MFAP5 positive synovial membrane lining layer stem cell for articular cartilage injury repair and identification method of MFAP5 positive synovial membrane lining layer stem cell

Abstract

The invention provides an MFAP5 positive synovial lining layer stem cell for articular cartilage injury repair and an identification method thereof, which are characterized in that a synovial sample in a cartilage defect animal model sample and a regenerated cartilage-like tissue in physical connection with the synovial sample are subjected to single-cell sequencing, and a bioinformatics analysis means is adopted to identify the MFAP5 positive synovial lining layer stem cell. According to the method, a cell subset, namely an MFAP5 positive subset, which plays a key role in cartilage-like tissue regeneration in synovial interstitial cells is found, an identification method of the cell subset is defined, and the cell subset can be used as a more effective source cell for promoting cartilage regeneration, so that the in-vitro cartilage differentiation time is expected to be greatly shortened, and the cartilage regeneration can be promoted. The method can be used for efficiently realizing in-vivo cartilage defect in-situ repair and has a wide clinical application prospect in cartilage repair and regeneration.

Description

Technical Field

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

[0002] Treatments to repair damaged or degenerated cartilage have been challenging, in part because of the complex phenotype of cartilage tissue and the conditions that natural cartilage must withstand. Microfracture was first introduced in 1959 and has been widely used since the 1990s. Microfracture generates fibrous repair tissue composed mainly of type I collagen, which has inferior mechanical properties and durability to natural hyaline cartilage tissue. Autologous chondrocyte implantation (ACI) was first used in 1994. The procedure involves two surgical procedures, first isolating cells from healthy tissue, then expanding them in vitro and reimplanting them into the same patient. This method was later developed into matrix-induced autologous chondrocyte implantation (MACI), which is to seed chondrocytes through a scaffold before implantation. The above two methods are the main methods currently used in clinical practice.

[0003] Currently approved cell-based tissue engineering cartilage products include Spherox (EU, Switzerland and UK); Bioseed-C and CaReS (part of European countries); Novocart 3D (Germany, Switzerland); MACI (USA); J-TecAutologous 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 suitable for OA (osteoarthritis) and RA (rheumatoid arthritis); except Cartistem, all of the above products use autologous chondrocytes; and there are no reports on approved products that prove that cartilage regeneration can completely and long-term restore joint function and relieve pain.

[0004] gMSC1 from TWOCELLS of Japan uses synovial mesenchymal stem cells (MSC) as seed cells to develop a three-dimensional scaffold-free tissue engineering construct (TEC), which is composed of synovial-derived MSC and cell-synthesized extracellular matrix. However, it needs to be cultured in a variety of different culture media for more than 5 generations to obtain a three-dimensional tissue with active contraction, which is called "gMSC1". However, in the selection of seed cells, this product did not pay attention to the stem cell subpopulation in the synovium that is directly related to chondrogenic differentiation, but only extracted cells from the synovium for culture under special technical conditions. The Phase III clinical trial of this product showed that there was no statistically significant improvement compared to microfractures. It can be seen that gMSC1, which is directly prepared by synovial tissue and cultured under multiple special technical conditions, is still very different from natural cartilage, and it is still difficult to repair articular cartilage loss and achieve ideal repair effects.

[0005] In summary, the current difficulties in articular cartilage regeneration and repair are: the 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 the existing cartilage tissue engineering technology, the repaired 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 source of seed cells for articular cartilage repair: the seed cells traditionally used for articular cartilage repair are mainly derived from bone marrow mesenchymal stem cells (BMSCs) and adipose mesenchymal stem cells (ADSCs), but these cells have low chondrogenic differentiation efficiency in vitro, are prone to calcification and excessive 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 subpopulations directly related to articular cartilage regeneration have not yet been clearly identified, especially the key stem / progenitor cell subpopulations with high chondrogenic differentiation potential; the lack of high-definition analysis of these cell subpopulations and systematic evaluation of cartilage regeneration and repair functions in vivo limits their application in the treatment of cartilage injuries.

