovial membrane-derived mesenchymal stem cells having a surface antigen of any one or more of integrin β1 or platelet-derived growth factor receptor β.
In sorting of synovial membrane-derived mesenchymal stem cells having a surface antigen of any one or more of integrin β1 and platelet-derived growth factor receptor β, for example, the level of integrin β1 or platelet-derived growth factor receptor β in the cells measured by the above method can be compared with a predetermined reference expression level to perform sorting. The reference expression level may be, for example, the expression level of integrin β1 or platelet-derived growth factor receptor β in a cell (positive control) that has been confirmed to have a certain quality, or the expression level of a cell (negative control) that has not been confirmed to have a certain quality.
By comparing the expression level of integrin beta 1 or platelet-derived growth factor receptor beta with the reference expression level, it is possible to sort out cells in which the expression level of integrin beta 1 or platelet-derived growth factor receptor beta in the cells is equal to or greater than the expression level of the positive control, and use the cells as an arthropathy therapeutic agent.
Furthermore, a cutoff value for the expression level of integrin β1 or platelet-derived growth factor receptor β may be preset, and the expression level of integrin β1 or platelet-derived growth factor receptor β in the measured cells may be compared with the cutoff value. For example, the cut-off value can be used as the expression level of integrin β1 or platelet-derived growth factor receptor β that imparts the desired therapeutic effect, based on a regression line that indicates the correlation between the expression amount of integrin β1 or platelet-derived growth factor receptor β and the therapeutic effect. For example, it is possible to sort out cells in which the expression level of integrin beta 1 or platelet-derived growth factor receptor beta is equal to or higher than a cutoff value, and use the cells as an arthropathy therapeutic agent.
The therapeutic agent for arthrosis of the present invention can be used for joint treatment. The joint treatment includes treatment of diseases accompanied by injury, damage or inflammation of joints, and includes treatment of joint diseases caused by modification of connective tissues such as cartilage and/or inflammation, or non-inflammatory joint diseases. Examples of the joint treatment include treatment of diseases selected from the group consisting of meniscus injury, traumatic cartilage injury, osteochondritis dissecans, aseptic osteonecrosis, osteoarthritis (e.g., knee osteoarthritis), rheumatoid arthritis (e.g., chronic rheumatoid arthritis), gout, reactive arthritis, psoriatic arthritis, juvenile arthritis, inflammatory arthritis, and articular cartilage defects, but the present invention is not limited to these diseases.
The method for treating a joint using the therapeutic agent for arthrosis of the present invention comprises the steps of:
A step of transplanting the therapeutic agent for arthrosis of the present invention so that the cartilage damaged portion or the meniscus damaged portion is covered with the synovial-derived mesenchymal stem cells; and
And a step of regenerating cartilage tissue in situ (in situ) at the cartilage injury part or meniscus injury part by differentiating synovial membrane-derived mesenchymal stem cells contained in the arthropathy therapeutic agent into chondrocytes.
When the therapeutic agent for arthrosis of the present invention is transplanted into a patient, it is preferable to apply 2.0X10 7～1.0×1011 or 2.5X10 7～1.0×1011 or 3.0X10 7～1.0×1011, 4.0X10 7～1.0×1011 or 2.5X10 7～1.0×1010 or 2.5X10 7～1.0×109 or 2.5X10 7～1.0×108 synovial mesenchymal stem cells or 2.0X10 7～1.0×108 mesenchymal stem cells to each cartilage injury portion or meniscus injury portion in order to effectively treat the cartilage injury portion or meniscus injury portion.
By transplanting the synovial-derived mesenchymal stem cells to the cartilage injury part or the meniscus injury part, the cartilage injury part or the meniscus injury part is covered with the synovial-derived mesenchymal stem cells. The transplantation of the synovial-derived mesenchymal stem cells can be performed by an open operation or by an arthroscopic operation. In order to minimize invasion, it is preferable to graft synovial-derived mesenchymal stem cells under arthroscopy.
The cartilage damaged portion or the meniscus damaged portion may be covered with a suspension of mesenchymal stem cells derived from synovium, or may be covered with a cell sheet of mesenchymal stem cells derived from synovium. For example, a bioabsorbable gel such as gelatin or collagen can be used as the gel-like substance. The mesenchymal stem cells from synovium have high adhesion capability to cartilage injury parts or meniscus injury parts.
In treating cartilage damage, the low invasive surgery of the present invention is characterized in that a cartilage damaged part is covered by synovial-derived mesenchymal stem cells, and comprises the steps of:
holding the body position so that the cartilage damaged part faces upward;
Resting a cell sheet of synovial-derived mesenchymal stem cells, a suspension of synovial-derived mesenchymal stem cells, or a gel-like substance comprising synovial-derived mesenchymal stem cells on the surface of the cartilage lesion; and
The body position is maintained for a specific time, thereby causing the synovial-derived mesenchymal stem cells to adhere to the surface of the cartilage damaged part.
In treating a meniscus injury, the low invasive procedure of the present invention is characterized by covering the meniscus injury with synovial-derived mesenchymal stem cells, and comprises the steps of:
Maintaining the posture so that the meniscus injury portion faces downward;
injecting a suspension of synovial-derived mesenchymal stem cells into the knee joint; and
The body position is kept for a specific time, so that the mesenchymal stem cells from the synovium are adhered to the damaged part of the meniscus.
In order to reliably adhere the synovial-derived mesenchymal stem cells to the surface of the cartilage injury portion or the meniscus injury portion, the transplanted synovial-derived mesenchymal stem cells are held on the surface of the cartilage injury portion or the meniscus injury portion for at least 10 minutes, preferably 15 minutes. To achieve this object, the posture is maintained for at least 10 minutes, preferably 15 minutes, with the aim of holding the cartilage damaged portion or the meniscus damaged portion upward and the synovial-derived mesenchymal stem cells in the cartilage damaged portion or the meniscus damaged portion upward.
In order to firmly adhere the synovial mesenchymal stem cells to the cartilage damaged portion or the meniscus damaged portion, the cartilage damaged portion or the meniscus damaged portion accompanied by the synovial mesenchymal stem cells may be further covered with a periosteum. After the synovial-derived mesenchymal stem cells were maintained on the surface of the cartilage injury part or the surface of the meniscus injury part for at least 10 minutes, the operation was completed.
In the present invention, the transplanted synovial-derived mesenchymal stem cells are differentiated into chondrocytes at a cartilage injury part or a meniscus injury part, and then cartilage tissue is regenerated in situ (in situ) at the cartilage injury part or the meniscus injury part.
