MFAP5-Positive Synovial Lining Layer Stem Cells for Repairing Articular Cartilage Injury and Their Identification Method

Abstract

The present invention provides an MFAP5-positive synovial lining layer stem cell for repairing articular cartilage injury and an identification method thereof. By performing single-cell sequencing on synovial samples in animal model samples with cartilage defects and the regenerated cartilage-like tissue that has a physical connection with the synovial samples, and through bioinformatics analysis means, a cell subset that plays a key role in the regeneration of cartilage-like tissue, namely the MFAP5-positive subset, is found in synovial interstitial cells, and an identification method for this cell subset is clarified. This cell subset can be used as a more effective cell source for promoting cartilage regeneration, is expected to greatly shorten the in vitro cartilage differentiation time, and can be used to efficiently achieve in-situ repair of cartilage defects in vivo, having broad clinical application prospects in cartilage repair and regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering. Specifically, the present invention relates to MFAP5-positive synovial lining layer stem cells for repairing articular cartilage injury and an identification method thereof. Background Art

[0002] Treatment methods for repairing damaged or degenerated cartilage have always been challenging, partly due to the complex phenotype of cartilage tissue and the conditions that natural cartilage needs to bear. Microfracture was first introduced in 1959. This method releases bone marrow by drilling into the subchondral bone to induce cartilage repair and has been widely used since the 1990s. However, the fibrous repair tissue generated by microfracture is mainly composed of type I collagen, and its mechanical properties and durability are inferior to those of natural hyaline cartilage tissue. Autologous chondrocyte implantation (ACI) was first used in 1994. This surgery includes two surgical procedures. First, cells are isolated from healthy tissue, and then after in vitro expansion, they are re-implanted into the same patient. This method has later evolved into matrix-induced autologous chondrocyte implantation (MACI), that is, chondrocytes are inoculated on a scaffold before implantation and then implanted into the body. The above two methods are the main methods currently used clinically.

[0003] Currently approved cell-based tissue engineering cartilage products mainly include Spherox (European Union, Switzerland, and the United Kingdom); Bioseed-C and CaReS (some European countries); Novocart 3D (Germany, Switzerland); MACI (USA); J-Tec Autologous Cultured Cartilage (JACC, Japan); Chondron, CartiLife, and Cartistem (South Korea); and OrthoACI (Australia). Most of the above products are only approved for focal cartilage defects in the knee joint and are not applicable to OA (osteoarthritis) and RA (rheumatoid arthritis); except for Cartistem, all of the above products use autologous chondrocytes; and there is no report on the approved products proving that joint function can be completely and permanently restored and pain can be relieved through cartilage regeneration.

[0004] gMSC1 of TWOCELLS Company in Japan uses synovium-derived mesenchymal stem cells (MSCs) as seed cells and develops a three-dimensional scaffold-free tissue engineering construct (TEC), which is composed of synovium-derived MSCs and cell-synthesized extracellular matrix. However, it is necessary to culture it in a variety of different culture media for more than 5 passages to obtain a three-dimensional tissue with active contraction, which is called "gMSC1". However, in the selection of seed cells for this product, the stem cell subset directly related to chondrogenic differentiation in the synovium is not noticed, and only the cells in the synovium are extracted for culture under special technical conditions. The phase III clinical trial of this product shows that there is no statistically significant improvement compared with microfracture. It can be seen that gMSC1 prepared by directly culturing synovial tissue under special technical conditions for many times still has a large difference from natural cartilage, and it is still difficult to achieve the repair of articular cartilage loss and obtain an ideal repair effect.

[0005] In summary, the current problems in articular cartilage regeneration and repair are as follows: Existing methods for treating osteoarthritis (OA), such as conservative treatment and surgical treatment, cannot achieve the self-regeneration of natural hyaline cartilage, resulting in unsatisfactory long-term repair effects. Although some progress has been made in existing cartilage tissue engineering technologies, the formed fibrocartilage tissue still faces problems such as low mechanical strength and poor integration with native cartilage. At the same time, there are limitations in the sources of seed cells for articular cartilage repair: Traditionally, the seed cells used for articular cartilage repair mainly come from bone marrow mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs). However, these cells have low chondrogenic differentiation efficiency in vitro, are prone to calcification and hypertrophy, have a long culture time, and their chondrogenic differentiation ability depends on specific materials and complex culture medium components. It can be seen that the endogenous stem / progenitor cell subsets directly related to articular cartilage regeneration have not been clearly defined, especially the key stem / progenitor cell subsets with high chondrogenic differentiation potential; the lack of high-definition analysis of these cell subsets and systematic evaluation of their in vivo cartilage regeneration and repair functions limit their application in the treatment of cartilage injury.

[0006] Therefore, it is urgent to find new sources of seed cells with high chondrogenic differentiation potential, so as to develop more efficient products and methods for articular cartilage injury repair, improve the efficiency and effect of cartilage repair, provide an efficient and safe new stem cell strategy for the treatment of articular cartilage injury, significantly improve the deficiencies of existing treatment methods, and promote the development of cartilage regenerative medicine. Summary of the Invention

[0007] To solve the above problems, the present invention provides an MFAP5-positive synovial lining layer stem cell for articular cartilage injury repair and its identification method. By performing single-cell sequencing on synovial samples in animal models of cartilage defects and the regenerated cartilage-like tissue that has a physical connection with them, and through bioinformatics analysis means, a cell subset that plays a key role in the regeneration of cartilage-like tissue, namely the MFAP5-positive subset, is found in synovial mesenchymal cells, and the identification method of this cell subset is clarified. This cell subset can be used as a more effective cell source for promoting cartilage regeneration, greatly shortening the in vitro cartilage differentiation time, and can be used to efficiently achieve in-situ repair of cartilage defects in vivo, having broad clinical application prospects in cartilage repair and regeneration.