[0006] Therefore, it is urgent to find new sources of seed cells with high chondrogenic differentiation potential, so as to develop more efficient articular cartilage damage repair products and methods, improve the efficiency and effectiveness of cartilage repair, provide efficient and safe new stem cell strategies for the treatment of articular cartilage damage, significantly improve the shortcomings 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 MFAP5-positive synovial lining layer stem cells for repairing articular cartilage damage and a method for identifying the same. By performing single-cell sequencing on synovial samples in cartilage defect animal model samples and regenerated cartilage-like tissue physically connected thereto, a cell subpopulation in synovial interstitial cells that plays a key role in the regeneration of cartilage-like tissue, namely, the MFAP5-positive subpopulation, is found through bioinformatics analysis. The identification method of the cell subpopulation is also clarified. The cell subpopulation can be used as a more effective source cell for promoting cartilage regeneration, greatly shortening the time of cartilage differentiation in vitro, and can be used to efficiently achieve in situ repair of cartilage defects in vivo, and has broad clinical application prospects in cartilage repair and regeneration.

[0008] In one aspect, the present invention provides a use of stem cells for preparing an agent for repairing articular cartilage damage or synthesizing new cartilage, wherein the stem cells are derived from synovial tissue and are positive for MFAP5.

[0009] The term “positive” as used in the present invention refers to the ability of the cell to express a protein, for example, “MFAP5 positive” refers to the ability of the cell to express MFAP5 protein.

[0010] The present invention proposes that there is a key mesenchymal stem cell subpopulation (MFAP5-positive cell subpopulation) with high chondrogenic differentiation potential in the synovial tissue, which can participate in articular cartilage repair and provide more accurate and efficient seed cell selection for cartilage regeneration and repair through local transplantation, 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 existing technology, the direct use of chondrocytes faces the problems of few cell sources, difficult separation, low proliferation potential, and easy hypertrophy. At present, bone marrow mesenchymal stem cells (BMSC) are mainly used for cartilage repair. Their cartilage differentiation ability depends on the structure of the material and scaffold, and they are prone to calcification and excessive hypertrophy. In addition, the efficiency of BMSC cartilage differentiation in vitro is low, and the culture time is long. It can only differentiate into mature chondrocytes after 21 days of co-culture with cartilage particles. Other sources of MSC include fat, umbilical cord, etc. Among them, adipose mesenchymal stem cells (ADSCs) are easier to obtain, but their cartilage differentiation potential is relatively lower.

[0012] In the previous animal cartilage defect model experimental study, the present invention found that obvious regenerative tissue appeared at the contact site between the defective cartilage and the synovial tissue, with obvious joint pulley-like structure. Therefore, the present invention believes that synovial mesenchymal cells are effective endogenous stem / progenitor cells that promote cartilage regeneration. The reason is that: on the one hand, synovial mesenchymal stem cells are more similar to chondrocytes in developmental relationship than other types of cells. In the early embryonic development, mesoderm mesenchymal stem cells (MSCs) will gather at the position where the joints will develop, and some of them will form the interarticular zone and express the Gdf5 gene in mice; with the development of the embryo, the MSCs around the interarticular zone further differentiate into fibroblasts and macrophages in the synovium; on the other hand, in adulthood, there are still proliferating cells with MSC phenotype in the synovium, and these stem cells can respond to damage, proliferate and differentiate to support cartilage repair.

[0013] In order to find the stem cell subpopulation with high cartilage differentiation potential in synovial tissue, the present invention performed single-cell RNA sequencing (scRNA-seq) on the synovial samples in the cartilage defect animal model samples and the regenerated cartilage-like tissue physically connected thereto, looking for similar cells that appear in both the synovial and neocartilage-like tissues, sorting them according to the degree of similarity, and determining 11 groups of cell clusters through integrated cluster analysis. Among them, a group of MFAP5-positive (specifically expressed) interstitial cell subpopulations from the sublining layer of regenerated cartilage-like and synovial tissues are closely related to typical interstitial cells and chondrocytes in their spatial position in the UMAP dimensionality reduction map. Subsequently, the present invention grouped the sequencing data of synovial tissue and neocartilage tissue separately, and found that the MFAP5 gene-positive cell group also showed strong specificity in a single tissue. The similarity comparison of the MFAP5 gene-positive cell groups of the two tissues also showed that the similarity of the interstitial cells of the MFAP5 gene-positive 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 cartilage differentiation and extracellular matrix synthesis. Pseudo-time analysis of newborn chondrocytes also showed that this cell group was at the front end of the chondrocyte differentiation trajectory, that is, upstream of the differentiation process. As pseudo-time passed, the expression of genes shifted from upstream development-related SFRP2, POSTN, etc. to downstream mature hyaline chondrocytes ACAN, COL2A1, etc.