In the cartilage formation process of synovial-derived mesenchymal stem cells in situ (in situ), cartilage tissue is regenerated according to local microenvironment (nutrient supply, cytokine environment, etc.), and thus, external manipulation is not required. As a result of in-situ cartilage formation of synovial-derived mesenchymal stem cells, cartilage tissue is regenerated at a cartilage injury portion or a meniscus injury portion to repair the injury, and in the case of cartilage injury, a bone region, a cartilage-to-bone boundary, a cartilage center portion, a surface region, and a region adjacent to the original cartilage are formed as the original cartilage tissue, or in the case of meniscus injury, meniscus cartilage is formed.
The present invention is further specifically illustrated by the following examples, but the present invention is not limited to the examples.
Examples
Example 1 > preparation of rat synovial-derived mesenchymal Stem cells
LEW/CrlCrlj rats were used in the establishment of rat synovial-derived mesenchymal stem cells. To a MEM no nucleosides (no riboside) (Gibco Cat. No. 12561056) medium, collagenase V (collagenase V) (Sigma Cat. No. C9263) was added to bring the concentration of synovial tissue harvested under isoflurane anesthesia to 2 or 3mg/mL and allowed to react at 37℃for 2 hours. The cooled medium was added to stop the reaction and the residual tissue was removed by a 40 μm cell filter. The recovered cells were inoculated into a cell culture flask, and cultured at a CO 2 concentration of 5% and 37℃with α MEM no nucleosides, which α MEM no nucleosides was added Fetal Bovine Serum (fetal bovine serum) (Gibco Cat. No. 10270106) to a final concentration of 20%, L-glutamine200mmol/L (Gibco Cat. # 25030081) to a final concentration of 1%, and Antibiotic-Antimycotic (antibiotic-antifungal) (100X) (Gibco Cat. No. 15240062) to a final concentration of 1%. After culturing for 8 days, the medium in the flask was discarded and washed 2 times with PBS (phosphate buffered saline), trypLE Express (Gibco Cat. No. 12604-013) was added, and left to stand in an incubator at 37℃for 5 minutes, and the cells were recovered as synovial-derived mesenchymal stem cells. A supernatant was discarded by centrifugation and replaced with COS-bank (COSMO BIO Cat. No. COS-CFM 01) to prepare a frozen stock of mesenchymal stem cells derived from rat synovial membrane.
Example 2 > inhibition of extracellular matrix adhesive Capacity of rat synovial-derived mesenchymal Stem cells based on inhibition of integrin beta 1
Preparation of rat-derived mesenchymal Stem cells in which integrin beta 1 was inhibited, the frozen stock solution of rat-derived mesenchymal Stem cells prepared in example 1 was resuscitated, and after culturing at a CO 2 concentration of 5% and 37℃for 1 week, the recovered cells were suspended in PBS containing 2% FBS as a reaction solvent, wherein Fetal Bovine Serum was added to a final concentration of 20%, L-glutamie 200mmol/L was added to a final concentration of 1%, and Antibiotic-Antimycotic (100X) was added to a final concentration of 1%. To 5X 10 6 cells per cell number, 12. Mu.g of Purified anti-mouse/rat (purified anti-mouse/rat) CD29 anti-body (Antibody) (BioLegend Cat. No. 102202) was added, and after allowing to react under ice-cooling for 1 hour, the cells (integrin. Beta.1-rSMSC) were recovered. As a control treatment without integrin β1 inhibition, purified ARMENIAN HAMSTER IGG Isotype Ctrl (purified hamster IgG isotype control) (BioLegend Cat. No. 400902) was reacted under ice-cooling for 1 hour, and then cells (IgG-rSMSC) were recovered. Untreated cells (Non-treated-rSMSC) having undergone the same reaction with only the reaction solvent were set and subjected to the following adhesion treatment.
In order to confirm that integrin β1 is inhibited, the function of adhesion of extracellular matrix, which is one of the functions of integrin β1, was confirmed. As an extracellular matrix adhesion reaction, washing was performed with 10mmol/L of PBS containing MgCl 2·6H2 O (hereinafter PBS (+)), and cells were suspended again with PBS (+) and adjusted to 1X 10 5 cells/10. Mu.L/well, and inoculated into Collagen TYPE ICELLWARE-Well Culture Slide (type I Collagen cell vessel 8-well culture slide) (Corning Cat. No. 354630). After standing at room temperature for 10 minutes, washing was performed with PBS (+). Then, observation was performed with a microscope (OLIMPUS Cat. No. IX 71) at a magnification of 10 times with an objective lens. Then, an image of 1 visual field in which the most adherent cells were observed was obtained, and the number of adherent cells was calculated.
The results are shown in fig. 1. Regarding the number of adhered cells, no-treated-rSMSC was 1637cells, igG-rSMSC was 1214cells, integrin beta 1-rSMSC was 194cells, and it was observed that the number of adhered cells was greatly reduced by inhibiting integrin beta 1. From this, it was confirmed that integrin β1 in rat synovial stem cells could be inhibited by treatment with Purified anti-mouse/rat CD29 Antibody.
Example 3 > inhibition of cell proliferation Capacity by PDGFRb-based inhibition of rat synovial-derived mesenchymal Stem cells
As preparation of PDGFRb-inhibited rat-derived mesenchymal stem cells, the frozen stock solution prepared in example 1 was resuscitated, and after culturing at a CO 2 concentration of 5% and 37℃for 1 week, the recovered cells were suspended in PBS containing 2% FBS as a reaction solvent, wherein Fetal Bovine Serum was added to a final concentration of 20%, L-glutamie 200mmol/L was added to a final concentration of 1%, and Antibiotic-Antimycotic (100X) was added to a final concentration of 1%.
For each cell number of 1X 10 6 cells, 40, 120. Mu.g of Anti-PDGF Receptor beta Human Goat-Poly (R & D Systems Cat. No. AF385) was reacted under ice-cooling for 1 hour, and after that, the cells were seeded at 1000cells/well on a 96-well plate (Corning Cat. No. 353072) and cultivation (PDGFRb-rSMSC) was started at a CO 2 concentration of 5% and 37 ℃. As a control treatment without PDGFRb inhibition, normal Goat IgG Control (normal goat IgG control) (R & D Systems Cat. No. AB-108-C) was reacted under ice-cooling for 1 hour and inoculated at 1000cells/well (IgG-rSMSC) on a 96-well plate. Furthermore, non-treated-rSMSC was also set, in which cells of 1000cells/well were inoculated only on a 96-well plate without performing the reaction.
In order to confirm that PDGFRb was inhibited, the ability of the PDGFRb to proliferate cells was confirmed as a function. The proliferation of cells was quantitatively evaluated by using ATPassay of CELL TITER Glo (Promega cat.no. g 7571) at 5% CO 2 concentration at 37 ℃ and on day 6 of the culture.