[0008] On the one hand, the present invention provides the use of a stem cell for preparing a reagent for articular cartilage injury repair or synthesis of new cartilage, wherein the stem cell is derived from synovial tissue and is positive for MFAP5.

[0009] As used in the present invention, "being positive" means that the cell can express a protein. For example, "being positive for MFAP5" means that the cell can express the MFAP5 protein.

[0010] The present invention proposes that there is a key mesenchymal stem cell subset with high cartilage differentiation potential (MFAP5-positive cell subset) in synovial tissue, which can participate in articular cartilage repair. By local transplantation, it can provide a more precise and efficient choice of seed cells for cartilage regeneration and repair, so as to solve the problem of low efficiency of existing cartilage repair and provide a new regeneration strategy for articular cartilage repair.

[0011] In the prior art, when directly using chondrocytes, there are problems such as few cell sources, difficult separation, low proliferation potential, and easy hypertrophy. Currently, bone marrow mesenchymal stem cells (BMSCs) are mainly used for cartilage repair, and their cartilage differentiation ability depends on the structure of materials and scaffolds, and there are tendencies such as easy calcification and excessive hypertrophy. In addition, the in vitro cartilage differentiation efficiency of BMSCs is low, the culture time is long, and it takes 21 days to differentiate into mature chondrocytes after co-culture with cartilage particles. Other sources of MSCs include adipose tissue, umbilical cord, etc. Among them, adipose-derived mesenchymal stem cells (ADSCs) are easier to obtain, but their cartilage differentiation potential is relatively lower.

[0012] In the previous experimental study on animal cartilage defect models, it was found that obvious regenerated tissues appeared at the contact site between the defective cartilage and synovial tissues, with obvious trochlear-like structures of joints. Therefore, the present invention believes that synovial mesenchymal cells are effective endogenous stem / progenitor cells for promoting cartilage regeneration, and the reasons are as follows: On the one hand, synovial mesenchymal stem cells are more similar to chondrocytes in terms of developmental relationship compared with other types of cells. In the early stage of embryonic development, mesenchymal stem cells (MSCs) in the mesoderm will aggregate at the position where joints will develop. A part of the MSCs form the interzone of the joint and express the Gdf5 gene in mice; with the development of the embryo, the MSCs around the interzone further differentiate into fibroblasts and macrophages in the synovium. On the other hand, in adulthood, there are still proliferative cells with the MSC phenotype in the synovium. These stem cells can respond to injuries, proliferate and differentiate to support cartilage repair.

[0013] In order to find the true stem cell subset with high chondrogenic differentiation potential in synovial tissues, the present invention performed single-cell RNA sequencing (scRNA-seq) on synovial samples in animal model samples with cartilage defects and the regenerated cartilage-like tissues that have physical connections with them, looking for similar cells that appear in both synovium and nascent cartilage-like tissues, sorting them according to the degree of similarity, and determining 11 cell clusters (Clusters) through integrated clustering analysis. One group is the MFAP5-positive (specifically expressed) mesenchymal cell subset from the regenerated cartilage-like and sublining layer of synovial tissues. Its spatial position in the UMAP dimensionality reduction map is closely related to both typical mesenchymal cells and chondrocytes. Subsequently, the present invention separately grouped the sequencing data of synovial tissues and nascent cartilage tissues and found that the MFAP5 gene-positive cell group also showed strong specificity in single tissues. The similarity comparison of the MFAP5 gene-positive cell groups in the two tissues also showed that the similarity of this MFAP5 gene-positive mesenchymal cell group between the two tissue sources was significantly higher than that of other cell groups. GO pathway analysis showed that this group specifically expressed biological pathways related to chondrogenic differentiation and extracellular matrix synthesis. The pseudotime analysis of nascent chondrocytes also showed that this cell group was at the front end of the chondrocyte differentiation trajectory, that is, at the upstream of the differentiation process. As the pseudotime progresses, the expressed genes change from upstream development-related SFRP2, POSTN, etc. to downstream mature articular cartilage cell-related ACAN, COL2A1, etc.

[0014] Based on the results of single-cell high-throughput sequencing, the technology of the present invention discovered and defined a key mesenchymal cell subset (MFAP5 gene-positive subset) with high chondrogenic differentiation potential through bioinformatics analysis means, and determined that it plays a key role in the regeneration of cartilage-like tissues, greatly shortening the in vitro chondrogenic differentiation time and being applicable to in situ repair of cartilage defects in vivo.

[0015] Previous studies have shown the existence of heterogeneous MFAP5-positive cell subsets in synovial stromal cells (such as Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes. Nature. 2023 Nov;623(7987):616 - 624.). However, it only revealed the existence of MFAP5-positive cell subsets in synovial tissue through transcriptome sequencing and did not find that MFAP5-positive cell subsets have the function of differentiating into cartilage. The present invention for the first time discovers that MFAP5-positive cell subsets play a key role in the regeneration of cartilaginous tissues and evaluates the potential and application prospects of MFAP5-positive synovial stem cells in chondrogenic differentiation.

[0016] On the other hand, the present invention provides a use of stem cells for preparing a reagent for repairing articular cartilage injury or synthesizing new cartilage, wherein the stem cells are derived from synovial tissue and are positive for CD34 and / or THY1.

[0017] Through single-cell data analysis and multi-database integration, the present invention determines the key surface markers of MFAP5-positive synovial lining stem cell subsets under different species and physiological conditions, namely CD34 and THY1, which can be used for the identification, sorting, and subsequent culture of cells in this subset, and clarifies the sorting and identification methods of MFAP5-positive synovial stem cell subsets.