[0014] The technology of the present invention is based on the results of single-cell high-throughput sequencing. Through bioinformatics analysis, it discovers and defines a key mesenchymal cell subpopulation with high chondrogenic differentiation potential (MFAP5 gene positive subpopulation), and determines that this cell subpopulation plays a key role in the regeneration of cartilage-like tissue, greatly shortening the in vitro chondrogenic differentiation time, and can be used for in situ repair of cartilage defects in vivo.

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

[0016] In another aspect, the present invention provides a use of stem cells for preparing an agent for repairing articular cartilage damage or synthesizing new cartilage, wherein the stem cells are derived from synovial tissue and are positive for CD34 and / or THY1.

[0017] The present invention determines the key surface markers of MFAP5-positive synovial lining layer stem cell subpopulations under different species and physiological conditions, namely CD34 and THY1, through single-cell data analysis and multi-database integration. These markers can be used for the identification, sorting and subsequent culture of the subpopulation cells, and clarifies the means for sorting and identifying the MFAP5-positive synovial stem cell subpopulations.

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

[0019] In another aspect, the present invention provides a method for identifying stem cells for repairing articular cartilage damage, by identifying whether the stem cell membrane protein is positive for CD34 and / or THY1, thereby determining whether it is the stem cell for repairing articular cartilage damage.

[0020] Studies have shown that CD34 and THY1 are membrane protein markers of MFAP5-positive synovial stem cells and can be used to identify MFAP5-positive synovial stem cells by flow cytometry.

[0021] Furthermore, by identifying whether the stem cell membrane proteins are positive for CD34 and THY1, it is determined whether they are stem cells for repairing articular cartilage damage.

[0022] Studies have shown that identifying CD34 or THY1 positivity alone is not conducive to the subsequent sorting of MFAP5-positive synovial stem cell subpopulations. Simultaneous identification of CD34 and THY1 positivity is required to accurately identify and sort the MFAP5-positive synovial stem cell subpopulation.

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

[0024] The accuracy of the identification of CD34 and THY1 was verified by analyzing whether the intracellular protein MFAP5 was positive.

[0025] Of course, it is also possible to directly identify whether the intracellular protein MFAP5 is positive by analyzing flow cytometry, but since the pre-treatment of intracellular protein analysis is cumbersome and will cause cell death, it is impossible to continue to sort out the MFAP5-positive synovial stem cell subpopulation. Therefore, it is necessary to identify the membrane proteins CD34 and THY1 to more directly and simply sort out the MFAP5-positive synovial stem cell subpopulation.

[0026] In another aspect, the present invention provides a method for sorting MFAP5-positive stem cells, wherein the method comprises taking a synovial sample, extracting primary cells, culturing to obtain a cell suspension, and then sorting out the MFAP5-positive stem cells through flow cytometric analysis.

[0027] Furthermore, the culture is suspension culture.

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

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

[0030] In another aspect, the present invention provides a method for preparing new cartilage by differentiating stem cells, wherein the stem cells are derived from synovial tissue and are positive for MFAP5.

[0031] In another aspect, the present invention provides a use of MFAP5-positive stem cells for preparing an agent for shortening the in vitro cartilage culture time and improving the consistency of cartilage differentiation, wherein the MFAP5-positive stem cells are derived from synovial tissue.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) Through single-cell sequencing technology, the MFAP5-positive stromal cell subpopulation was identified and applied for the first time, which promoted the in-depth study of cartilage regeneration cell subpopulations and provided new ideas and directions for subsequent cartilage repair research;

[0034] (2) The present invention has identified the key surface markers of MFAP5-positive subpopulation cells under different species and physiological conditions, namely CD34 and THY1, through the integration of multiple databases, and can be used for the sorting and subsequent culture of the subpopulation cells; through flow cytometry, combined with the MFAP5-positive subpopulation surface markers CD34 and THY1, the MFAP5-positive stromal cell subpopulation with high chondrogenic differentiation potential can be accurately identified and sorted from the synovial tissue, and the cell subpopulation can be efficiently separated and purified, providing a high-quality cell source for subsequent chondrogenic differentiation and regeneration; compared with simply extracting cells from the synovium, the cell sorting technology used in the present invention can ensure the consistency and efficiency of the obtained cell population in terms of chondrogenic differentiation potential;