The results are shown in fig. 2a. A significant inhibition of cell proliferation was observed by PDGFRb inhibition with an ATP concentration of 3.78.+ -. 0.84. Mu. Mol/L for 40. Mu.g/mL PDGFRb-rSMSC, 4.12.+ -. 1.29. Mu. Mol/L, igG-rSMSC for 120. Mu.g/mL PDGFRb-rSMSC and 6.08.+ -. 0.63. Mu. Mol/L, non-treated-rSMSC for 6.81.+ -. 0.82. Mu. Mol/L. From this, it was confirmed that proliferation of rat synovial stem cells can be inhibited by treatment with Anti-PDGF Receptor beta Human Goat-Poly.
To confirm ligand specificity of PDGFRb, the lyophilized solution prepared in example 1 was resuscitated by adding Fetal Bovine Serum to a final concentration of 20%, adding L-glutamie mmol/L to a final concentration of 1%, and adding Antibiotic to Antimycotic (100X) to a final concentration of 1% α MEM no nucleosides, and culturing the cells at 5% CO 2 concentration for 1 week at 37 ℃ to suspend the recovered cells in a reaction solvent PBS containing 2% fbs.
For each cell number of 1X 10 6 cells, 10, 20, 40. Mu.g of Anti-PDGF Receptor. Beta. Human Goat-Poly (R & D Systems Cat. No. AF385) was reacted under ice-cold for 1 hour, and then the cells were seeded at 1000cells/well on a 96-well plate (Corning Cat. No. 353072) and the culture (PDGFRb-rSMSC) was started at a CO 2 concentration of 5% and 37 ℃. As a control treatment without PDGFRb inhibition, normal Goat IgG Control (R & D Systems Cat. No. AB-108-C) was reacted under ice-cooling for 1 hour, and inoculated at 1000cells/well (IgG-rSMSC) on a 96-well plate. Furthermore, non-treated-rSMSC was also set, in which cells of 1000cells/well were inoculated only on a 96-well plate without performing the reaction.
The following day of cell seeding, the culture supernatant was discarded and the medium was replaced with a medium of a MEM no nucleosides (Gibco Cat. No. 10270106) to which a MEM no nucleosides was added Fetal Bovine Serum to a final concentration of 0.5%, L-glutamie 200mmol/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 4ng/mL. For the experimental level, anti-PDGF Receptor beta Human Goat-Poly (R & D Systems Cat. No. AF385) was added to give final concentrations of 10, 20, 40. Mu.g/mL, respectively, to give a total medium of 100. Mu.L. For positive control, normal Goat IgG Control (R & D Systems Cat. No. AB-108-C) was added to make the total amount of medium 100. Mu.L. For negative control, 100. Mu.L of medium was added without adding antibody. On day 6 of culture, proliferation of cells was quantitatively evaluated by using ATP assay of CELL TITER Glo (Promega Cat. No. G7571).
The results are shown in fig. 2 b. A significant reduction in proliferation of IgG-rSMSC cells by 40. Mu.g/mL PDGFRb-rSMSC was observed, with an ATP concentration of 10. Mu.g/mL PDGFRb-rSMSC of 0.62.+ -. 0.12. Mu. Mol/L, an ATP concentration of 20. Mu.g/mL PDGFRb-rSMSC of 0.65.+ -. 0.05. Mu. Mol/L, an ATP concentration of 40. Mu.g/mL PDGFRb-rSMSC of 0.24.+ -. 0.05. Mu. Mol/L, an IgG-rSMSC of 0.49.+ -. 0.17. Mu. Mol/L, and a Non-treated-rSMSC of 0.24.+ -. 0.11. Mu. Mol/L. From this, it was confirmed that PDGFRb in rat synovial stem cells could be inhibited in a ligand-specific manner by treatment with Anti-PDGF Receptor beta Human Goat-Poly.
Example 4 > preparation of Col2A 1-deleted rat synovial-derived mesenchymal Stem cells
The mesenchymal stem cells derived from the rat sliding film prepared in example 1 were subjected to deletion of the Col2A1 gene, and deletion of the Col2A1 gene was confirmed by Sanger sequencing analysis (Sanger sequencing analysis). The Col2A1 base sequences of the rat synovial stem cells of the Col2A1 gene wild type (Col 2A1 WT-rSMSC) and the deletion type (Col 2A1 KO-rSMSC) are shown in FIGS. 3, 4, 5 and 6, and the amino acid sequences translated based on the sequences are shown in FIGS. 7, 8 and 9. The Col2A1 base sequence of the rat synovial stem cell of the Col2A1 gene wild type (Col 2A1 WT-rSMSC) is shown in SEQ ID NO. 1, the Col2A1 base sequence of one chromosome of the rat synovial stem cell of the Col2A1 gene deleted type (Col 2A1 KO-rSMSC) is shown in SEQ ID NO. 2, and the Col2A1 base sequence of the other chromosome of the rat synovial stem cell of the Col2A1 gene deleted type (Col 2A1 KO-rSMSC) is shown in SEQ ID NO. 3. The amino acid sequence of the wild type Col2A1 gene (Col 2A1 WT-rSMSC) is shown in SEQ ID NO. 4, and the amino acid sequences of the deleted type Col2A1 gene (Col 2A1 KO-rSMSC) are shown in SEQ ID NO. 5 and SEQ ID NO. 6. As a result, it was found that, in Col2A1KO-rSMSC, an abnormal frameshift mutant having a DNA from which 55 th to 62 th bases have been deleted and having a DNA inserted into 59 th bases was obtained starting from the amino acid translation initiation codon ATG. One of the alleles was mutated from the 19 th amino acid sequence by deletion of the base, and a stop codon was inserted at the 29 th amino acid sequence, so that the nucleotide sequence of the mutant which was originally 1419 amino acid but was now translated into 28 amino acid residues was found. The other allele was mutated from the 20 th amino acid sequence by base insertion and inserted with a stop codon at the 50 th amino acid sequence, and thus found a base sequence which was originally 1419 amino acids but was now translated into a mutant of 49 amino acid residues. Since the nucleotide sequence showing this mutant was not translated in the 133 th amino acid to 1146 th amino acid sequence which is the triple helix region that is the important functional region of Col2A1, it was determined that synovial stem cells (Col 2A1 KO-rSMSC) having the gene sequence with the deleted Col2A1 function could be obtained.
