CN119235920A

CN119235920A - MFAP5 positive synovial lining stem cells for repairing articular cartilage injury and identification method thereof

Info

Publication number: CN119235920A
Application number: CN202411783882.3A
Authority: CN
Inventors: 欧阳宏伟; 张显著; 黄博玮; 张家楷; 王飞
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2024-12-06
Filing date: 2024-12-06
Publication date: 2025-01-03

Abstract

The invention provides MFAP5 positive synovial lining stem cells for repairing articular cartilage injury and an identification method thereof, wherein single-cell sequencing is carried out on synovial samples in cartilage defect animal model samples and regenerated cartilage-like tissues which are physically connected with the synovial samples, a cell subgroup-MFAP 5 positive subgroup which plays a key role in cartilage-like tissue regeneration is found in synovial interstitial cells through a biological information analysis means, and an identification method of the cell subgroup is clarified, and the cell subgroup can be used as more effective cartilage regeneration-promoting source cells, is hopeful to greatly shorten in-vitro cartilage differentiation time, can be used for efficiently realizing in-situ repair of in-vivo cartilage defects, and has wide clinical application prospect in cartilage repair regeneration.

Description

MFAP5 positive synovial lining stem cells for repairing articular cartilage injury and identification method thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to MFAP5 positive synovial lining stem cells for repairing articular cartilage damage and an identification method thereof.

Background

Methods of treatment to repair damaged or degenerated cartilage have been challenging, in part, due to the complex phenotype of cartilage tissue and conditions that natural cartilage must withstand. Microfracture was first introduced in 1959 and this method was widely used since the 90 s of the 20 th century by drilling into subchondral bone to release bone marrow and induce cartilage repair. However, the fibrous repair tissue produced by microfracture surgery is composed mainly of type I collagen, and has inferior mechanical properties and durability to natural hyaline cartilage tissue. Autologous Chondrocyte Implantation (ACI) was used for the first time in 1994, and this procedure involved two surgical procedures, first to isolate cells from healthy tissue, then to expand in vitro and then to re-implant the same patient. The method has later evolved into matrix-induced autologous chondrocyte implantation (MACI), i.e., implantation into the body after seeding chondrocytes through a scaffold prior to implantation. The two methods are the main methods used clinically at present.

Cell-based tissue engineering cartilage products currently approved are mainly Spherox (European Union, switzerland and England), bioseed-C and CaReS (part of European countries), novocart D (Germany, switzerland), MACI (U.S.), J-Tec Autologous Cultured Cartilage (JACC, japan), chondron, cartiLife and CARTISTEM (Korea), and OrthoACI (Australia). Most of the above products are only approved for focal cartilage defects in knee joints, are not suitable for OA (osteoarthritis) and RA (rheumatoid arthritis), all of the above products use autologous chondrocytes except CARTISTEM, and no report on approved products demonstrates that the function of joints can be completely restored and pain can be reduced for a long period of time by cartilage regeneration.

GMSC1 of Japanese TWOCELLS, inc., developed a three-dimensional scaffolds-free Tissue Engineering Construct (TEC) consisting of synovially derived MSCs and extracellular matrix synthesized by cells using synovially derived MSCs (MSCs) as seed cells. However, it requires more than 5 generations of culture in a variety of different media to obtain three-dimensional tissue with active shrinkage, called "gMSC" 1. However, the product does not pay attention to stem cell subpopulations in synovium, which are directly related to chondrogenic differentiation, on the selection of seed cells, but only the cells in the synovium are extracted for culture under special technical conditions. Phase III clinical trials of this product showed no statistically significant improvement over microfracture. It can be seen that gMSC a prepared by directly adopting synovial tissue and culturing for many times under special technical conditions has larger difference from natural cartilage, and the repair of the loss of articular cartilage is still difficult to realize, and the ideal repair effect cannot be obtained.

In summary, the existing method for treating Osteoarthritis (OA), such as conservative treatment and surgical treatment, cannot realize self-regeneration of natural hyaline cartilage, so that the long-term repair effect is not ideal. Although some progress has been made in the existing cartilage tissue engineering technology, the fibrocartilage tissue formed by repair still has the problems of low mechanical strength, poor integration effect with the original cartilage and the like. Meanwhile, there are limitations in the sources of seed cells for articular cartilage repair, in that seed cells for articular cartilage repair have been conventionally mainly derived from bone marrow mesenchymal stem cells (BMSCs) and adipose mesenchymal stem cells (ADSCs), however, these cells have low in-vitro cartilage differentiation efficiency, are liable to undergo calcification and excessive hypertrophy, have long culture time, and their cartilage differentiation ability depends on specific materials and complex medium components. It can be seen that there is currently no clear direct correlation of articular cartilage regeneration with endogenous stem/progenitor cell subsets, in particular key stem/progenitor cell subsets with high chondrogenic differentiation potential, and that the lack of high definition resolution of these cell subsets and systematic assessment of cartilage regeneration repair function in vivo has limited their application in cartilage injury treatment.

Therefore, it is urgently needed to find a new seed cell source with high cartilage differentiation potential, so as to develop more efficient articular cartilage injury repair products and methods, improve the efficiency and effect of cartilage repair, provide a novel stem cell strategy with high efficiency and safety for articular cartilage injury treatment, remarkably improve the defects of the existing treatment methods, and promote the development of cartilage regeneration medicine.