[0035] (3) The MFAP5-positive stromal cells of the present invention can differentiate into chondrocytes in a relatively short period of time, significantly shortening the in vitro culture time, and improving the maturity of cartilage differentiation, thereby increasing 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 cartilage differentiation potential, and can be efficiently expanded and differentiated in vitro; they can also avoid the disadvantage that autologous chondrocyte transplantation requires two operations, reduce the patient's pain and surgical risks, and provide 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 cartilage differentiation, and are less prone to calcification and excessive hypertrophy. The chondrogenic differentiation potential is more efficient and specific, and is expected to achieve true regeneration of functional cartilage tissue.

[0038] Detailed description

[0039] 1. Cartilage 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 essential for the formation of cartilage in the embryonic period and the repair of cartilage damage in adult individuals. Chondrogenic differentiation is usually regulated by signals from the TGF-β family, including early cell aggregation, differentiation into chondrocytes, and production of extracellular matrix.

[0041] The cartilage differentiation in the present invention also includes the differentiation of the 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 and elastin) and polysaccharides (such as glycosaminoglycans), which provide physical support and biochemical signals to help maintain the function, shape and structure of cells. ECM not only supports the physical structure of tissues, but also regulates cell behavior, affecting cell proliferation, differentiation, migration and other processes. In cartilage tissue, the extracellular matrix is ​​composed of a large amount of collagen and proteoglycans, which give cartilage elasticity and pressure-bearing capacity.

[0044] 3. Hyaline Cartilage

[0045] Hyaline cartilage is a widely distributed type of cartilage with a smooth, translucent appearance. It is mainly found in joints, ribs, noses, trachea, etc. It is composed 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, which provide 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 tougher type of cartilage that contains a large amount of crude fibrous collagen. It usually appears in areas that need to withstand greater mechanical stress, such as intervertebral discs, articular discs, and bone joints. The collagen fibers of fibrocartilage are more densely arranged, with stronger tensile and compressive properties, and are a key tissue type for structural support and cushioning.

[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 to identify and classify different types of cells, especially in stem cell biology and immunology research. The expression of each cell surface marker is usually closely related to the function, differentiation state and development process of the cell.

[0050] In the present invention, the sorting of MFAP5-positive stromal cell subpopulations relies on specific cell surface markers, such as CD34, and is used to separate cell subpopulations 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 the potential for self-renewal and multidirectional differentiation, which can differentiate into a variety of 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, which have strong chondrogenic 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 subpopulation is used as a marker of synovial stromal cells, which has high chondrogenic differentiation potential and can promote cartilage repair.

[0058] 9. Collagen Type II (COL2A1)

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

[0060] 10. Aggrecan (ACAN)

[0061] Aggrecan is one of the main proteins produced by chondrocytes. It combines with 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 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] BMSCs are mesenchymal stem cells derived from bone marrow, which have multidirectional differentiation potential and can differentiate into various cell types such as cartilage, bone, fat, etc. BMSCs are often used in cartilage regeneration research, but their cartilage differentiation efficiency is relatively low.

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

[0067] ADSCs are mesenchymal stem cells extracted from adipose tissue, which have the potential for self-renewal and differentiation. ADSCs are increasingly used in cartilage regeneration, but their cartilage differentiation potential is slightly lower than that of BMSCs.

[0068] 14. Flow Cytometry

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

[0070] In the present invention, flow cytometry is used to sort out MFAP5-positive stromal cell subpopulations by 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 measure gene expression profiles in individual cells. It enables researchers to analyze heterogeneous cell populations in complex tissues and identify the gene expression signature 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 was used to analyze the cell types in synovial tissue and neocartilage-like tissue, and to identify the key cell subpopulation involved in cartilage regeneration - MFAP5-positive stromal cells.

[0074] 16. Pseudotime Analysis

[0075] Pseudo-time analysis is an analysis method for single-cell RNA sequencing data that can infer the continuous trajectory of cells from an undifferentiated state to a differentiated state. Through this technology, researchers can reconstruct the development process of cells and observe changes in gene expression of cells at different differentiation stages.