[0018] On yet another aspect, the present invention provides a product for repairing articular cartilage injury or preparing new cartilage, the product comprising stem cells, wherein the stem cells are derived from synovial tissue and are positive for MFAP5.

[0019] On yet another aspect, the present invention provides a method for identifying stem cells for repairing articular cartilage injury. By identifying whether the stem cell membrane protein is positive for CD34 and / or THY1, it is determined whether the stem cell is the stem cell for repairing articular cartilage injury.

[0020] Research has proven that CD34 and THY1 are membrane protein markers of MFAP5-positive synovial stem cells and can be used for identifying MFAP5-positive synovial stem cells by flow cytometry.

[0021] Furthermore, by identifying whether the stem cell membrane protein is positive for CD34 and THY1, it is determined whether the stem cell is the stem cell for repairing articular cartilage injury.

[0022] Research has shown that solely identifying positive CD34 or THY1 is not conducive to the subsequent sorting of the MFAP5-positive synovial stem cell subset. It is necessary to identify both CD34 and THY1 as positive to accurately identify and sort out the MFAP5-positive synovial stem cell subset.

[0023] In some ways, flow cytometry is used to analyze whether the intracellular protein MFAP5 is positive to verify whether it is the stem cell for articular cartilage injury repair.

[0024] By analyzing whether the intracellular protein MFAP5 is positive, the accuracy of identifying CD34 and THY1 is thus verified.

[0025] Of course, it is also possible to directly identify by analyzing whether the intracellular protein MFAP5 is positive through flow cytometry. However, since the pretreatment of intracellular protein analysis is cumbersome and can lead to cell death, making it impossible to continue sorting out the MFAP5-positive synovial stem cell subset, it is therefore more direct and convenient to sort out the MFAP5-positive synovial stem cell subset by identifying CD34 and THY1 through membrane proteins.

[0026] On the other hand, the present invention provides a method for sorting MFAP5-positive stem cells. The method involves taking a synovial sample, extracting primary cells, obtaining a cell suspension through culture, and then sorting out MFAP5-positive stem cells through flow cytometry analysis.

[0027] Furthermore, the culture is suspension culture.

[0028] Theoretically, any culture method, such as adherent culture or suspension culture, as long as it can culture the extracted primary cells, can be used to prepare and analyze MFAP5-positive stem cells. Research of the present invention has shown that suspension culture is more conducive to sorting out MFAP5-positive stem cells.

[0029] Furthermore, during the flow cytometry analysis, two surface markers, THY1 and CD34, of MFAP5-positive stem cells need to be analyzed simultaneously.

[0030] On the other hand, the present invention provides a method for preparing new cartilage. The method is obtained through the differentiation of stem cells, and the stem cells are derived from synovial tissue and are positive for MFAP5.

[0031] On the other hand, the present invention provides the use of MFAP5-positive stem cells for preparing a reagent that shortens the in vitro culture time of cartilage and improves the consistency of cartilage differentiation. The MFAP5-positive stem cells are derived from synovial tissue.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1)By means of single-cell sequencing technology, the MFAP5-positive stromal cell subset was first identified and applied, promoting in-depth research on chondrogenic regenerative cell subsets and providing new ideas and directions for subsequent cartilage repair research;

[0034] (2)Through multi-database integration, the present invention determined the key surface markers of MFAP5-positive subset cells under different species and physiological conditions, namely CD34 and THY1, which can be used for sorting and subsequent culture of this subset of cells; by flow cytometry, combined with the surface markers CD34 and THY1 of the MFAP5-positive subset, the MFAP5-positive stromal cell subset with high chondrogenic differentiation potential was accurately identified and sorted from synovial tissue, enabling efficient separation and purification of this subset of cells and providing a high-quality cell source for subsequent chondrogenic differentiation and regeneration; compared with simply extracting cells from the synovium, the cell sorting technology utilized in the present invention can ensure the consistency and efficiency of the obtained cell population in chondrogenic differentiation potential;

[0035] (3)The MFAP5-positive stromal cells of the present invention can differentiate into chondrocyte-like cells in a relatively short time, significantly shortening the in vitro culture time, enhancing the maturity of chondrogenic differentiation, improving the efficiency of cartilage regeneration and the feasibility of clinical application;

[0036] (4)The MFAP5-positive stromal cells of the present invention do not need to rely on autologous chondrocytes and have high chondrogenic differentiation potential, and can be efficiently amplified and differentiated in vitro; it can also avoid the disadvantage of two surgeries required for autologous chondrocyte transplantation, reducing the pain and surgical risks of patients, and providing a safer, long-term and effective solution for cartilage repair;

[0037] (5)Compared with traditional BMSCs and ADSCs, the MFAP5-positive stromal cells of the present invention have a closer differentiation relationship with articular cartilage, show more efficient differentiation ability during chondrogenic differentiation, and are not prone to problems such as calcification and overhypertrophy, and their chondrogenic differentiation potential is more efficient and specific, promising to achieve the true regeneration of functional cartilage tissue.

[0038] Detailed description

[0039] 1. Chondrogenic differentiation

[0040] Chondrogenic differentiation refers to the process by which undifferentiated mesenchymal stem cells (such as bone marrow mesenchymal stem cells or embryonic stem cells) gradually differentiate into chondrocytes through specific developmental signals and environments. This process is crucial for the formation of cartilage during embryogenesis and the repair of cartilage damage in adult individuals. Chondrogenic differentiation is usually regulated by signals of the TGF-β family and includes steps such as initial cell aggregation, differentiation into chondrocytes, and production of extracellular matrix.

[0041] The chondrogenic differentiation in the present invention also includes the differentiation of MFAP5-positive stromal cells provided by the present invention into chondrocytes.