Disclosure of Invention

In order to solve the problems, the invention provides MFAP5 positive synovial lining stem cells for repairing articular cartilage injury and an identification method thereof, wherein single-cell sequencing is carried out on synovial samples in cartilage defect animal model samples and regenerated cartilage-like tissues which are physically connected with the synovial samples, a cell subgroup which plays a key role in cartilage-like tissue regeneration, namely MFAP5 positive subgroup, is found out from synovial interstitial cells through a bioinformatics analysis means, and the identification method of the cell subgroup is clarified.

In one aspect, the invention provides the use of stem cells derived from synovial tissue and positive for MFAP5 for the preparation of an agent for repair of articular cartilage damage or synthesis of neocartilage.

"Positive" as used herein means that the cell is capable of expressing a protein, such as "positive for MFAP 5", and means that the cell is capable of expressing MFAP5 protein.

The invention provides that a key mesenchymal stem cell subgroup (MFAP 5 positive cell subgroup) with high cartilage differentiation potential exists in synovial tissue, can participate in articular cartilage repair, and can provide more accurate and efficient seed cell selection for cartilage regeneration repair through local transplantation so as to solve the problem of lower cartilage repair efficiency in the prior art and provide a new regeneration strategy for articular cartilage repair.

In the prior art, the direct use of chondrocytes has the problems of less cell sources, difficult separation, low proliferation potential, easy generation of hypertrophy and the like. At present, bone marrow mesenchymal stem cells (BMSCs) are mainly applied to cartilage repair, and the cartilage differentiation capacity of the BMSCs depends on the structures of materials and scaffolds and tends to be calcified, excessively hypertrophic and the like. In addition, BMSCs have low in-vitro cartilage differentiation efficiency and long culture time, and can be differentiated into mature chondrocytes after 21 days of co-culture with cartilage particles. Other sources of MSCs are fat, umbilical cord, etc., where adipose mesenchymal stem cells (ADSCs) are more readily available, but their cartilage differentiation potential is relatively lower.

In the experimental study of the early animal cartilage defect model, the invention discovers that obvious regeneration tissues appear at the contact part of the defect cartilage and synovial tissue, and has obvious joint pulley-like structure. The present invention therefore considers that synovial mesenchymal cells are potent endogenous stem/progenitor cells promoting cartilage regeneration, in that, on the one hand, synovial mesenchymal stem cells are more similar in developmental relationship to chondrocytes than other types of cells, and in early embryonic development, mesodermal Mesenchymal Stem Cells (MSCs) aggregate at the site where they will develop into joints, with a portion of MSCs forming the internode region, and the Gdf5 gene being expressed in mice, and that, as the embryo develops, MSCs around the internode zone further differentiate into fibroblasts and macrophages in the synovium, and, on the other hand, after adulthood, there are still proliferative cells in the synovium with the phenotype of MSCs that can proliferate and differentiate in response to injury to support cartilage repair.

In order to find stem cell subsets with high cartilage differentiation potential in synovial tissue, the invention carries out single-cell RNA sequencing (scRNA-seq) on the synovial sample in a cartilage defect animal model sample and regenerated cartilage-like tissue which is physically connected with the synovial sample, finds similar cells which are simultaneously present in the synovial tissue and the regenerated cartilage-like tissue, sequences according to the similarity, and determines 11 cell clusters (Clusters) through integrated cluster analysis. Wherein a population of MFAP5 positive (specifically expressed) subpopulations of mesenchymal cells from regenerating cartilage-like and synovial tissue sub-lining layers (Sublining Layer) are closely related to typical mesenchymal and chondrocytes at the spatial location in the UMAP dimensionality map. Then, the sequencing data of synovial tissue and nascent cartilage tissue are separately clustered, and the MFAP5 gene positive cell population is found to have stronger specificity in a single tissue, and the similarity comparison of the MFAP5 gene positive cell population of two tissues also shows that the similarity of the MFAP5 gene positive cell population is obviously higher than that of other cell populations between two tissue sources. GO pathway analysis showed that this population specifically expressed biological pathways associated with cartilage differentiation and extracellular matrix synthesis. In contrast, the pseudo-timing analysis of the nascent chondrocytes also shows that the cell population is at the front end of the chondrocyte differentiation locus, that is, upstream of the differentiation process, and that the expression genes are changed from upstream development-related SFRP2, POSTN, etc. to downstream mature hyaline chondrocyte ACAN, COL2A1, etc. over the pseudo-timing.

The technology of the invention is based on single-cell high-throughput sequencing results, discovers and defines a key mesenchymal cell subset (MFAP 5 gene positive subset) with high cartilage differentiation potential through a bioinformatics analysis means, determines that the key mesenchymal cell subset plays a key role in regenerating cartilage-like tissues, greatly shortens in-vitro cartilage differentiation time, and can be used for in-situ repair of in-vivo cartilage defects.

Studies have shown that there is a heterogeneous MFAP 5-positive cell subpopulation in synovial stromal cells (e.g., Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes. Nature. 2023 Nov;623(7987):616- 624.）, but it only discloses the presence of MFAP 5-positive cell subpopulations in synovial tissue by transcriptome sequencing, and MFAP 5-positive cell subpopulations were not found to have the function of differentiating into cartilage. The present invention first found that MFAP 5-positive cell subpopulations play a key role in cartilage-like tissue regeneration, evaluating the potential and application prospects of MFAP 5-positive synovial stem cells in chondrogenic differentiation.

In another aspect, the invention provides the use of stem cells derived from synovial tissue and positive for CD34 and/or THY1 for the preparation of an agent for repair of articular cartilage damage or synthesis of neocartilage.