Comparative example 1 > production of mouse synovial membrane-derived mesenchymal Stem cells with CD120a deficiency
The mesenchymal stem cells derived from the rat sliding film prepared in example 1 were subjected to deletion operation of the CD120a gene, and deletion of the CD120a gene was confirmed by Sanger sequencing analysis. The nucleotide sequence of CD120a of the rat synovial stem cell of the wild type (CD 120 aWT-rSMSC) and the deletion type (CD 120 aKO-rSMSC) of the CD120a gene and the amino acid sequence translated based on the same are shown in FIGS. 10 and 11. The CD120a base sequence of the rat synovial stem cells of the wild type (CD 120 aWT-rSMSC) and the deleted type (CD 120 aKO-rSMSC) of the CD120a gene is shown in SEQ ID NO. 7, the amino acid sequence of the wild type (CD 120 aWT-rSMSC) of the CD120a gene is shown in SEQ ID NO. 8, and the amino acid sequence of the deleted type (CD 120 aKO-rSMSC) of the CD120a gene is shown in SEQ ID NO. 9. As a result, it was found that in CD120aKO-rSMSC, it was a frameshift mutant having a DNA with the 16 th base deleted starting from the ATG of the amino acid translation initiation codon. Since the sequence was mutated from the 6 th amino acid by deletion of the base and the stop codon was inserted at the 19 th amino acid, the base sequence of the mutant which was originally 461 amino acid but which was now translated into 19 amino acid residues was found. Since the nucleotide sequence of the mutant was not translated in the protein region constituting CD120a, it was determined that synovial stem cells (CD 120 aKO-rSMSC) having the gene sequence with the function of CD120a deleted could be obtained.
Comparative example 2 > production of mouse synovial membrane-derived mesenchymal Stem cells with CD106 deficiency
The mesenchymal stem cells derived from the rat sliding film prepared in example 1 were subjected to deletion operation of the CD106 gene, and deletion of the CD106 gene was confirmed by Sanger sequencing analysis. The nucleotide sequences of CD106 of rat synovial stem cells of the wild type (CD 106 WT-rSMSC) and the deletion type (CD 106 KO-rSMSC) of the CD106 gene and the amino acid sequences translated based on the sequences are shown in FIGS. 12 and 13. The CD106 base sequence of the rat synovial stem cells of the wild type CD106 gene (CD 106 WT-rSMSC) and the deleted type (CD 106 KO-rSMSC) is shown in SEQ ID NO. 10, the amino acid sequence of the wild type CD106 gene (CD 106 WT-rSMSC) is shown in SEQ ID NO. 11, and the amino acid sequence of the deleted type CD106 gene (CD 106 KO-rSMSC) is shown in SEQ ID NO. 12. As a result, it was found that, in CD106KO-rSMSC, it was a frameshift mutant having DNA from 1059 th to 1076 th bases deleted starting from the amino acid translation initiation codon ATG. Since the sequence was mutated from the 354 th amino acid by deletion of the base and the stop codon was inserted at 356 th amino acid, the base sequence of a mutant which was originally 739 amino acids but which was now translated into 355 amino acid residues was found. Since the nucleotide sequence of the mutant was not translated from 699 amino acids to 720 amino acids, which is the transmembrane region of CD106, it was determined that a synovial stem cell (CD 106 KO-rSMSC) having a gene sequence with a deleted CD106 function could be obtained.
Example 5 > inhibition of cartilage differentiation Capacity in rat synovial-derived mesenchymal Stem cells deleted for Col2A1
Col2A1 is one of the cartilage constituent components. To confirm the absence of Col2A1 at the cellular function level, the cartilage differentiation ability of Col2A1KO-rSMSO was examined. Col2A1KO-rSMSC 2.5X10 5 cells prepared in example 4 were suspended in DMEM high glucose (high sugar) (Thermo Cat. No. 11965092) and centrifuged with 450g for 10 minutes, and then cultured at CO 2 concentration of 5% at 37℃and induced cartilage differentiation, TGF-. Beta.3 (R & D Systems Cat. No. 243-B3-002) was added to DMEM high glucose to a final concentration of 10ng/mL, dexamethasone (dexamethasone) (Wako Cat. No. 041-18861) to a final concentration of 3.92. Mu.g/mL, L-Ascorbic Acid-phosphate (L-ascorbic acid 2-phosphate) (CAYMAN CHEMICAL Cat. No. 57) to a final concentration of 50. Mu.g/mL, L-proline (MP biomedical. No. 728) Cat. 16483 to a final concentration of 40. Mu.g/mL, sodium (Invro Cat. No. 041-18861) to a final concentration of 35. Mu.2-35 g/mL, and a final concentration of 35-35% to a final concentration of 35% to which additional (Wako Cat. No. 35) was added to 35.6.1-35.6). As a control cell, cartilage differentiation was induced in the same manner as in Col2A1 WT-rSMSC. After 3 weeks of culture, the diameter and weight of the cell mass were measured, and the cartilage differentiation ability was evaluated based on the tissue staining of the cell mass, and the results are shown in fig. 14. In Col2A1WT-rSMSC, the minor diameter was 1.55.+ -. 0.14mm, the major diameter was 2.04.+ -. 0.25mm, and the weight was 1.9.+ -. 0.26mg, whereas in Col2A1KO-rSMSC, the minor diameter was 0.54.+ -. 0.04mm, the major diameter was 0.76.+ -. 0.18mm, and the weight was 0.85.+ -. 0.4mg, and significant reductions in cartilage size and weight were observed. Col2A1 is a cartilage constituent, and therefore, it can be seen that the size and weight of cartilage are reduced due to the deficiency of Col2A 1. Furthermore, it was found that in Col2A1KO-rSMSC, the staining properties of safranin O-fast green staining and type II collagen immunostaining were disappearing. That is, it was revealed that the absence of Col2A1 not only caused the disappearance of the type II collagen production ability, but also affected the production of mucopolysaccharide cartilage matrix. This suggests that type II collagen not only contributes to the formation of bone lattice in cartilage tissue, but also contributes to the induction of cartilage differentiation/matrix production.
Comparative example 3 > inhibition of cartilage differentiation ability in rat synovial-derived mesenchymal stem cells having deleted CD120a
The cartilage differentiation ability of CD120aKO-rSMSO produced in comparative example 1 was examined. Cartilage differentiation induction was performed under the same differentiation medium and culture conditions as in example 5. As a control cell, cartilage differentiation was induced in the same manner as in CD120 aWT-rSMSC. After 3 weeks of culture, the diameter and weight of the cell mass were measured, and the cartilage differentiation ability was evaluated based on the tissue staining of the cell mass, and the results are shown in fig. 15. In CD120aWT-rSMSC, the short diameter was 1.55.+ -. 0.14mm, the long diameter was 2.04.+ -. 0.25mm, and the weight was 1.9.+ -. 0.26mg, whereas in CD120aKO-rSMSC, the short diameter was 1.19.+ -. 0.17mm, the long diameter was 1.53.+ -. 0.04mm, and the weight was 1.55.+ -. 1.20mg, and no significant decrease in cartilage size and weight was observed. Furthermore, no difference in staining of safranin O-fast green, type II collagen immunostaining caused by CD120a deletion was observed, so CD120a is not considered a molecule contributing to the cartilage differentiation ability of cells.