[0076] In the present invention, pseudo-time series analysis was used to study the differentiation trajectory of MFAP5-positive cells and to speculate how these cells gradually differentiated 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 use cells and scaffold materials to build functional tissues to replace or repair damaged tissues or organs.

[0079] In cartilage regeneration, tissue engineering technology combines 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 form a three-dimensional structure to support tissue regeneration by adsorbing or implanting cells.

[0080] 18. Osteoarthritis (OA)

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

[0082] 19. Cartilage Injury

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

[0084] 20. Osteochondritis Dissecans (OCD)

[0085] Osteochondrosis is a disease in which cartilage and bone tissue separate due to insufficient blood supply to the bone ends, which 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, consisting of the femur, tibia and patella, and the joint surface is covered with hyaline cartilage. Knee cartilage damage is the main feature of diseases such as osteoarthritis, and cartilage repair is crucial to the recovery of knee joint function.

[0088] 22. Cartilage Injury Model

[0089] Cartilage injury models are experimental models 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 These are photos of the new cartilage formation in the animal model in Example 1, wherein the left photo is an animal model with a defect, and the right photo is the new cartilage formation in the animal model;

[0091] Figure 2 The dimensionality reduction and Mfap5+ subpopulation distribution results of multi-tissue single-cell sequencing in Example 1;

[0092] Figure 3 The results of separate clustering analysis of sequencing data of synovial tissue and neonatal cartilage tissue in Example 1, including (ad) UMAP dimensionality reduction and Mfap5 gene expression of single-cell sequencing of synovial tissue and neonatal cartilage, S1 and E2 are Mfap5+ subgroups; (e) Pseudo-time series analysis in neonatal cartilage tissue shows that the Mfap5+ subgroup differentiates toward cartilage; (fg) The GO pathways of S1 and E2 subgroups show similarity, with cartilage development and extracellular matrix synthesis entries, where the horizontal axis (bar length) represents the gene ratio, and the number marked on the right is the enrichment P value; (h) Comparison of subgroup similarity between tissues, S1 and E2 subgroups show strong similarity.

[0093] Figure 4The results of cross-species integration analysis of the MFAP5-positive cell population in Example 1, where (ab) are the single-cell sequencing results of human OA and RA samples, and it was found that there were also MFAP5+ subpopulations with similar expression patterns, namely, OA-1 subpopulation and RA-4 subpopulation; (cd) are the results of cross-species integration of human and rat synovial data, and after integration, the overlap between species in group 1 is high; (e) is the expression of MFAP5 gene in the cell population after integration, and group 1 shows specific expression of MFAP5, and 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 the human synovial samples in Example 2 and the staining results of different culture methods; (a) H&E staining of human synovium, LL is Lining Layer, SL is Sublining Layer; (b) Immunofluorescence staining of MFAP5+ subpopulation in human synovial samples (Alexa Fluor 488 / 546 labeling); (c) Sox9 / Col2 immunofluorescence staining of synovial mixed cells induced chondrocytes, confirming that some synovial cells have the ability to differentiate into chondrocytes under induced conditions; (d) Fluorescence staining of adherent cultured cells; (e) Under suspension culture conditions, MFAP5+ cells exist and most of them co-express their surface markers;

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

[0097] Figure 8 The staining and fluorescence images of the rat MFAP5 positive cell population inducing chondrocytes in Example 3;

[0098] Fig. 9 These are the staining and fluorescence images of rat BMSCs cells inducing chondrocytes in Example 3. DETAILED DESCRIPTION

[0099] The present invention is further described in detail below in conjunction with the examples. It should be noted that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on them. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer, and the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

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

[0101] In this example, single-cell RNA sequencing (scRNA-seq) technology combined with bioinformatics analysis was used to conduct an in-depth study of the synovium and regenerated cartilage-like tissue, and a key cell subpopulation with high chondrogenic differentiation potential was identified.

[0102] 1. Obvious new cartilage phenomenon appears in the animal cartilage defect model

[0103] In the previous animal cartilage defect model experiment, about four weeks after the defect modeling, obvious regeneration tissue appeared on the side of the accidental joint, especially at the attachment site of the patellar ligament, with obvious joint pulley-like structure. The SO (safranin-fast green) staining of the tissue detected that it was bright red transparent cartilage tissue, and a large number of cartilage pits could be observed in the tissue cells of the cartilage layer area, such as Figure 1 As shown in the figure, histological staining results show that the new cartilage has formed a complete osteochondral structure, and the surface cartilage is hyaline cartilage. This phenomenon is different from the more common synovial tumor tissue in clinical practice. Hemosynovial tumor has a large amount of blood vessels, fat, and fibrous tissue, while the new tissue is all hyaline cartilage components, so it is considered that there is a new cartilage phenomenon.