[0042] 2. Extracellular Matrix (ECM)

[0043] The extracellular matrix is a complex network structure in tissues, mainly composed of proteins (such as collagen, elastin) and polysaccharides (such as glycosaminoglycans), providing physical support and biochemical signals to help maintain cell functions, shapes, and tissue structures. The ECM not only supports the physical structure of tissues but also regulates cell behavior, affecting processes such as cell proliferation, differentiation, and migration. In cartilage tissue, the extracellular matrix is composed of a large amount of collagen and proteoglycans, endowing cartilage with elasticity and pressure-bearing capacity.

[0044] 3. Hyaline Cartilage

[0045] Hyaline cartilage is a widely distributed type of cartilage with a smooth, translucent appearance, mainly present in joints, ribs, nose, trachea, etc. It consists of a rich extracellular matrix and a small number of chondrocytes. The extracellular matrix of hyaline cartilage contains a large amount of type II collagen and proteoglycans, providing pressure resistance and a certain degree of elasticity. Its main function is to reduce friction between bones, support, and buffer pressure.

[0046] 4. Fibrocartilage

[0047] Fibrocartilage is a relatively tough type of cartilage containing a large amount of thick fibrous collagen. It usually appears in areas that need to withstand greater mechanical stress, such as intervertebral discs, joint discs, and bone junctions. The collagen fibers in fibrocartilage are arranged more closely, having strong tensile and compressive properties, and it is a key tissue type for structural support and buffering.

[0048] 5. Cell Surface Markers

[0049] Refers to specific protein or glycoprotein molecules, usually located on the cell membrane surface, which can achieve cell recognition and separation by binding to specific antibodies. Cell surface markers are widely used in biomedical research for identifying and classifying different types of cells, especially in stem cell biology and immunology research. The expression of each cell surface marker is usually closely related to cell functions, differentiation states, and developmental processes.

[0050] In the present invention, the sorting of the MFAP5-positive stromal cell subset relies on specific cell surface markers, such as CD34, for isolating the cell subset with high chondrogenic differentiation potential by flow cytometry.

[0051] 6. Mesenchymal Stem Cells (MSCs)

[0052] Mesenchymal stem cells are a type of adult stem cells with self-renewal and multi-directional differentiation potential, capable of differentiating into various cell types such as bone, cartilage, and fat. Common sources include bone marrow, adipose tissue, synovium, etc.

[0053] 7. Synovial Mesenchymal Stem Cells (sMSCs)

[0054] Synovial mesenchymal stem cells are mesenchymal stem cells derived from the synovium of joints and have strong cartilage differentiation potential. Compared with MSCs from other sources, sMSCs show higher efficiency in cartilage regeneration.

[0055] The MFAP5-positive stromal cells provided by the present invention also belong to a type of synovial mesenchymal stem cells.

[0056] 8. MFAP5 (Microfibril-Associated Glycoprotein 5)

[0057] MFAP5 is a glycoprotein associated with extracellular matrix microfibrils. In the present invention, the MFAP5-positive cell subset is used as a marker for synovial stromal cells, has high cartilage differentiation potential, and can promote cartilage repair.

[0058] 9. Collagen Type II (COL2A1)

[0059] COL2A1 is a collagen specifically expressed in cartilage tissue and constitutes the main component of the cartilage matrix. The expression of COL2A1 is an important landmark event during cartilage differentiation and is used to evaluate the success rate of chondrocyte differentiation.

[0060] 10. Aggrecan (ACAN)

[0061] Aggrecan is one of the main proteins produced by chondrocytes. It binds to hyaluronic acid to form a large molecular complex in the cartilage matrix, which is responsible for maintaining the elasticity and structure of cartilage.

[0062] 11. Fibroblasts

[0063] Fibroblasts are the main cell type in connective tissue and are involved in the production of the extracellular matrix. In synovial tissue, fibroblasts have a certain developmental relationship with chondrocytes and may play a role in cartilage repair.

[0064] 12. Bone Marrow Mesenchymal Stem Cells (BMSC)

[0065] BMSC are mesenchymal stem cells derived from bone marrow, with the potential for multi-directional differentiation and can differentiate into various cell types such as cartilage, bone, and fat. BMSC are commonly used in cartilage regeneration research, but their cartilage differentiation efficiency is relatively low.

[0066] 13. Adipose-Derived Mesenchymal Stem Cells (ADSC)

[0067] ADSC are mesenchymal stem cells extracted from adipose tissue, with self-renewal and differentiation potential. The application of ADSC in cartilage regeneration is becoming increasingly widespread, but their cartilage differentiation potential is slightly lower than that of BMSC.

[0068] 14. Flow Cytometry

[0069] Flow cytometry is a technique used for the rapid analysis and sorting of cell populations. This technique suspends cells in a flowing liquid and then passes them one by one through a laser beam, using fluorescent labels to detect specific molecules on the cell surface or inside the cell. The characteristics of each cell (such as size, shape, fluorescence intensity of surface markers) are recorded, thus enabling the classification and counting of cells.

[0070] In the present invention, flow cytometry is used to sort out the MFAP5-positive stromal cell subset through specific cell surface markers (such as CD34). These cells have high differentiation potential in cartilage regeneration and repair.

[0071] 15. Single-Cell RNA Sequencing (scRNA-seq)

[0072] Single-cell RNA sequencing is a technique used to determine the gene expression profile in a single cell. It enables researchers to analyze heterogeneous cell populations in complex tissues and identify the gene expression characteristics of each cell. This technique is particularly suitable for studying cell differentiation, developmental trajectories, and cell interactions.