The invention determines key surface markers of the MFAP5 positive synovial lining stem cell subgroup under different species and physiological conditions, namely CD34 and THY1, through single cell data analysis and multi-database integration, and can be used for the identification, separation and subsequent culture of the subgroup cells, thereby defining separation and identification means of the MFAP5 positive synovial stem cell subgroup.

In yet another aspect, the invention provides a product for repair of articular cartilage damage or preparation of neocartilage comprising stem cells derived from synovial tissue and positive for MFAP 5.

In yet another aspect, the invention provides a method of identifying stem cells for repair of articular cartilage damage by identifying whether stem cell membrane proteins are positive for CD34 and/or THY1, thereby determining whether the stem cells are such stem cells for repair of articular cartilage damage.

The research proves that CD34 and THY1 are membrane protein markers of MFAP5 positive synovial stem cells, and can be used for identifying the MFAP5 positive synovial stem cells by using flow cells.

Further, it was determined whether the stem cell for repair of articular cartilage damage was the stem cell by identifying whether the stem cell membrane protein was positive for CD34 and THY 1.

The study proves that the identification of CD34 or THY1 is positive alone, which is unfavorable for the separation of the subsequent MFAP5 positive synovial stem cell subgroup, and the identification and separation of the MFAP5 positive synovial stem cell subgroup can be accurately achieved only by simultaneously identifying CD34 and THY1 to be positive.

In some embodiments, the stem cells for repair of articular cartilage damage are validated by flow cytometry for the presence of intracellular protein MFAP5 positive.

The intracellular protein MFAP5 was analyzed for positivity, thus verifying the accuracy of identifying CD34 and THY 1.

Of course, the identification can also be directly performed by analyzing the flow cell to identify whether the intracellular protein MFAP5 is positive, but because the pretreatment of the intracellular protein analysis is complicated, the cell death is caused, and the MFAP5 positive synovial stem cell subgroup cannot be continuously separated, the MFAP5 positive synovial stem cell subgroup needs to be more directly and simply separated by identifying the CD34 and THY1 through the membrane protein.

In yet another aspect, the invention provides a method of sorting MFAP5 positive stem cells by taking a synovial sample, extracting primary cells, culturing to obtain a cell suspension, and sorting MFAP5 positive stem cells by flow cytometry.

Further, the culture is a suspension culture.

In theory, any culture method, such as adherent culture or suspension culture, can be used to prepare and analyze MFAP 5-positive stem cells, provided that the extracted primary cells can be cultured. The invention proves that the suspension culture is more favorable for separating MFAP5 positive stem cells.

Further, in the flow cytometry analysis, two surface markers, namely THY1 and CD34, of the MFAP5 positive stem cells need to be analyzed simultaneously.

In yet another aspect, the invention provides a method of preparing neocartilage obtained by differentiation of stem cells derived from synovial tissue and positive for MFAP 5.

In yet another aspect, the invention provides the use of MFAP 5-positive stem cells derived from synovial tissue for the preparation of an agent that shortens the time to culture cartilage in vitro and improves consistency of cartilage differentiation.

Compared with the prior art, the invention has the beneficial effects that:

(1) Through single cell sequencing technology, MFAP5 positive matrix cell subsets are identified and applied for the first time, so that deep research on cartilage regeneration cell subsets is promoted, and a new thought and direction are provided for subsequent cartilage repair research;

(2) The invention determines key surface markers of MFAP5 positive subgroup cells under different species and physiological conditions, namely CD34 and THY1, and can be used for sorting and subsequent culture of the subgroup cells, and by combining with the surface markers of the MFAP5 positive subgroup CD34 and THY1, the MFAP5 positive matrix cell subgroup with high cartilage differentiation potential is precisely identified and sorted from synovial tissues through flow cytometry, so that the efficient separation and purification of the cell subgroup can be realized, and a high-quality cell source is provided for subsequent cartilage differentiation and regeneration;

(3) The MFAP5 positive matrix cells can be differentiated into cartilage-like cells in a short time, so that the in-vitro culture time is obviously shortened, the maturity of cartilage differentiation is improved, and the efficiency of cartilage regeneration and the feasibility of clinical application are improved;

(4) The MFAP5 positive stromal cells do not need to depend on autologous chondrocytes, have high-efficiency cartilage differentiation potential, can be subjected to in vitro high-efficiency amplification and differentiation, can avoid the defect that autologous chondrocyte transplantation requires two operations, reduce pain and operation risks of patients, and provide a safer and long-term effective solution for cartilage repair;

(5) Compared with traditional BMSC and ADSC, the MFAP5 positive matrix cells have closer differentiation relation with articular cartilage, have higher efficient differentiation capacity in the cartilage differentiation process, are not easy to cause calcification and excessive hypertrophy, have higher and specific chondrogenic differentiation potential, and are expected to realize the real regeneration of functional cartilage tissues.

Detailed Description

1. Cartilage differentiation

Cartilage 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 critical for the formation of embryonic cartilage and repair of cartilage damage in adult individuals. Cartilage differentiation is usually regulated by signals of the TGF- β family, and involves the steps of early cell aggregation, differentiation into chondrocytes, production of extracellular matrix, etc.

Cartilage differentiation in the present invention also includes differentiation of MFAP 5-positive stromal cells provided by the present invention into chondrocytes.