[0104] 2. Complete single-cell sequencing analysis of regenerated cartilage and synovial tissue to identify chondrogenesis-related mesenchymal cell subpopulations

[0105] Therefore, in this example, cells from two tissues, synovium and new cartilage, 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 from the animal model. First, wash the sample, then enzymatically hydrolyze the synovial tissue to obtain the primary cell suspension.

[0107] Preparation of synovial tissue cell suspension: Add synovial tissue samples to culture medium containing digestive enzymes and incubate at 37°C for 1 hour. Afterwards, terminate digestion and centrifuge at 1200 rps to obtain cell pellets, which are resuspended in complete culture medium to obtain synovial tissue cell suspension for single-cell sequencing.

[0108] Preparation of neocartilage tissue cell suspension: neocartilage tissue samples were added to a culture medium containing digestive enzymes and incubated at 37°C for 1 hour. Afterwards, digestion was terminated and centrifuged at 1200 rps to obtain cell pellets, which were resuspended in complete culture medium to obtain neocartilage tissue cell suspension for single-cell sequencing.

[0109] 2. Single-cell RNA sequencing

[0110] Cell capture and library construction: 10x Genomics platform was used for cell capture and cDNA library construction. After the sample was processed with single cell suspension, the single cell was encapsulated in oil droplets and efficiently captured by microfluidics. After that, cDNA was synthesized by reverse transcription reaction and sequencing library was generated by PCR amplification.

[0111] 3. Sequencing and data processing

[0112] The sequencing platform was Illumina NovaSeq. After the sequencing data was quality controlled, data analysis was performed using software such as Cell Ranger to ultimately obtain a gene expression matrix.

[0113] 4. Data analysis and cell subpopulation identification

[0114] Cluster analysis: Seurat and other tools were used to perform cluster analysis on the sequencing data, integrating the single-cell data of synovium and regenerated cartilage-like tissue, and identifying 11 cell clusters (Clusters), see Figure 2 In the figure, a is a group of cells that are similar in the synovial and neocartilaginous tissues. One of the groups of Mfap5-positive interstitial cells from the sublining layer of the regenerated cartilaginous and synovial tissues is closely related to typical interstitial cells and chondrocytes in the spatial position of the UMAP dimensionality reduction map. In the Umap integration, it was found that MFAP5 is a marker specifically expressed in the adjacent area of ​​the synovial tissue and the neocartilaginous tissue ( Figure 2 c3 in c), showing significant aggregation, indicating that it plays an important role in synovial membrane and regenerated cartilage-like tissue.

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

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

[0117] GO pathway analysis showed that the Mfap5 gene-positive mesenchymal cell population specifically expressed biological pathways related to cartilage differentiation and extracellular matrix synthesis ( Figure 3 f in Figure 3 g in), where ( Figure 3 a~ Figure 3 d) UMAP dimensionality reduction and Mfap5 gene expression of single-cell sequencing of synovial tissue and new cartilage, found that S1 and E2 are Mfap5+ subpopulations; ( Figure 3 e) Pseudo-time series analysis in the newly formed cartilage tissue shows that the Mfap5+ subpopulation differentiates towards cartilage; ( Figure 3 f~ Figure 3 g) The GO pathways of S1 and E2 subgroups show similarity, with entries for cartilage development and extracellular matrix synthesis; ( Figure 3 h) Comparison of similarities between tissue subgroups, S1 and E2 subgroups show strong similarities.

[0118] The pseudo-time analysis of newborn 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 the pseudo-time passed, the expression of genes shifted from upstream development-related SFRP2, POSTN, etc. to downstream mature hyaline chondrocytes ACAN, COL2A1, etc. ( Figure 3 e in the figure). It can be determined that the Mfap5 gene-positive mesenchymal cells are in the precursor stage of cartilage differentiation, have cartilage differentiation potential, and contribute to the formation of new cartilage.

[0119] The above analysis confirmed that the functions of MFAP5-positive cell populations were mainly related to extracellular matrix synthesis and cartilage differentiation.