Comparative example 4 > inhibition of cartilage differentiation ability in CD 106-deleted rat synovial-derived mesenchymal stem cells
The cartilage differentiation ability of CD106KO-rSMSO produced in comparative example 2 was examined. The cartilage differentiation induction of CD106KO-rSMSC 2.5X10 5 cells was performed under the same differentiation medium and culture conditions as in example 5. As a control cell, cartilage differentiation was induced in the same manner as in CD106 WT-rSMSC. After 3 weeks of culture, the diameter and weight of the cell mass were measured, and the cartilage differentiation ability was evaluated based on the tissue staining of the cell mass, and the results are shown in fig. 16. In CD106WT-rSMSC, the minor axis was 1.55.+ -. 0.14mm, the major axis was 2.04.+ -. 0.25mm, and the weight was 1.9.+ -. 0.26mg, whereas in CD106KO-rSMSO, the minor axis was 1.58.+ -. 0.56mm, the major axis was 1.75.+ -. 0.52mm, and the weight was 2.38.+ -. 1.54mg, and no significant decrease in cartilage size and weight was observed. Furthermore, no difference in staining of safranin O-fast green, type II collagen immunostaining caused by CD106 deletion was observed, so CD106 is not considered to be a molecule contributing to the cartilage differentiation ability of cells.
Example 6 > confirmation of the Effect of meniscus regeneration of rat synovial-derived mesenchymal Stem cells in which integrin beta 1 was inhibited
As a preparation of rat synovial membrane-derived mesenchymal stem cells with integrin β1 inhibited, the preparation was performed as described in example 2. The frozen stock was resuscitated and after 1 week of incubation, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. To 5X10 6 cells per cell number, 12. Mu.g of Purified anti-mouse/rat integrin beta 1 anti-body was added, and after reacting for 1 hour under ice-cooling, recovered for transplantation (integrin beta 1-rSMSC). Then, purified ARMENIAN HAMSTER IGG Isotype Ctrl was reacted under ice-cooling for 1 hour as a control treatment without inhibition, and then recovered for transplantation (IgG-rSMSC).
LEW/CrlCrlj rats were used in the preparation of a meniscus injury model for evaluation of meniscus regeneration effect. In the method of meniscus injury and mesenchymal stem cell transplantation, knee joint skin is incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule under the knee was exposed, and the cartilage at the distal end of the femur was exposed by longitudinal incision with a scalpel. The medial meniscus was peeled off the synovium, exposing the medial meniscus, and about 2/3 of the total was resected. The knee tendon and synovium were sutured, and then the muscle was sutured to make a model of meniscus injury. Then, the treated animals were divided into 3 groups, and synovial stem cells (integrin β1-rSMSC) 5×10 6 cells, which had been treated with non-inhibitory controls (IgG-rSMSC) 5×10 6 cells, and only solvent were injected into the joint capsule, respectively. The following day of cell injection, purified anti-mouse/rat integrin beta 1 anti-body, ARMENIAN HAMSTER IGG Isotype Ctrl or solvent was injected into the joint capsule at 12 μg per knee. After treatment, all rats were returned to the cage, and allowed free movement and food intake.
After 3 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 17. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. In the group of IgG-rSMSC as a positive control, the meniscus regeneration to the anterior segment was more frequent, and the observed meniscus regeneration was more frequent. On the other hand, in the group of integrin β1-rSMSC, which are cells whose molecules are inhibited or have been deleted, a smaller portion of meniscus regeneration can be observed as compared to the positive control.
To quantitatively evaluate the visual observation result of the meniscus regenerating portion of fig. 17, the area of the meniscus regenerating portion (within the dotted line) was calculated by the following formula using ImageJ (version 1.52).
Meniscus regeneration partial area (mm 2) =number of pixels of meniscus regeneration partial area/number of pixels per 1mm 2
The mean, standard deviation and statistical analysis of the regenerated partial areas of each group were performed with Microsoft Excel 2007 (Microsoft corp.). For statistical analysis, 2 Student's T-Test (Student t Test) was performed on the positive control group and the molecular inhibition group and the solvent group, and P-values were calculated, respectively. Since the assay was repeated, a value obtained by applying Bonferroni correction and multiplying the calculated P value by the number of assays (2) was used. Regarding the significance level, 5% (α=0.05) was set as a difference, and the results thereof are shown in table 1.
Regarding the average area values of the meniscus regenerating portions described in Table 1, 2.1mm 2 of the integrin beta 1-rSMSC group was significantly reduced relative to 3.4mm 2 of IgG-rSMSC. On the other hand, the integrin beta 1-rSMSC group had the same extent of regeneration area relative to 1.7mm 2 of the solvent group. From this, it was confirmed that integrin β1 molecules in synovial stem cells are important molecules contributing to meniscus regeneration.
TABLE 1
Table 1: area of meniscus regeneration section (mean ± standard deviation)
| Group of | Area (mm 2) |
| Integrin beta 1-rSMSC | 2.1±0.5* |
| IgG-rSMSC | 3.4±1.0 |
| Solvent(s) | 1.7±0.9 |
* Group P < 0.05vs IgG-rSMSC
Example 7 > confirmation of the Effect of PDGFRb on regeneration of menisci in rat synovial-derived mesenchymal Stem cells
The preparation of the rat synovial-derived mesenchymal stem cells with suppressed PDGFRb was performed as described in example 3. The frozen stock was resuscitated and after 1 week of incubation, the recovered cells were suspended in PBS containing 2% FBS as the reaction solvent. For 5X10 6 cells per cell number, 12. Mu.g of Anti-PDGF Receptor. Beta. Human Goat-Poly (R & D Systems Cat. No. AF385) was recovered for transplantation (PDGFRb-rSMSC) after 1 hour of reaction under ice-cooling. Then, nomal Goat IgG Control (R & D Systems Cat. No. AB-108-C) was reacted under ice-cooling for 1 hour as a control treatment without inhibition, and recovered for transplantation (IgG-rSMSC).
A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in example 6. Then, the treated animals were divided into 3 groups, and 5×10 6 cells in which PDGFRb was inhibited (PDGFRb-rSMSC), 5×10 6 cells in which non-inhibition control treatment was performed (IgG-rSMSC), and only solvent were injected into joint capsules, respectively. The next day of cell injection, 12 μg of Anti-PDGF Receptor beta Human Goat-Poly, nomal Goat IgG Control or solvent per knee is injected into the joint capsule. After treatment, all rats were returned to the cage, and allowed free movement and food intake.
After 3 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 18. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. In the group of IgG-rSMSC as a positive control, the meniscus regeneration to the anterior segment was more frequent, and the observed meniscus regeneration was more frequent. On the other hand, in the group of PDGFRb-rSMSC, which are cells in which the molecule is inhibited or has been deleted, the observed portion of meniscus regeneration is smaller than that of the positive control.