2. Extracellular matrix (Extracellular Matrix, ECM)

The extracellular matrix is a complex network structure in tissues, mainly composed of proteins (e.g., collagen, elastin) and polysaccharides (e.g., glycosaminoglycans), which provide physical support and biochemical signals that help maintain the function, shape and structure of the cells. ECM not only supports the physical structure of tissue, but also regulates the behavior of cells, affecting the processes of proliferation, differentiation, migration, etc. of cells. In cartilage tissue, the extracellular matrix is composed of a large amount of collagen and proteoglycans, giving cartilage elasticity and bearing capacity.

3. Hyaline cartilage (HYALINE CARTILAGE)

Hyaline cartilage is a widely distributed cartilage type with a smooth, translucent appearance, and is mainly found 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 proteoglycan, and provides pressure resistance and certain elasticity, and the main functions are to reduce inter-bone friction, support and buffer pressure.

4. Fibrocartilage (Fibrocartilage)

Fibrocartilage is a relatively tough cartilage type containing large amounts of crude fibrous collagen, often found in locations where greater mechanical stress is required, such as intervertebral discs, articular discs and skeletal joints. The collagen fibers of the fibrocartilage are arranged more tightly, have stronger tensile and compressive properties, and are key tissue types for structural support and buffering.

5. Cell surface marker (Cell Surface Markers)

Refers to a specific protein or glycoprotein molecule, typically located on the surface of a cell membrane, that can be recognized and isolated by binding to a specific antibody. Cell surface markers are widely used in biomedical research for identifying and classifying different types of cells, particularly in stem cell biology and immunology research. The expression of each cell surface marker is generally closely related to the function, differentiation state and developmental process of the cell.

In the present invention, the sorting of MFAP 5-positive stromal cell subpopulations relies on specific cell surface markers, such as CD34, for isolating cell subpopulations with high cartilage differentiation potential by flow cytometry.

6. Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCS)

Mesenchymal stem cells are a class of adult stem cells with self-renewal and multipotent differentiation potential, capable of differentiating into various cell types such as bone, cartilage, fat, etc. Common sources include bone marrow, adipose tissue, synovium, and the like.

7. Synovial mesenchymal stem cells (Synovial MESENCHYMAL STEM CELLS, SMSCS)

The synovial mesenchymal stem cells are derived from joint synovial mesenchymal stem cells and have stronger cartilage differentiation potential. sMSCs showed higher efficiency in cartilage regeneration compared to MSCs from other sources.

The MFAP5 positive stromal cells provided by the invention also belong to synovial mesenchymal stem cells.

8、MFAP5 (Microfibril-Associated Glycoprotein 5)

MFAP5 is a glycoprotein associated with extracellular matrix microfibrils. In the present invention, MFAP5 positive cell subsets are used as markers for synovial stromal cells, have high cartilage differentiation potential, and are capable of promoting cartilage repair.

9. Collagen II (Collagen Type II, COL2A 1)

COL2A1 is collagen specifically expressed in cartilage tissues, and constitutes the main component of cartilage matrix. The expression of COL2A1 is an important marker event in the cartilage differentiation process, and is used to evaluate the differentiation success rate of chondrocytes.

10. Glycan protein (Aggrecan, ACAN)

Aggrecan is one of the major proteins of chondrocyte production, which binds to hyaluronic acid to form a large molecular complex in the cartilage matrix responsible for maintaining the elasticity and structure of cartilage.

11. Fibroblast (Fibroblasts)

Fibroblasts are the major cell types in connective tissue and are involved in the production of extracellular matrix. In synovial tissue, fibroblasts have a developmental relationship with chondrocytes, and may play a role in cartilage repair.

12. Bone Marrow mesenchymal stem cells (Bone Marrow MESENCHYMAL STEM CELLS, BMSC)

BMSCs are mesenchymal stem cells derived from bone marrow, have multidirectional differentiation potential, and can differentiate into various cell types such as cartilage, bone, fat and the like. BMSCs are commonly used in cartilage regeneration studies, but their cartilage differentiation efficiency is relatively low.

13. Adipose-derived mesenchymal stem cells (Adipose-DERIVED MESENCHYMAL STEM CELLS, ADSC)

ADSCs are mesenchymal stem cells extracted from adipose tissue and have self-renewal and differentiation potential. ADSC has been used more and more widely in cartilage regeneration, but its cartilage differentiation potential is slightly lower than BMSC.

14. Flow Cytometry (Flow Cytometry)

Flow cytometry is a technique for rapidly analyzing and sorting cell populations. This technique detects specific molecules on the cell surface or inside the cell by suspending the cell in a flowing liquid and then passing the laser beam one by one, using fluorescent markers. The characteristics of each cell (e.g., size, shape, fluorescence intensity of the surface markers) are recorded, thereby allowing classification and counting of the cells.

In the present invention, flow cytometry is used to sort MFAP 5-positive stromal cell subsets by specific cell surface markers (e.g., CD 34). These cells have a potent differentiation potential in cartilage regeneration repair.

15. Single cell RNA sequencing (Single-Cell RNA Sequencing, scRNA-seq)

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

In the present invention, single cell RNA sequencing is used to resolve cell types in synovial tissue and neocartilage-like tissue, identifying the critical cell subpopulation involved in cartilage regeneration, MFAP5 positive stromal cells.

16. Quasi-timing analysis (Pseudotime Analysis)

The pseudo-timing analysis is an analysis method for single-cell RNA sequencing data, capable of presuming a continuous trajectory of cells from an undifferentiated state to a differentiated state. By this technique, researchers can reconstruct the developmental process of cells and observe the changes in gene expression of cells at different differentiation stages.