[0073] In the present invention, single-cell RNA sequencing is used to analyze cell types in synovial tissue and neo-chondroid tissue, and to identify a key cell subset involved in cartilage regeneration - MFAP5-positive stromal cells.

[0074] 16. Pseudotime Analysis

[0075] Pseudotime analysis is an analytical method for single-cell RNA sequencing data, which can infer the continuous trajectory of cells from an undifferentiated state to a differentiated state. Through this technique, researchers can reconstruct the developmental process of cells and observe the gene expression changes of cells at different differentiation stages.

[0076] In the present invention, pseudotime analysis is used to study the differentiation trajectory of MFAP5-positive cells and to infer how these cells gradually differentiate into mature chondrocytes during cartilage regeneration.

[0077] 17. Tissue Engineering

[0078] Tissue engineering is a technology that creates or repairs tissues by combining cells, engineering materials, and biological factors. Its goal is to construct functional tissues using cells and scaffold materials for replacing or repairing damaged tissues or organs.

[0079] In cartilage regeneration, tissue engineering techniques combine seed cells (such as MFAP5-positive stromal cells) with scaffold materials to promote cartilage regeneration. Common tissue engineering methods include using natural or synthetic scaffold materials to adsorb or implant cells to form a three-dimensional structure to support tissue regeneration.

[0080] 18. Osteoarthritis (OA)

[0081] Osteoarthritis is a common degenerative joint disease, mainly manifested as cartilage degeneration, joint pain, and dysfunction. Due to the limited regenerative ability of cartilage, the treatment of OA usually relies on symptom management or surgical repair.

[0082] 19. Cartilage Injury

[0083] Cartilage injury refers to the damage or degeneration of articular cartilage due to trauma, degenerative diseases, or other pathological factors. The repair of cartilage injury is relatively difficult because the self-repair ability of cartilage tissue is low.

[0084] 20. Osteochondritis Dissecans (OCD)

[0085] Osteochondrosis is a disease caused by insufficient blood supply to the epiphysis, resulting in the separation of cartilage and bone tissue, and is common in the knee joint. OCD is one of the main pathological models in cartilage repair research.

[0086] 21. Knee Joint

[0087] The knee joint is the largest joint in the human body, composed of the femur, tibia, and patella, and the joint surface is covered with hyaline cartilage. Cartilage injury in the knee joint is a major feature of diseases such as osteoarthritis, and cartilage repair is crucial for the restoration of knee joint function.

[0088] 22. Cartilage Injury Model

[0089] The cartilage injury model is an experimental model used to study cartilage repair and regeneration. Animal models (such as mice, rabbits, or pigs) are usually used to simulate the pathological process of human articular cartilage injury. Description of the Drawings

[0090] Figure 1 It is a photograph of the appearance of new cartilage in the animal model in Example 1. The left figure is the animal model with a defect site, and the right figure is the new cartilage that appears in the animal model;

[0091] Figure 2 It is the result of dimensionality reduction and Mfap5+ subset distribution of multi-tissue single-cell sequencing in Example 1;

[0092] Figure 3 It is the result of separate clustering analysis of the sequencing data of synovial tissue and new cartilage tissue in Example 1. Among them, (a-d) are the UMAP dimensionality reduction and Mfap5 gene expression of synovial tissue and new cartilage single-cell sequencing, and S1 and E2 are Mfap5+ subsets; (e) pseudotime analysis in the new cartilage tissue shows that the Mfap5+ subset differentiates towards the cartilage direction; (f-g) the GO pathways of S1 and E2 subsets show similarity, with cartilage development and extracellular matrix synthesis entries. The horizontal axis (bar length) represents the gene ratio, and the numbers marked on the right are the enrichment P values; (h) comparison of subset similarity between tissues, and S1 and E2 subsets show strong similarity.

[0093] Figure 4Results of cross-species integration analysis of the MFAP5-positive cell population in Example 1, where (a-b) are single-cell sequencing results of human OA and RA samples, and it is found that there is also an MFAP5+ subset with similar expression patterns, namely the OA-1 subset and the RA-4 subset; (c-d) are the results of cross-species integration of synovial data of humans and rats, and the overlap of Cluster 1 is high among species; (e) is the expression of the MFAP5 gene in the integrated cell population, and Cluster 1 shows specific expression of MFAP5, and the surface markers CD34 and THY1 are also positive;

[0094] Figure 5 The technical flow chart of cell culture and sorting in Example 2;

[0095] Figure 6 The staining analysis of human synovial samples and the staining results of different culture methods in Example 2; where (a) is the H&E staining of human synovium, LL is the Lining Layer, and SL is the Sublining Layer; (b) is the immunofluorescence staining of the MFAP5+ subset in human synovial samples (labeled with Alexa Fluor 488 / 546); (c) is the immunofluorescence staining of Sox9 / Col2 in chondrospheres induced by synovial mixed cells to confirm that some synovial cells have the ability of chondrogenic differentiation under induction conditions; (d) is the fluorescence staining of adherent cultured cells; (e) under suspension culture conditions, MFAP5+ cells exist and most co-express their surface markers;

[0096] Figure 7 The results of cell flow cytometry analysis after two weeks of three-dimensional culture of synovial fibroblasts in Example 2;

[0097] Figure 8 The staining and fluorescence images of chondrocytes induced by the MFAP5-positive cell population of rats in Example 3;

[0098] Figure 9 The staining and fluorescence images of chondrocytes induced by rat BMSCs cells in Example 3. Detailed implementation manners

[0099] The present invention will be further described in detail below with reference to the examples. It should be noted that the following examples are intended to facilitate the understanding of the present invention and do not limit it in any way. Those conditions not specified in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0100] Example 1. Identification of synovial stem cell subsets involved in articular cartilage loss repair

[0101] In this example, through single-cell RNA sequencing (scRNA-seq) technology and combined with bioinformatics analysis, in-depth research was carried out on synovium and regenerated cartilage-like tissue, and a key cell subset with high chondrogenic potential was identified.