To quantitatively evaluate the visual observation result of the meniscus regenerating portion of fig. 18, the area of the meniscus regenerating portion (within the broken line) was measured as described in example 6. The results are shown in Table 2. Regarding the average area value of the meniscus regenerating portion, PDGFRb-rSMSC group of 2.2mm 2 was significantly reduced relative to 3.2mm 2 of IgG-rSMSC. On the other hand, the PDGFRb-rSMSC group was significantly increased relative to 1.3mm 2 of the solvent group. From this, it was confirmed that PDGFRb molecules in synovial stem cells are molecules contributing to meniscus regeneration.
TABLE 2
Table 2: area of meniscus regeneration section (mean ± standard deviation)
* Group P < 0.05vs IgG-rSMSC
Solvent set
Comparative example 5 > confirmation of the meniscus regeneration effect of CD 44-inhibited rat synovial-derived mesenchymal Stem cells
Preparation of rat synovial-derived mesenchymal stem cells with suppressed CD44, resuscitating the frozen stock, and after culturing for 1 week, suspending the recovered cells in PBS containing 2% fbs as a reaction solvent. For 5X10 6 cells per cell number, 12. Mu.g of Anti-CD44 Rabbit IgG clone Hermes-1 (Absolute Antibody Cat. No. Ab00628-23.0) was recovered for transplantation (CD 44-rSMSC) after 30 minutes of reaction under ice-cooling. Then, rabbit IgG Isotype Control (rabbit IgG isotype control) (invitrogen Cat. No. 10500C) was reacted under ice-cooling for 1 hour as a control treatment without inhibition, and recovered for transplantation (IgG-rSMSC).
A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in example 6. The treated animals were then divided into 3 groups, and CD 44-inhibited synovial stem cells (CD 44-rSMSC) 5X10 6 cells, uninhibited control treated synovial stem cells (IgG-rSMSC) 5X10 6 cells, and solvent only were injected into the joint capsule, respectively. The following day of cell injection, 12 μg of Anti-CD44 Rabbit IgG clone (Rabbit IgG clone) Hermes-1, rabbit IgG Isotype Control or solvent per knee was injected into the joint capsule. After treatment, all rats were returned to the cage, and allowed free movement and food intake.
After 4 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 19. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. In the group of IgG-rSMSC and the group of CD44-rSMSC, which are positive controls, the meniscus regeneration to the anterior segment was more frequent, and the observed meniscus regeneration was more frequent.
To quantitatively evaluate the visual observation result of the meniscus regenerating portion of fig. 19, the area of the meniscus regenerating portion (within the broken line) was measured as described in example 6. The results are shown in Table 3. Regarding the average area value of the meniscus regenerating portion, there was no significant difference in the 3.4mm 2 of the CD44-rSMSC group compared to the 4.0mm 2 of IgG-rSMSC, to the same extent. On the other hand, the CD44-rSMSC group was significantly increased relative to the 2.7mm 2 group of the solvent group. It is thus believed that CD44 in synovial stem cells is not a molecule that contributes to meniscus regeneration.
TABLE 3
Table 3: area of meniscus regeneration section (mean ± standard deviation)
Solvent set
Example 8 > confirmation of the meniscus regeneration Effect of rat synovial-derived mesenchymal Stem cells (Col 2A1 KO-rSMSC) deleted Col2A1
As described in example 4, the rat synovial-derived mesenchymal stem cells in which Col2A1 was deleted were used for transplantation by performing an expansion culture adjustment after confirming the Col2A1 deletion (Col 2A1 KO-rSMSC). After confirming the Col2A1 wild-type sequence as a positive control, the sequence was adjusted by expansion culture and used for transplantation (Col 2A1 WT-rSMSC).
A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in example 6. The treated animals were then divided into 3 groups, and Col2A1KO-rSMSC, col2A1WT-rSMSC X10 6 cells, and solvent only were injected into the joint capsule, respectively.
After 3 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 20. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. In the group Col2A1WT-rSMSC as a positive control, the meniscus regeneration to the anterior segment was more exemplified, and the observed meniscus regeneration portion was larger. On the other hand, in the Col2A1KO-rSMSC group, the observed meniscus regeneration fraction was smaller compared to the positive control.
To quantitatively evaluate the visual observation result of the meniscus regenerating portion of fig. 20, the area of the meniscus regenerating portion (within the broken line) was measured as described in example 6. The results are shown in Table 4. Regarding the average area value of the meniscus regenerating portion, col2A1WT-rSMSC was significantly increased to 3.8mm 2 as compared to 2.9mm 2 of the Col2A1KO-rSMSC group. On the other hand, the Col2A1KO-rSMSC group was significantly increased compared to 2.1mm 2 of the solvent group. From this, it was confirmed that the Col2A1 molecule in the synovial stem cell was a molecule contributing to meniscus regeneration.
TABLE 4
Table 4: area of meniscus regeneration section (mean ± standard deviation)
* Group P < 0.05vs IgG-rSMSC
Solvent set
Comparative example 6 > confirmation of the meniscus regeneration effect of rat synovial-derived mesenchymal Stem cells (CD 120 aKO-rSMSC) deleted for CD120a
As described in comparative example 1, the rat synovial-derived mesenchymal stem cells with CD120a deleted were used for transplantation (CD 120 aKO-rSMSC) by performing an expansion culture after confirming the deletion of CD120 a. After confirming the CD120a wild-type sequence as a positive control, it was adjusted by expansion culture for transplantation (CD 120 aWT-rSMSC).
A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in example 6. The treated animals were then divided into 3 groups, and CD120aKO-rSMSC, CD120aWT-rSMSC 5x10 6 cells, and solvent only were injected into the joint capsule, respectively.
After 3 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 21. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. On the other hand, in the group of CD120aWT-rSMSC and the group of CD120aKO-rSMSC, which are positive controls, the meniscus regeneration to the anterior segment was more frequently observed, and the observed meniscus regeneration portion was larger than that in the solvent group.
To quantitatively evaluate the visual observation result of the meniscus regenerating portion of fig. 21, the area of the meniscus regenerating portion (within the broken line) was measured as described in example 6. The results are shown in Table 5. Regarding the average area value of the meniscus regenerating portion, no significant difference was observed with CD120aWT-rSMSC of 3.3mm 2 compared to 3.1mm 2 of CD120aKO-rSMSC group. On the other hand, the CD120aKO-rSMSC group was significantly increased compared to 1.8mm 2 for the solvent group. It is thus believed that CD120a in synovial stem cells is not a molecule that contributes to meniscus regeneration.