In the present invention, a pseudo-temporal analysis was used to study the differentiation trajectories of MFAP 5-positive cells, and it was speculated how these cells gradually differentiated into mature chondrocytes during cartilage regeneration.

17. Tissue engineering (Tissue Engineering)

Tissue engineering is a technique that creates or repairs tissue by combining cells, engineering materials, and biological factors. The goal is to construct functional tissue using cellular and scaffold materials for replacement or repair of damaged tissues or organs.

In cartilage regeneration, tissue engineering techniques combine seed cells (e.g., MFAP5 positive stromal cells) with scaffold materials to promote cartilage regeneration. Common tissue engineering methods include the use of natural or synthetic scaffold materials to form three-dimensional structures by adsorbing or implanting cells to support tissue regeneration.

18. Osteoarthritis (Osteoarthritis, OA)

Osteoarthritis is a common degenerative joint disease, mainly manifested by cartilage degeneration, joint pain and dysfunction. Treatment of OA generally relies on symptomatic management or surgical repair due to the limited ability of cartilage to regenerate.

19. Cartilage damage (CARTILAGE INJURY)

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

20. Osteochondrosis (Osteochondritis Dissecans, OCD)

Osteochondrosis is a disease in which cartilage and bone tissue separate due to insufficient blood supply at the bone ends, commonly found in the knee joint. OCD is one of the major pathological models in cartilage repair studies.

21. Knee Joint (Knee Joint)

The knee joint is the largest joint in the human body, and consists of a femur, a tibia and a patella, and the joint surface is covered with hyaline cartilage. Cartilage damage in the knee is a major feature of diseases such as osteoarthritis, and cartilage repair is critical for restoration of knee function.

22. Cartilage injury model (CARTILAGE INJURY MODEL)

Cartilage damage models are experimental models used to study cartilage repair and regeneration, and animal models (e.g., mice, rabbits, or pigs) are commonly used to simulate the pathological processes of human articular cartilage damage.

Drawings

FIG. 1 is a photograph showing the appearance of a new cartilage phenomenon in an animal model of example 1, wherein the left image is an animal model with a defect site, and the right image is new cartilage appearing in the animal model;

FIG. 2 shows the results of multi-tissue single-cell sequencing dimension reduction and Mfap5+ subgroup distribution in example 1;

FIG. 3 shows the results of single-cluster analysis of sequencing data of synovial tissue and neocartilage tissue in example 1, wherein (a-d) synovial tissue, neocartilage single-cell sequencing UMAP for dimension reduction and Mfap gene expression, S1 and E2 are Mfap5+ subgroups, (E) pseudo-time series analysis in neocartilage tissue shows Mfap5+ subgroups to differentiate in cartilage direction, and (f-g) GO pathways of S1 and E2 subgroups show similarity, cartilage development and extracellular matrix synthesis entries exist, wherein the horizontal axis (bar length) represents gene proportion, the right-hand number represents an enriched P value, and (h) subgroup similarity comparison between tissues, S1 and E2 subgroups show strong similarity.

FIG. 4 shows the cross-species integration analysis results of the MFAP 5-positive cell population in example 1, wherein (a-b) shows the single cell sequencing results of human OA and RA samples, and the MFAP5+ sub-population is found to exist similarly, and the expression patterns are similar, namely, the OA-1 sub-population and the RA-4 sub-population, (c-d) shows the cross-species integration results of the synovial data of human and rat, the overlap ratio of the integrated 1 population between species is high, (e) shows the MFAP5 gene expression condition of the integrated cell population, the 1 population shows specific expression of MFAP5, and the surface markers CD34 and THY1 show positive;

FIG. 5 is a technical flow chart of cell culture and sorting in example 2;

FIG. 6 shows the results of staining analysis and staining of different culture formats for human synovial samples of example 2, wherein (a) human synovial H & E stain, LL is LINING LAYER, SL is Sublining Layer, (b) MFAP5+ subpopulation immunofluorescence stain (Alexa Fluor 488/546 marker) in human synovial samples, (c) synovial mixed cell induced cartilage ball Sox9/Col2 immunofluorescence stain, confirming that part of synovial cells have chondrogenic differentiation ability under induction conditions, (d) adherent cultured cell fluorescent stain, (E) MFAP5+ cells exist and most of them co-express their surface markers under suspension culture conditions;

FIG. 7 shows the results of cell flow analysis of example 2 after two weeks of three-dimensional culture of synovial fibroblasts;

FIG. 8 is a staining and fluorescence pattern of rat MFAP 5-positive cell populations induced chondrocytes in example 3;

FIG. 9 is a staining and fluorescence pattern of rat BMSCs cells in example 3 induced chondrocytes.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are intended to facilitate the understanding of the present invention without any limitation thereto. The specific conditions not noted in the examples were carried out according to the conventional conditions or the conditions suggested by the manufacturer, and the reagents or instruments used, not noted by the manufacturer, were conventional products available commercially.

Example 1 identification of synovial Stem cell subpopulations involved in repair of articular cartilage loss

This example conducted intensive studies on synovial and regenerated cartilage-like tissue by single cell RNA sequencing (scRNA-seq) technology, in combination with bioinformatic analysis, to identify a critical cell subset with high cartilage differentiation potential.