[0102] I. Obvious new cartilage phenomenon appears in animal cartilage defect model

[0103] In the previous research on animal cartilage defect model experiments, about four weeks after the defect modeling, obvious regenerated tissue unexpectedly appeared on the side of the joint, especially obvious at the attachment site of the patellar ligament, with an obvious trochlear joint-like structure. After histological staining with SO (safranin-fast green), it was found to be bright red transparent cartilage tissue. A large number of cartilage lacunae could be observed in the tissue cells in the cartilage layer area, as Figure 1 shown. The histological staining results showed that the newly formed cartilage formed a complete osteochondral structure, and the surface cartilage was transparent cartilage. This phenomenon was different from the synovial tumor tissue commonly seen clinically. Hemangiomatous synovial tumors had a large amount of blood vessels, fat, and fibrous tissue, while this newly formed tissue was all composed of transparent cartilage components. Therefore, it was considered that there was a new cartilage phenomenon.

[0104] II. Complete single-cell sequencing analysis of regenerated cartilage and synovial tissue, and determine the chondrogenesis-related mesenchymal cell subset

[0105] Therefore, in this example, cells from two tissues, synovium and regenerated cartilage-like, were extracted and single-cell RNA sequencing (scRNA-seq) was performed respectively:

[0106] 1. Sample extraction: Extract synovial tissue and regenerated cartilage tissue from the cartilage defect site of the animal model. First, wash the samples, and then enzymatically digest the synovial tissue to obtain a primary cell suspension.

[0107] Preparation of synovial tissue cell suspension: Add the synovial tissue sample to a medium containing digestive enzymes and incubate at 37°C for 1 hour. Then, terminate the digestion and centrifuge at 1200 rps to obtain a cell pellet, which is resuspended in complete medium to obtain a synovial tissue cell suspension for single-cell sequencing.

[0108] Preparation of regenerated cartilage tissue cell suspension: Add the regenerated cartilage tissue sample to a medium containing digestive enzymes and incubate at 37°C for 1 hour. Then, terminate the digestion and centrifuge at 1200 rps to obtain a cell pellet, which is resuspended in complete medium to obtain a regenerated cartilage tissue cell suspension for single-cell sequencing.

[0109] 2. Single-cell RNA sequencing

[0110] Cell Capture and Library Construction: The 10x Genomics platform was used for cell capture and cDNA library construction. After the sample was processed into a single-cell suspension, individual cells were encapsulated in oil droplets, and high-efficiency single-cell capture was performed through microfluidic technology. Subsequently, cDNA was synthesized through reverse transcription reaction, and a sequencing library was generated through PCR amplification.

[0111] 3. Sequencing and Data Processing

[0112] The sequencing platform was Illumina NovaSeq. After the sequencing data passed quality control, software such as Cell Ranger was used for data analysis, and finally a gene expression matrix was obtained.

[0113] 4. Data Analysis and Cell Subset Identification

[0114] Clustering Analysis: Tools such as Seurat were used to perform clustering analysis on the sequencing data, integrating single-cell data from synovium and regenerated cartilage-like tissue, and 11 cell clusters (Clusters) were determined, as shown in Figure 2 a in. These are relatively similar cell clusters in the synovium and neonatal cartilage-like tissues. One group of Mfap5 gene-positive mesenchymal cells from the sublining layer of the regenerated cartilage-like and synovial tissues is closely related to typical mesenchymal cells and chondrocytes in terms of their spatial position in the UMAP dimensionality reduction map. It was found in the Umap integration that MFAP5 is a marker specifically expressed in the adjacent region of synovial tissue and neonatal cartilage tissue ( Figure 2 c3 in c of ), showing significant aggregation, indicating its important role in synovial and regenerated cartilage-like tissues.

[0115] Specific Marker Analysis: Through differential expression analysis, it was found that the MFAP5-positive cell group specifically expressed marker genes related to cartilage differentiation, such as COL2A1 and ACAN, and its potential in cartilage differentiation was confirmed. In addition, the MFAP5-positive cell group also highly expressed genes related to extracellular matrix synthesis, such as SFRP2 and POSTN.

[0116] Subsequently, in this example, the sequencing data of synovial tissue and neonatal cartilage tissue were grouped separately, and it was found that this cell subset of Mfap5 gene-positive mesenchymal cells also showed strong specificity in single tissues. The similarity comparison of the Mfap5+ cell groups (S1, E2 groups) in the two tissues ( Figure 3 h in ) also showed that the similarity of this group of mesenchymal cells between the two tissue sources was significantly higher than that of other cell groups.

[0117] GO pathway analysis showed that this Mfap5 gene-positive mesenchymal cell group specifically expressed biological pathways related to cartilage differentiation and extracellular matrix synthesis (Figure 3 f in Figure 3 g) in Figure 3 a~ in Figure 3 d) Synovial tissue, single-cell sequencing UMAP dimensionality reduction of neonatal cartilage and Mfap5 gene expression in Figure 3 e) Pseudotime analysis in neonatal cartilage tissue showed that the Mfap5+ subset differentiated towards cartilage Figure 3 f~ in Figure 3 g) The GO pathways of the S1 and E2 subsets showed similarity, with entries related to cartilage development and extracellular matrix synthesis Figure 3 h) Comparison of subset similarity between tissues showed strong similarity between the S1 and E2 subsets