TABLE 5
Table 5: area of meniscus regeneration section (mean ± standard deviation)
Solvent set
Comparative example 7 > confirmation of the meniscus regeneration effect of rat synovial-derived mesenchymal Stem cells (CD 106 aKO-rSMSC) deleted for CD106
As described in comparative example 1, the rat synovial-derived mesenchymal stem cells with CD106 deleted were used for transplantation (CD 106 KO-rSMSC) by performing an expansion culture after confirming the deletion of CD 106. After confirming the CD106 wild-type sequence as a positive control, it was adjusted by expansion culture for transplantation (CD 106 WT-rSMSC).
A meniscus injury model for evaluating the meniscus regeneration effect was prepared as described in example 6. The treated animals were then divided into 3 groups, and CD106KO-rSMSC, CD106WT-rSMSC X10 6 cells, and solvent only were injected into the joint capsule, respectively.
After 3 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 22. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. On the other hand, in the CD106WT-rSMSC group and the CD106KO-rSMSC group, which are positive controls, the meniscus regeneration to the anterior segment was more likely to occur, and the observed meniscus regeneration was greater than that in the solvent group.
To quantitatively evaluate the visual observation result of the meniscus regenerating portion of fig. 22, the area of the meniscus regenerating portion (within the broken line) was measured as described in example 6. The results are shown in Table 6. Regarding the average area value of the meniscus regenerating portion, CD106WT-rSMSC was 3.3mm 2, compared to 3.7mm 2 of CD106KO-rSMSC group, and no significant difference was observed. On the other hand, the CD106KO-rSMSC group was significantly increased compared to 1.7mm 2 of the solvent group. It is thus believed that CD106 in synovial stem cells is not a molecule that contributes to meniscus regeneration.
TABLE 6
Table 6: area of meniscus regeneration section (mean ± standard deviation)
Solvent set
Example 9 > establishment procedure of rat synovial membrane-derived mesenchymal Stem cells, treatment time difference at cell recovery, and integrin beta 1 and PDGFRb expression Rate
In the same manner as in example 1, cells were isolated from a rat synovial source and cultured for 8 days to obtain mesenchymal stem cells from the rat synovial source. After the medium in the flask was discarded and washed 2 times with PBS, trypLE Express (Gibco Cat. No. 12604-013) was added, and the cells were peeled off and recovered by leaving the medium in an incubator at 37℃for 5, 30, 60 and 120 minutes, respectively.
10 6 Cells recovered under each condition were suspended in 500. Mu.L of PBS. For dead cell staining, LIVE/DEAD Fixable Aqua DEAD CELL STAIN KIT (Invitrogen Cat. No. L34957) was added to 0.5. Mu.L of cell suspension and incubated for 30 min at room temperature. After centrifugation, the supernatant was discarded, and 1mL of FACS buffer (buffer) (final concentration 2mmol/L EDTA.2Na, PBS containing 1%bovine serum albumin (bovine serum albumin)) was added to suspend the cells therein. To determine the expression rate of integrin β1, PE anti-mouse/rat integrin β1anti (Biolegend cat.no. 102207) or PE ARMENIAN HAMSTER IGG Isotype Ctrl Antibody (Biolegend cat.no. 400907) were added 5 μl and allowed to react for 30 min at 4 ℃. Then, the supernatant was discarded by centrifugation, suspended in 1mL of FACS buffer, and then the supernatant was discarded by centrifugation again, suspended in 500. Mu.L of FACS buffer, and supplied to the assay. To determine the PDGFRb expression rate, 5. Mu.L of Anti-PDGF Receptor beta, human, goat-Poly (R & DSsystems Cat. No. AF385) or Nomal Goat IgG Control (R & D Systems Cat. No. AB-108-C) was added and reacted at 4℃for 30 minutes. Then, the supernatant was discarded by centrifugation and suspended in 1mL of FACS buffer. Furthermore, donkey anti-Goat IgG (Donkey anti-goat IgG) (H+L) Cross-Adsorbed Secondary Antibody (Cross-adsorbed secondary antibody) was added thereto, and FITC (invitrogen Cat. No. A16006) was reacted at 4℃for 30 minutes in 1. Mu.L. Then, the supernatant was centrifuged to suspend it in 1mL of FACS buffer, and then centrifuged again to suspend it in 500. Mu.L of FACS buffer, and supplied to a flow Cytometer (Attune NxT, autoFocusing Cytometer model: AFC2, invitrogen) to measure the integrin beta 1 and PDGFRb expression rates.
The expression rates of integrin β1 and PDGFRb based on the differences in the time of the separation treatment of the synovial-derived mesenchymal stem cells are shown in table 7. Even if the peeling treatment time is prolonged from usual 5 minutes to 120 minutes, the expression rate of integrin beta 1 is maintained at 90% or more. On the other hand, the expression rate of PDGFRb was decreased in a time-dependent manner, 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, and in the cell separation treatment time, when the PDGFRb expression rate exceeds half, the meniscus regeneration effect can be further expected, and therefore the treatment time is preferably 60 minutes or less, and the required molecule expression rate can be set to 60% or more.
TABLE 7
Table 7: time of stripping treatment in cell recovery and expression rate of integrin beta 1 and PDGFRb
Example 10 > establishing days of culture and integrin beta 1 expression Rate for cell recovery of rat synovial-derived mesenchymal Stem cells
In the same manner as in example 1, cells were isolated from a rat synovial source, and 7.5x10 4 cells were inoculated into a flask having an area of 75cm 2 and cultured for 8, 21, and 28 days, respectively, to obtain mesenchymal stem cells from a rat synovial source. After discarding the medium in the flask and washing 2 times with PBS, trypLE Express (Gibco Cat. No. 12604-013) was added, and left to stand in an incubator at 37℃for 120 minutes, cells were peeled off and recovered. Then, dead cells and integrin β1 were stained in the same manner as in example 9, and supplied to measurement.
The expression rate of integrin β1 based on the difference in culture days of synovial-derived mesenchymal stem cells is shown in table 8. The integrin β1 expression rate of synovial-derived mesenchymal stem cells decreased in a day-dependent manner, 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, so that more than half of integrin β1 positive rate cells are preferred. Further, as shown in example 7, the molecular expression rate required for meniscus regeneration can be set to 60% or more, so that the number of days of culture is preferably 21 days or less.
TABLE 8
Table 8: culture time and integrin beta 1 expression rate after cell separation
Example 11 >
The positive rate of integrin beta 1 and PDGFRb, which are protein molecules necessary for drug efficacy, was also determined by flow cytometry on human synovial-derived stem cells (Cryopreserved Synoviocytes, normal, model P1: CDD-H-2910-N, batch: ST1414, ST1420, ST1434, ST 1462) purchased from Articular Engineering. The measurement apparatus used was Attune NxT, autoFocusing Cytometer (model: AFC2, invitrogen). As antibodies, integrin beta 1 (APC Mouse Anti-Human CD29 Cat: 559883), PDGFRb (Anti-PDGF Receptor beta, human, goat-Poly Cat: AF 385) were used, respectively.