[0120] 3. Cross-species integrated analysis preliminarily confirmed the strong conservation of this subgroup

[0121] In the clinical pathological process of osteoarthritis, there is no new occurrence of trochlear cartilage, but more fibrotic pathological synovial tumors. This example uses published literature data to analyze single-cell RNA sequencing data from the synovium of clinical samples in healthy and arthritic states. The results are as follows: Figure 4 As shown, Figure 4 a~ Figure 4 As can be seen in b, MFAP5+ subpopulations also exist in single-cell sequencing of human OA and RA samples, and their expression patterns are similar, namely OA-1 subpopulation and RA-4 subpopulation, confirming that MFAP5+ interstitial cells also exist in human synovium, and their gene expression patterns are similar ( Figure 4 a in the figure), proving the conservation of this subgroup in different species and pathological environments. Cross-species data integration analysis also found that MFAP5-positive subgroups in different species overlap in UMAP dimensionality reduction ( Figure 4 b) in the above.

[0122] Figure 4 c~ Figure 4 In the figure, human and rat synovial data were integrated across species, and it was found that the overlap between species in group 1 after integration was high; Figure 4 The expression of MFAP5 gene in the cell groups after integration in e. Group 1 showed specific expression of MFAP5.

[0123] After verification in human synovial tissue, analysis of single-cell RNA sequencing data of human synovial samples revealed the existence of MFAP5-positive cell subpopulations, and their gene expression patterns were highly similar to those of MFAP5-positive cell populations in animal models, indicating that this cell subpopulation is conserved among different species. This lays the foundation for the clinical application of MFAP5-positive cell populations.

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

[0125] This example completes the histological verification of the specific markers of the MFAP5-positive cell subpopulation, and determines the culture and flow cytometry analysis methods of the cells.

[0126] Technical process such as Figure 5 As shown: Knee synovial samples were taken from patients (or healthy volunteers), primary cells were extracted by type I collagenase, and flow cytometry was performed with the help of special cell surface markers to obtain MFAP5 positive subpopulations. This cell subpopulation was then used as seed cells with high chondrogenic potential for subsequent applications and cell culture.

[0127] In this example, immunofluorescence staining of MFAP5 protein and subpopulation surface markers was performed in human synovial samples ( Figure 6 ), where the results of H&E staining of human synovium are shown in Figure 6 a in the figure is used to observe the basic tissue structure of the synovium, including LL and SL, LL stands for Lining Layer, and SL stands for Sublining Layer; the results of immunofluorescence staining of MFAP5+ subpopulation in human synovial samples (labeled with Alexa Fluor 488 / 546) are shown in Figure 6 b in the figure verifies the existence of subpopulations and the feasibility of sorting them, and also provides a certain feasibility for the analysis and sorting of subpopulations.

[0128] In this example, primary cells (synovial mixed cells) were directly used to induce chondrocytes (the induction method was chondrocyte induction medium, and the cell suspension was centrifuged into spheroid culture medium. The composition of the medium was: DMEM high glucose medium + 1x Sodiumpyruvate + 1% ITS + 10^(-7)M dexamethasone + 50μg / mL Vc + 10ng / mL TGF-β3 + 1x P / S), and Sox9 / Col2 immunofluorescence staining was performed. The results are shown in Figure 6 (c) confirmed that some synoviocytes have the ability to differentiate into chondrocytes under induced conditions.

[0129] In order to culture and sort the corresponding synovial cell subpopulations, this example tried various methods such as cell adherent culture and suspension culture, and determined the culture scheme ( Figure 6 In d, Figure 6 e in the above figure).

[0130] The specific process of cell suspension culture is as follows:

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

[0132] 2) Cell culture: 6 mL of cell suspension was inoculated into a low-adsorption 6-well plate, and microspheres (mesoporous mineralized silk fibroin methacrylamide microspheres, patent 2024111118570 applied for) were added. The cells were cultured in a cell culture incubator (37°C, 5% CO2), the medium was changed every other day, and the cells were expanded for 2 weeks.