1. Obvious new cartilage phenomenon in animal cartilage defect model

In the research of the early animal cartilage defect model experiment, the obvious regeneration tissue appears at the side direction of the unexpected joint around after the defect modeling, especially the patellar ligament attachment position is obvious, and the structure of the joint pulley is obvious. The clear red cartilage tissue was detected by staining with tissue SO (safranin-fast green) and a large number of cartilage pits were observed in the tissue cells in the area of the cartilage layer, as shown in FIG. 1. Histological staining revealed that the nascent cartilage formed a complete osteochondral structure and the superficial cartilage was hyaline cartilage. This phenomenon is different from the clinically common synovial tumor tissue, and the synovial tumor has a large amount of vascular, adipose and fibrous tissues, and the new tissue is all hyaline cartilage components, so that the new cartilage phenomenon is considered.

2. Single cell sequencing analysis of regenerated cartilage and synovial tissue was completed to determine chondrogenesis-related mesenchymal cell subsets

Thus, in this example, cells of both synovial tissue and neocartilage tissue were extracted, and single-cell RNA sequencing (scRNA-seq) was performed:

1. Sample extraction, namely extracting synovial tissue and regenerated cartilage tissue of the cartilage defect part from the animal model. Firstly, washing a sample, and then, carrying out enzymolysis on synovial tissue to obtain primary cell suspension.

Synovial tissue cell suspensions were prepared by adding synovial tissue samples to a medium containing digestive enzymes and incubating for 1 hour at 37 ℃. Thereafter, digestion was terminated and 1200rps centrifugation was performed to obtain a cell pellet, resuspended in complete medium to obtain a synovial tissue cell suspension, and single cell sequencing was prepared.

The suspension of the cartilage tissue cells is prepared by adding the cartilage tissue sample to a medium containing digestive enzymes and incubating at 37 ℃ for 1 hour. Thereafter, digestion was terminated and 1200rps centrifugation was performed to obtain a cell pellet, resuspended in complete medium to obtain a suspension of neo-cartilage tissue cells, and single cell sequencing was prepared.

2. Single cell RNA sequencing

Cell capturing and library construction cell capturing and cDNA library construction were performed using a 10X Genomics platform. After the sample is processed by single cell suspension, single cells are wrapped in oil drops, and high-efficiency single cell capturing is carried out by a microfluidic technology. Thereafter, cDNA was synthesized by reverse transcription reaction, and a sequencing library was generated by PCR amplification.

3. Sequencing and data processing

The sequencing platform is Illumina NovaSeq. And after quality control of the sequencing data, carrying out data analysis by utilizing CELL RANGER and other software, and finally obtaining a gene expression matrix.

4. Data analysis and cell subpopulation identification

Cluster analysis, in which sequencing data are subjected to cluster analysis by using Seurat and other tools, single cell data of synovial tissue and regenerated cartilage-like tissue are integrated, and 11 cell clusters (Clusters) which are similar in the synovial tissue and the regenerated cartilage-like tissue are determined, see a in fig. 2. Wherein a population of Mfap gene positive mesenchymal cells from the sub-lining layer (Sublining Layer) of regenerated cartilage-like and synovial tissue, the spatial position in the UMAP dimension-reducing map was closely related to typical mesenchymal and chondrocytes, MFAP5 was found to be Marker (c 3 in fig. 2) of specific expression of the adjacent region of synovial tissue and regenerated cartilage tissue in Umap integration, showing significant aggregation, indicating that it plays an important role in synovial and regenerated cartilage-like tissue.

Specific marker analysis by differential expression analysis, MFAP 5-positive cell populations were found to specifically express marker genes associated with cartilage differentiation, such as COL2A1 and ACAN, and their potential in cartilage differentiation was confirmed. In addition, MFAP5 positive cell populations also highly express genes associated with extracellular matrix synthesis, such as SFRP2 and POSTN.

Subsequently, the present example separately clusters the sequencing data of synovial tissue and neocartilage tissue, and found that this cell subset of Mfap gene positive stromal cells also exhibited a strong specificity in a single tissue, and the similarity comparison of Mfap5+ cell populations (S1, E2 populations) of both tissues (h in fig. 3) also showed that the stromal cells of this population were significantly more similar between the two tissue sources than the other cell populations.

GO pathway analysis shows that the Mfap gene positive interstitial cell population specifically expresses biological pathways related to cartilage differentiation and extracellular matrix synthesis (f in fig. 3 and g in fig. 3), wherein (a-d) in fig. 3) synovial tissues, nascent cartilage single cell sequencing UMAP dimensionality reduction and Mfap gene expression find that S1 and E2 are Mfap + subgroups, (E) pseudo-time sequence analysis in nascent cartilage tissues in fig. 3) Mfap5+ subgroups differentiate in the cartilage direction, f in fig. 3-g) S1 and E2 subgroups GO pathways in fig. 3 show similarity, cartilage development and extracellular matrix synthesis entries exist, and h) tissue-tissue subgroup similarity comparison is carried out, and S1 and E2 subgroups show strong similarity.

A pseudo-temporal analysis of the nascent chondrocytes was performed to monitor their continuous trajectory from an undifferentiated state to a chondrocyte differentiated state. The results showed that MFAP5 positive cell population was at an early stage of chondrocyte differentiation trace, and that over pseudo time, expression genes were turned from upstream development-related SFRP2, POSTN, etc. to downstream mature hyaline chondrocyte ACAN, COL2A1, etc. (e in fig. 3). From this, it was confirmed that Mfap gene-positive mesenchymal cells were in the precursor stage of differentiation into cartilage, had the potential for cartilage differentiation, and contributed to the formation of new cartilage.