As a result, the surface antigen positive rate of integrin beta 1 was 99.4%, 99.6% and 99.5% in each batch. The surface antigen positive rates of PDGFRb were found to be 92.2%, 90.7%, 95.2%, and 85.9%, respectively, for each batch. Thus, it was found that when the human synovial stem cells have therapeutic effects as an arthropathy therapeutic agent, it is appropriate to control integrin β1 to 90% or more and PDGFRb to 80% or more as standard values.
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, indicating that these can be set as effective quality control items of cells.
Example 12 > confirmation of meniscus regeneration effect of FGFR 3-inhibited rat synovial-derived Stem cells
As preparation of rat synovial-derived stem cells in which FGFR3 was inhibited, the frozen stock solution of the rat synovial-derived stem cells prepared in example 1 was resuscitated, and the cells were suspended in PBS containing 2% fbs as a reaction solvent after culturing for 1 week at 5% CO 2 concentration and 37 ℃ by adding Fetal Bovine Serum to give a final concentration of 20%, adding L-glutamie mmol/L to give a final concentration of 1%, and adding Antibiotic-Antimycotic (100×) to give a final concentration of α MEM no nucleosides of 1%. To 5X 10 6 cells per cell number, 100. Mu.g of FGFR3 Polyclonal Antibody (Invitrogen Cat. No. PA5-34574) was added, and after 1 hour of reaction under ice-cooling, the cells (FGFR 3-rSMSC) were recovered. As a control treatment without FGFR3 inhibition, rabbit IgG Isotype Control (Thermo FISHER SCIENTIFIC Cat. No. 10500C) was allowed to react under ice-cooling for 1 hour, and then cells (IgG-rSMSC) were recovered.
LEW/CrlCrlj rats were used in the preparation of a meniscus injury model for evaluation of meniscus regeneration effect. In the method of meniscus injury and mesenchymal stem cell transplantation, knee joint skin is incised under isoflurane anesthesia to expose the knee joint. The medial joint capsule under the knee was exposed, and the cartilage at the distal end of the femur was exposed by longitudinal incision with a scalpel. The medial meniscus was peeled away from the synovium, exposing the medial meniscus, and resected about 2/3 from anterior. The knee tendon and synovium were sutured, and then the muscle was sutured to make a model of meniscus injury. Then, the treated animals were divided into 3 groups, and 5×10 6 cells in which FGFR3 was inhibited (FGFR 3-rSMSC), 5×10 6 cells in which uninhibited control treatment was performed (IgG-rSMSC), and only solvent were injected into joint capsules, respectively. After treatment, all rats were returned to the cage, and allowed free movement and food intake.
After 3 weeks of treatment, animals were euthanized under isoflurane anesthesia by exsanguinating the lower aorta by severing it. Then, the meniscus was exposed from the knee joint, and the medial meniscus was removed and photographed. An image of the medial meniscus of the two knee joints removed is shown in fig. 23. The regenerated portion is determined based on the differences in hue and shape from the normal meniscus and is encircled with a dashed line. In the solvent group as a negative control, the meniscus had a majority of shapes to the middle node. In the group of IgG-rSMSC as a positive control, the meniscus regeneration to the anterior segment was more frequent, and the observed meniscus regeneration was more frequent. On the other hand, in FGFR3-rSMSC group, which is a cell whose molecule is inhibited or has been deleted, the observed meniscus regeneration portion is smaller than that of the positive control.
To quantitatively evaluate the visual observation result of the meniscus reproducing portion of fig. 23, the area of the meniscus reproducing portion (within the broken line) was calculated by the following formula using Image J (version 1.52).
Meniscus regeneration partial area (mm 2) =number of pixels of meniscus regeneration partial area/number of pixels per 1mm 2
The mean, standard deviation and statistical analysis of the regenerated partial areas of each group were performed with Microsoft Excel 2007 (Microsoft corp.). For statistical analysis, 2 Student's T-Test (Student t Test) was performed on the positive control group and the molecular inhibition group and the solvent group, and P-values were calculated, respectively. When the significance level was 5% (α=0.05), the significance level obtained by dividing the correction by Bonferroni by the number of assays (2 times) was 2.5% (α=0.025), and the results are shown in table 9.
Regarding the average area values of the meniscus regenerative portions described in Table 9, 1.2mm 2 of FGFR3-rSMSC group was significantly reduced relative to 1.9mm 2 of IgG-rSMSC. On the other hand, FGFR3-rSMSC groups had the same extent of regeneration area relative to 1.0mm 2 of the solvent group. From this, it was confirmed that FGFR3 molecules in synovial stem cells are important molecules contributing to meniscus regeneration.
TABLE 9
Table 9: area of meniscus regeneration section (mean ± standard deviation)
* Group P < 0.025vs IgG-rSMSC
A solvent group. /(I)
Claims (10)
1. An arthropathy therapeutic agent comprising synovial-derived mesenchymal stem cells having a surface antigen of any one or more of integrin beta 1 or platelet-derived growth factor receptor beta.
2. The therapeutic agent for arthrosis according to claim 1, wherein,
The synovial-derived mesenchymal stem cells have surface antigens of both integrin beta 1 and platelet-derived growth factor receptor beta.
3. The therapeutic agent for arthrosis according to 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 therapeutic agent according to claim 1 or 2, which has a surface antigen of FGFR 3.
5. The therapeutic agent for arthrosis according to claim 1 or 2, wherein,
The ratio of synovial membrane-derived mesenchymal stem cells having a surface antigen of at least one of integrin beta 1 and platelet-derived growth factor receptor beta relative to all cells contained in the therapeutic agent for arthrosis is at least 30%.
6. A method of manufacturing the arthropathy therapeutic agent of claim 1, comprising:
Step A, treating synovial tissue with enzyme;
Step B, cleaning the enzyme-treated mixture;
Step C, culturing the mesenchymal stem cells of synovial origin contained in the cleaned mixture on the substrate; and
And step D, separating the cultured mesenchymal stem cells from the substrate.
7. The method of claim 6, wherein,
The step B is a step of washing the enzyme-treated mixture until the residual enzyme concentration in the supernatant becomes 0.5ng/mL or less.
8. The method according to claim 6 or 7, wherein,
In the step C, the period of culturing the synovial-derived mesenchymal stem cells is 28 days or less.
9. The method according to claim 6 or 7, wherein,
In the step D, the mesenchymal stem cells are allowed to act on the cell-separating liquid for a period of time of 120 minutes or less.
10. The method according to claim 6 or 7, further comprising a step of sorting the synovial-derived mesenchymal stem cells having the surface antigen of any one or more of integrin β1 and platelet-derived growth factor receptor β.
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