[0118] Pseudotime analysis of neonatal chondrocytes monitored their continuous trajectory from the undifferentiated state to the chondrocyte differentiation state. The results showed that the MFAP5-positive cell population was in the early stage of the chondrocyte differentiation trajectory. As pseudotime progressed, the expressed genes changed from upstream development-related SFRP2, POSTN, etc. to downstream mature hyaline chondrocyte ACAN, COL2A1, etc. ( Figure 3 e) in

[0119] The above analysis confirmed that the function of the MFAP5-positive cell population was mainly related to extracellular matrix synthesis and cartilage differentiation entries

[0120] III. Cross-species integration analysis preliminarily confirmed the strong conservation of this subset

[0121] During the pathological process of clinical osteoarthritis, there is no phenomenon of newly formed trochlear cartilage. Instead, there are more fibrotic pathological synovial tumors. In this example, the published literature data was used to analyze the single-cell RNA sequencing data of synovium from clinical samples in healthy and arthritic states. The results are as Figure 4 shown, where Figure 4 a~ in Figure 4 b in Figure 4 showed that the MFAP5+ subset also existed in the single-cell sequencing of human OA and RA samples, and the expression patterns were similar, namely the OA-1 subset and the RA-4 subset, confirming that the MFAP5+ mesenchymal cells also existed in human synovium and the gene expression patterns were similar ( Figure 4 a) in

[0122] Figure 4 c~ in Figure 4 Cross-species integration of synovial data from humans and rats for d in Figure 4 revealed a high degree of overlap among 1 group after integration; Figure 4 For the expression of MFAP5 gene in the integrated cell population e in Figure 4 , 1 group showed specific expression of MFAP5.

[0123] After validation in human synovial tissue, by analyzing single-cell RNA sequencing data of human synovial samples, it was found that there was also a MFAP5-positive cell subset, and its gene expression pattern was highly similar to that of the MFAP5-positive cell population in the animal model, indicating the conservation of this cell subset among different species. This laid the foundation for the clinical application of the MFAP5-positive cell population.

[0124] Example 2: Flow cytometry analysis of MFAP5-positive cell population

[0125] This example completed the histological validation of specific markers of the MFAP5-positive cell subset and determined the culture and flow cytometry analysis methods for this cell.

[0126] The technical process is as Figure 5 shown: Knee joint synovial samples were taken from patients ( / healthy volunteers), primary cells were extracted by enzymatic digestion with type I collagenase, and flow cytometry sorting was performed with the help of special cell surface markers to obtain the MFAP5-positive subset. Then, this cell subset was used as seed cells with high chondrogenic potential for subsequent applications and cell culture.

[0127] In this example, immunofluorescence staining was first performed on MFAP5 protein and subset surface markers in human synovial samples ( Figure 6 ), and the results of H&E staining of human synovium are shown in Figure 6 a in Figure 6 to observe the basic tissue structure of the synovium, including LL and SL. LL is the Lining Layer, and SL indicates the Sublining Layer; the results of immunofluorescence staining (Alexa Fluor 488 / 546-labeled) of the MFAP5+ subset in human synovial samples are shown in Figure 6 b in Figure 6 , thus verifying the existence and sorting feasibility of the subset and providing certain feasibility for the analysis and sorting of the subset.

[0128] In this example, chondrospheres were directly obtained by inducing primary cells (synovial mixed cells) (the induction method was chondrogenic induction medium, and the components of the cell suspension centrifuged into spheres culture medium were: high-glucose DMEM medium + 1x Sodium pyruvate + 1% ITS + 10^(-7) M dexamethasone + 50 μg / mL Vc + 10 ng / mL TGF-β3 + 1x P / S), and Sox9 / Col2 immunofluorescence staining was performed on them. The results are shown in Figure 6 c in

[0129] To culture and sort the corresponding synovial cell subsets, this example tried various methods such as cell adherent culture and suspension culture, and determined the culture protocol ( Figure 6 d in Figure 6 e in

[0130] Among them, the specific process of cell suspension culture is as follows:

[0131] 1) Cell extraction: Take a clinical synovial sample (taking the size of a soybean as an example), first wash it (washing solution: dilute PS to 2% with PBS) and roughly cut the sample. Put the sample into a 1.5 mL centrifuge tube, add 1 mL of enzyme solution (2 μg / mL type I collagenase) and cut it into pieces. The sample is digested at 37 °C for 4 hours. Transfer the sample to a centrifuge tube, centrifuge at 1200 rps for 6 min, discard the supernatant, resuspend it with the medium (formula of low-glucose DMEM medium + 10% FBS + 1x P / S), and centrifuge at 1200 rps for 6 min, discard the supernatant. Finally, resuspend the cells with 6 mL of medium.

[0132] 2) Cell culture: Inoculate 6 mL of the cell suspension into a low-attachment 6-well plate, add microspheres (mesoporous mineralized silk methacrylamide microspheres, patent applied for 2024111118570), and culture it in a cell incubator (37 °C, 5% CO2), change the medium every other day, and expand for 2 weeks.

[0133] Fluorescent staining was performed on adherent-cultured cells and suspension-cultured cells respectively. The results are shown in Figure 6 d in Figure 6 e in Figure 6 d in Figure 6 is the result of fluorescent staining of adherent-cultured cells, and

[0134] For the cell suspension obtained by suspension culture, flow cytometry analysis of cell surface markers was performed in this example: Based on the single-cell sequencing results, we confirmed the specific surface marker CD34 of the MFAP5-positive cell population and the synovial sublining marker THY1. Therefore, in this example, flow cytometry was used to perform flow cytometry analysis of the MFAP5-positive cell population with surface markers THY1 and CD34 as markers.