The above analysis determined that the function of MFAP 5-positive cell populations was primarily related to extracellular matrix synthesis, to cartilage differentiation entries, and the like.

3. Cross-species integration analysis initially established strong conservation of this subpopulation

In the clinical osteoarthritis pathology, there is no new occurrence of pulley-like cartilage, more of which is fibrotic pathological synovial tumor. The single cell RNA sequencing data of synovial membranes in the healthy and arthritic states of clinical samples are analyzed by using published literature data, and the results are shown in FIG. 4, wherein a in FIG. 4-b in FIG. 4 can be seen, MFAP5+ subgroups are also present in single cell sequencing of human OA and RA samples, and the expression patterns are similar, namely OA-1 subgroups and RA-4 subgroups, so that the fact that MFAP5+ mesenchymal cells are also present in human synovial membranes and the gene expression patterns are similar (a in FIG. 4) is proved, and the conservation of the subgroups under different species and pathological environments is proved. Data integration analysis across species also found that there was an overlap in UMAP dimension reduction in MFAP 5-positive subpopulations between different species (b in fig. 4).

And c in fig. 4-d in fig. 4 are integrated across species in human and rat synovial data, and the fact that the overlap ratio of the integrated group 1 among species is high is found, and the cell group MFAP5 gene expression condition of the integrated group e in fig. 4 is shown, wherein the group 1 shows specific expression of MFAP5.

Through verification in human synovial tissue, single-cell RNA sequencing data of a human synovial sample are analyzed, and the fact that the MFAP5 positive cell subset exists is found, and the gene expression pattern of the MFAP5 positive cell subset is highly similar to that of the MFAP5 positive cell population in an animal model, so that the cell subset has conservation among different species is shown. This lays a foundation for clinical application of MFAP5 positive cell populations.

Example 2 flow analysis of MFAP 5-positive cell populations

This example completes histological verification of specific markers for MFAP5 positive cell subsets and determines culture and flow analysis methods for the cells.

The technical flow is shown in figure 5, a knee joint synovial membrane sample is taken from the body of a patient (/ healthy volunteer), primary cells are extracted by enzymolysis of type I collagenase, and flow cell sorting is carried out by means of a special cell surface marker, so that an MFAP5 positive subgroup is obtained. Then the cell subset is used as seed cells with high-efficiency cartilage forming potential to enter subsequent application and cell culture.

In the embodiment, immunofluorescence staining is firstly carried out on MFAP5 protein and a subgroup surface Marker in a human synovial sample (fig. 6), wherein the human synovial H & E staining result is shown as a in fig. 6 and is used for observing the basic tissue structure of the synovial, the human synovial H & E staining result comprises LL and SL, LL is LINING LAYER (lining layer), SL shows Sublining Layer (lower layer), and the MFAP5+ subgroup immunofluorescence staining (Alexa Fluor 488/546 marking) result in the human synovial sample is shown as b in fig. 6, so that the existence and sorting feasibility of the subgroup is verified, and a certain feasibility is provided for the analysis and sorting of the subgroup.

In this example, cartilage balls were also obtained by direct induction with primary cells (synovial mixed cells) (the induction method is cartilage induction medium, the cell suspension was centrifuged to form ball medium components: DMEM high sugar medium +1x Sodium pyruvate+1%ITS+10 (-7) M dexamethasone +50. Mu.g/mL Vc +10ng/mL TGF-. Beta.3 +1XP/S), and were subjected to Sox9/Col2 immunofluorescent staining, as shown in FIG. 6, and as a result, c, it was confirmed that part of synovial cells had chondrogenic differentiation ability under induction conditions.

In order to culture and sort the corresponding synovial cell subpopulations, this example tried various methods such as cell attachment culture, suspension culture, etc., and determined culture protocols (d in fig. 6, e in fig. 6).

The specific flow of the cell suspension culture is as follows:

1) Cell extraction, taking a clinical synovial membrane sample (taking soybean size as an example), firstly cleaning (cleaning solution: PBS diluted PS to 2%) and roughly cutting the sample. The sample was placed in a 1.5mL centrifuge tube, 1mL of enzyme solution (2. Mu.g/mLI type collagenase) was added and sheared, and the sample was digested at 37℃for 4 hours. The samples were transferred to a centrifuge tube, centrifuged at 1200rps for 6min, the supernatant was discarded, the medium (DMEM low-sugar medium+10% FBS+1x P/S formulation) was added for resuspension, and the supernatant was discarded after centrifugation at 1200rps for 6 min. Finally, the cells were resuspended in 6mL of medium.

2) Cell culture 6mL of the cell suspension was inoculated into a low adsorption 6-well plate, microspheres (medium Kong Kuanghua silk fibroin methacrylamide microspheres, filed with patent 2024111118570) were added, and the cells were cultured in a cell incubator (37 ℃ C., 5% CO 2) and the solution was changed every other day and amplified for 2 weeks.

The surface markers of MFAP5+ cells are present and mostly co-expressed under the suspension culture condition, and compared with the surface markers of the MFAP5+ cells, the suspension culture can obtain more MFAP5+ cells, and the fluorescence intensity is higher. Therefore, suspension culture is preferably used to sort MFAP 5-positive cell populations.

For the cell suspension obtained by suspension culture, the flow analysis of cell surface markers was performed in this example, based on single cell sequencing results, we confirmed the specific surface marker CD34 and synovial sublining marker THY1 of MFAP5 positive cell population. This example therefore uses flow cytometry to perform a flow assay of MFAP5 positive cell populations using the surface markers THY1 and CD34 as markers.