[0133] Fluorescence staining was performed on adherent culture cells and suspension culture cells, and the results are shown in Figure 6 In d, Figure 6 e in which Figure 6 The d in the figure is the result of fluorescence staining of adherent cultured cells. Figure 6 In the figure e, MFAP5+ cells exist under suspension culture conditions and most of them co-express their surface markers. It can be seen that compared with adherent culture, suspension culture can obtain more MFAP5+ cells with higher fluorescence intensity. Therefore, it is preferred to use suspension culture to sort and obtain MFAP5-positive cell populations.

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

[0135] Flow cytometry sorting process:

[0136] 1) Take 1-2 x 10 6 Single cells obtained from suspension culture were resuspended in flow cytometry staining buffer. Two fluorescent conjugated antibodies (anti-CD34, THY1, PE and APC were used for conjugation of fluorescence) were added to the cell suspension at the recommended dilution ratio (1:50-1:200). After mixing, incubate at 4℃ in the dark for 30 minutes. Wash the cells with FACS buffer, centrifuge at 300g for 5 minutes, discard the supernatant, and resuspend in FACS buffer.

[0137] 2) Add an appropriate amount of dead cell dye, such as Fixable Viability Dye, according to the product instructions, and wash once after incubation. Dead cell dye is used to exclude dead cell signals in flow cytometry. Resuspend the cells in an appropriate amount of FACS buffer and prepare for flow cytometry.

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

[0139] 4) Exclude cell debris and impurities based on forward scatter (FSC) and side scatter (SSC) graphs. Exclude dead cells based on the signal of dead cell dye. Select CD34⁺THY1 on the flow cytometer + The expression of cell markers is determined by the fluorescence signal of the antibody.

[0140] Since MFAP5 is an intracellular protein, while CD34 and THY1 are membrane proteins, direct use of MFAP5 for flow sorting requires breaking the membrane and destroying the cell structure, which affects the subsequent cell sorting application; and although CD34 can specifically mark the MFAP5 subpopulation in fibroblasts, CD34 is also a marker for vascular endothelial cells. Using only CD34 as a marker for flow cytometry analysis cannot accurately screen the MFAP5-positive cell population, and vascular endothelial cells may be introduced. THY1 is a marker for synovial sublining fibroblasts, and using only THY1 cannot accurately screen the MFAP5-positive cell population. When THY1 and CD34 are used as markers at the same time, the MFAP5-positive cell population can be sorted more accurately.

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

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

[0143] After the MFAP5-positive cell population was sorted out, this example further performed in vitro expansion and functional verification on it.

[0144] The rat MFAP5 positive cell group obtained by screening in Example 2 was collected into a centrifuge tube, the supernatant was discarded after centrifugation, 3 mL of cartilage 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) was added, and cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium was replaced every other day, and the induced culture was carried out for 3 weeks to successfully form cartilage particles (see Figure 7 for staining and fluorescence images). In addition, this example also compares the cartilage particle induction effect of 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 Figures 7 and 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 that they have stronger chondrogenic ability and better differentiation consistency. Fluorescence staining detected the expression levels of SOX9 and type II collagen, among which SOX9 is an important transcription factor related to cartilage matrix formation, 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 performed poorly in chondrogenesis, with only a small number of cells showing fluorescent staining. In addition, synovial mesenchymal stem cells can differentiate into chondrogenic cells in a shorter time, significantly shortening the in vitro culture time and improving the maturity and consistency of cartilage 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 shall 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 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 are derived from synovial tissue and are positive for CD34 and / or THY1.

3. A product for repairing articular cartilage damage or preparing new cartilage, characterized in that: Included are stem cells that are derived from synovial tissue and are positive for MFAP5.

4. A method for identifying stem cells for repairing articular cartilage damage, characterized in that: By identifying whether the stem cell membrane protein is positive for CD34 and / or THY1, it can be determined whether it is the stem cell for repairing articular cartilage damage.

5. The identification method according to claim 4, characterized in that: By identifying whether the stem cell membrane proteins are positive for CD34 and THY1, it can be determined whether they are stem cells for repairing articular cartilage damage.

6. A method for sorting MFAP5-positive stem cells, characterized in that: Synovial samples were taken, primary cells were extracted, cell suspension was obtained by culture, and MFAP5-positive stem cells were sorted out by flow cytometry analysis.

7. The method according to claim 6, characterized in that The culture is a suspension culture.

8. The method according to claim 6, characterized in that During the flow cytometric analysis, two markers, CD34 and THY1, of MFAP5-positive stem cells need to be analyzed simultaneously.

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

10. 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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