[0135] Flow cytometry sorting process:

[0136] 1) Take 1 - 2 x 10 6 single cells obtained by suspension culture and resuspend them in flow cytometry staining buffer. Add two fluorescently conjugated antibodies (anti-CD34, THY1, conjugated with PE and APC fluorescence respectively) to the cell suspension according to the recommended dilution ratio (1:50 - 1:200). After mixing, incubate at 4°C in the dark for 30 minutes. Wash the cells with FACS buffer, centrifuge at 300g for 5 minutes, discard the supernatant, and then resuspend in FACS buffer.

[0137] 2) Add an appropriate amount of dead cell dye, such as Fixable Viability Dye, according to the product manual, incubate and then wash once. The dead cell dye is used to exclude dead cell signals in flow cytometry. Then resuspend the cells in an appropriate amount of FACS buffer and prepare for flow cytometry detection.

[0138] 3) Set the laser channels of the flow cytometer according to the fluorescence labeling of the antibodies. Use PE fluorescence to detect CD34 protein, use APC fluorescence to detect THY1 protein, and set fluorescence compensation using single-stained cell samples to ensure that signals between different fluorescence channels do not interfere with each other.

[0139] 4) According to the forward scatter (FSC) and side scatter (SSC) plots, exclude cell debris and impurities. Then exclude dead cells according to the signal of the dead cell dye. Select the CD34⁺THY1 + positive cell subset on the flow cytometer. Determine the expression of cell markers through the fluorescence signals of the antibodies.

[0140] Since MFAP5 is an intracellular protein, while CD34 and THY1 are membrane proteins, directly using MFAP5 for flow sorting requires cell membrane permeabilization to disrupt the cell structure, which affects subsequent cell sorting applications. Although CD34 can preferably and specifically label the MFAP5 subset in fibroblasts, CD34 is also a marker for vascular endothelial cells. Using only CD34 as a marker for flow cytometry analysis cannot accurately screen for the MFAP5-positive cell population and may introduce vascular endothelial cells. THY1 is a marker for synovial sublining fibroblasts. Using only THY1 also cannot accurately screen for the MFAP5-positive cell population. When THY1 and CD34 are used simultaneously as markers, the MFAP5-positive cell population can be sorted more accurately.

[0141] Figure 7 This is the result of flow cytometry analysis of cells after two weeks of three-dimensional culture of synovial fibroblasts. Q2-1 is the CD34+THY1+ double-positive region (about 31%). After screening and immunofluorescence staining identification, the double-positive region is indeed the MFAP5-positive cell population.

[0142] Example 3. In vitro functional verification of MFAP5-positive cells

[0143] After sorting out the MFAP5-positive cell population, this example further amplifies and verifies its functions in vitro.

[0144] Collect the rat MFAP5-positive cell population obtained by screening in Example 2 into a centrifuge tube. After centrifugation and precipitation, discard the supernatant, add 3 mL of chondrogenic differentiation medium (high-glucose DMEM + 1x Sodium Pyruvate + 1% ITS + 10^(-7) M dexamethasone + 50 μg / mL vitamin C + 10 ng / mL TGF-β3 + 1x antibiotic P / S), and culture in a cell incubator at 37°C and 5% CO2. Replace the medium every other day and induce culture for 3 weeks to successfully form cartilage particles (staining and fluorescence images are shown in Figure 7). In addition, this example also compares the chondrogenic particle induction effects of using rat bone marrow mesenchymal stem cells (BMSCs) and synovial mesenchymal stem cells (SMSCs) under the same conditions (see Figure 8).

[0145] In SO staining, red represents the degree of staining into the cartilage matrix. The darker the color, the more significant the effect of chondrogenic differentiation. By comparing Figure 7 and Figure 8, it can be seen that after 21 days of induction, the red staining of synovial mesenchymal stem cells is deeper than that of BMSCs, indicating stronger chondrogenic ability and better differentiation consistency. Fluorescence staining was used to detect the expression levels of SOX9 and type II collagen. Among them, SOX9 is an important transcription factor related to the formation of cartilage matrix, and type II collagen is the main component of cartilage matrix. The results showed that the fluorescence staining of synovial mesenchymal stem cells was strongly positive, while BMSCs showed poor performance in chondrogenesis, and only a small number of cells showed fluorescence staining. In addition, synovial mesenchymal stem cells can differentiate into chondrocyte-like cells in a shorter time, significantly shortening the in vitro culture time and improving the maturity and consistency of chondrogenic differentiation.

[0146] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A method for preparing a reagent for repairing articular cartilage damage or synthesizing new cartilage by using stem cells, characterized in that: The stem cells were derived from synovial tissue and were positive for MFAP5.

2. A method for preparing a reagent for repairing articular cartilage damage or synthesizing new cartilage using stem cells, characterized in that: The stem cells were derived from synovial tissue and were positive for CD34 and THY1.

3. A method for identifying synovial stem cells for repairing articular cartilage damage, characterized in that: By identifying whether the stem cell membrane proteins are positive for CD34 and THY1, it can be determined whether they are synovial stem cells for repairing articular cartilage damage.

4. A method for sorting MFAP5-positive stem cells, characterized in that: A synovial sample is taken, primary cells are extracted, a cell suspension is obtained by culture, and then MFAP5-positive stem cells are sorted out by flow cytometry analysis; during the flow cytometry analysis, two markers, CD34 and THY1, of the MFAP5-positive stem cells need to be analyzed simultaneously.

5. The method according to claim 4, characterized in that The culture is a suspension culture.

6. A method for preparing new cartilage, characterized in that: Obtained by differentiation of stem cells derived from synovial tissue and positive for MFAP5.

7. Use of MFAP5-positive stem cells for preparing a reagent for shortening the in vitro cartilage culture time and improving the consistency of cartilage differentiation, characterized in that: The MFAP5-positive stem cells are derived from synovial tissue.

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