Flow cytometry sorting process:

1) 1-2 x10 ⁶ single cells obtained by suspension culture are taken and resuspended in flow cytometry buffer. Two fluorescent conjugated antibodies (anti-CD 34, THY1, conjugated fluorescence PE, APC respectively) were added to the cell suspension at the recommended dilution ratio (1:50-1:200). After mixing, incubation was carried out at 4℃for 30 minutes in the absence of light. Cells were washed with FACS buffer, centrifuged at 300g for 5min, the supernatant discarded and resuspended in FACS buffer.

2) An appropriate amount of dead cell dye, such as Fixable Viability Dye, was added according to the product instructions and washed once after incubation. Dead cell dyes are used to exclude dead cell signals in flow cytometry. The cells were resuspended in an appropriate amount of FACS buffer and ready for flow cytometric detection.

3) The laser channel of the flow cytometer is set according to the fluorescent label of the antibody. The CD34 protein is detected by PE fluorescence, the THY1 protein is detected by APC fluorescence, and fluorescence compensation is set by using a single-stained cell sample, so that signals of different fluorescent channels are ensured not to interfere with each other.

4) Cell debris and impurities are removed from Forward Scatter (FSC) and Side Scatter (SSC) patterns. Dead cells were then excluded based on the signal of the dead cell dye. A positive cell subset of cd34+thy1 ⁺ was selected on a flow cytometer. Expression of the cell markers is determined by fluorescent signal of the antibody.

Since MFAP5 is intracellular protein and CD34 and THY1 are membrane proteins, direct use of MFAP5 for flow separation requires rupture of membranes to destroy cell structure, affecting subsequent cell separation application, while CD34 can better specifically mark MFAP5 subpopulation in fibroblasts, CD34 is also a marker of vascular endothelial cells, and only CD34 is used as a marker for flow cytometry analysis, thus screening of MFAP5 positive cell population cannot be accurately achieved, and vascular endothelial cells may be introduced. THY1 is a synovial sub-lining layer fibroblast marker, and screening of MFAP5 positive cell groups cannot be accurately realized only by using THY1, and when THY1 and CD34 are used as markers at the same time, the MFAP5 positive cell groups can be more accurately sorted.

FIG. 7 shows the results of a two week three-dimensional culture of synovial fibroblasts followed by cell flow analysis, Q2-1 being the CD34+THY1+ biscationic region (about 31%), which was then screened and immunofluorescent-stained to identify that the biscationic region was indeed a MFAP5 positive cell population.

Example 3 in vitro functional validation of MFAP5 positive cells

After the MFAP5 positive cell population was isolated, it was further amplified and functionally validated in vitro in this example.

The rat MFAP5 positive cell population obtained by the screening in example 2 was collected into a centrifuge tube, the supernatant was discarded after centrifugation and precipitation, 3mL cartilage differentiation medium (high sugar DMEM+ x Sodium Pyruvate +1% ITS+10 (-7) M dexamethasone+50. Mu.g/mL vitamin C+10 ng/mL TGF-. Beta.3+1X antibiotic P/S) was added, and the mixture was placed in a cell incubator at 37℃with 5% CO ₂ for culture, and the medium was changed every other day, and induction culture was performed for 3 weeks, thereby successfully forming cartilage particles (staining and fluorescent pattern are shown in FIG. 7). In addition, this example also compares the cartilage particle induction effect under the same conditions using rat bone marrow mesenchymal stem cells (BMSCs) and Synovial Mesenchymal Stem Cells (SMSCs) (see fig. 8).

In SO staining, red represents the extent of staining into the cartilage matrix, the darker the color, the more pronounced the effect of chondrogenic differentiation. As can be seen by comparing fig. 7 and 8, after 21 days of induction, the synovial mesenchymal stem cells were stained deeper than BMSCs, indicating that they were more chondrogenic and more consistent in differentiation. Fluorescence staining examined the expression levels of SOX9, an important transcription factor involved in cartilage matrix formation, and type II collagen, a major component of cartilage matrix. The results showed that the fluorescent staining of synovial mesenchymal stem cells was strongly positive, whereas BMSCs showed poor chondrogenesis with only a few cells exhibiting fluorescent staining. In addition, the synovial mesenchymal stem cells can be differentiated into cartilage-like cells in a shorter time, so that the in-vitro culture time is obviously shortened, and the differentiation maturity and consistency of cartilage are improved.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

Claims

1. Use of stem cells for preparing an agent for repairing articular cartilage damage or synthesizing new cartilage, characterized in that the stem cells are derived from synovial tissue and are positive for MFAP5.

2. Use of stem cells for preparing an agent for repairing articular cartilage damage or synthesizing new cartilage, 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 it comprises stem cells, wherein the stem cells 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 whether the stem cell is the stem cell for repairing articular cartilage damage is determined by identifying whether the stem cell membrane protein is positive for CD34 and/or THY1.

5. The identification method according to claim 4, characterized in that whether the stem cell is a stem cell for repairing articular cartilage damage is determined by identifying whether the stem cell membrane protein is positive for CD34 and THY1.

6. 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 culturing, and then MFAP5-positive stem cells are sorted out by flow cytometric analysis.

7. The method according to claim 6, wherein 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 it is obtained by differentiation of stem cells, wherein the stem cells are derived from synovial tissue and are 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.