Synovial membrane stem cell subpopulation with spontaneous chondrogenic differentiation capacity as well as separation method, identification method and application of synovial membrane stem cell subpopulation

Abstract

The invention provides a synovial membrane stem cell subgroup with spontaneous chondrogenic differentiation capability and a separation method, an identification method and application thereof, the cell surface of the synovial membrane stem cell subgroup with spontaneous chondrogenic differentiation capability simultaneously expresses CDH13 and CD90, and the expression of NOTCH3 is deleted or the expression level of NOTCH3 is lower than a preset threshold value. The synovial stem cell subgroup with the spontaneous chondrogenic differentiation capacity is obtained and can be spontaneously differentiated into chondrocytes under the basic culture condition without adding growth factors, and better seed cells are provided for articular cartilage injury repair; the problems of inaccurate cartilage regeneration seed cell source, tedious induction steps and the like in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity, and its isolation, identification, and applications. Background Technology

[0002] Articular cartilage injury and degeneration are common clinical challenges. Because cartilage tissue itself lacks blood vessels and nerves, its self-repair capacity is extremely limited. Traditional treatments, such as microfracture surgery, involve drilling holes in the subchondral bone to release bone marrow matrix and stimulate cartilage repair, but the resulting tissue is primarily fibrocartilage, whose mechanical properties and durability are far inferior to hyaline cartilage. Autologous chondrocyte transplantation (ACI) and its modified technique, MAI, offer another approach, but suffer from problems such as donor site damage, limited cell numbers, and the risk of chondrocyte phenotype loss during in vitro expansion. In recent years, various cell-based cartilage tissue engineering products have emerged, including tissue-engineered constructs using mesenchymal stem cells (such as gMSC1 from Twocells, Japan). However, these methods often require complex in vitro induction and culture steps, such as repeated passages in various culture media, to obtain cells or tissues with chondrogenic capacity.

[0003] Synovial stem cells (MSCs) have long been considered for cartilage repair due to their excellent multi-directional differentiation potential. However, it remains difficult to identify specific cell subpopulations within this population that are highly efficient at chondrogenesis and possess significant application value. In current technologies, chondrogenesis by stem cells typically relies on exogenous inducing factors such as bone morphogenetic protein-2 (BMP-2), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), and insulin-like growth factor-1 (IGF-1). When exogenous inducing factors are lacking, traditional bone marrow MSCs or adipose-derived MSCs generally struggle to spontaneously form cartilage tissue. Notably, human synovial tissue may contain a unique cell subpopulation with chondrogenic potential. As early as the 2000s, researchers discovered chondrogenic progenitor cells capable of forming cartilaginous nodules in the synovium of patients with synovial chondromatosis, suggesting the potential presence of chondrogenic progenitor cells within the synovium itself. Normal synovial tissue contains various cellular components, including synovial fibroblast-like cells (FLS), and different subpopulations exhibit functional heterogeneity. In recent years, single-cell sequencing and other technologies have revealed that synovial stromal cells can be further subdivided into different subpopulations. For example, Fan Zhang et al. identified a synovial stromal cell subpopulation highly expressing MFAP5 in a single-cell transcriptome analysis of synovial tissue from rheumatoid arthritis. Subsequent studies demonstrated for the first time that this MFAP5-positive subpopulation is closely related to cartilage regeneration: flow cytometry sorting of CD34 and THY1 (CD90) double-positive synovial cells indirectly enriched MFAP5-positive synovial stem cells, thereby improving cartilage differentiation efficiency and shortening cartilage formation time. This finding indicates that sorting synovial stem cell subpopulations using specific markers is an effective strategy for improving cartilage regeneration. However, even MFAP5-positive synovial stem cells may still require specific induction conditions (such as the addition of exogenous TGF-β-dependent factors) or stimulation from an injury environment for chondrogenic differentiation in vitro; they have not been reported to autonomously form mature cartilage tissue without external induction. Chinese invention patent application CN105392489A discloses a cartilage injury treatment agent and its manufacturing method, requiring a serum-free culture medium containing FGF, PDGF, TGF-β, HGF, EGF, at least one phospholipid, and at least one fatty acid in the mesenchymal stem cell proliferation step. This method is characterized by high cost, poor stability, and safety risks associated with in vivo application. Chinese invention patent application CN115521909A only optimizes the culture medium composition to efficiently and conveniently prepare synovial mesenchymal stem cells, without addressing specific biomarkers. Chinese invention patent application CN113755433A discloses a modified mesenchymal stem cell culture suspension, but it still requires the addition of growth factors such as FGF-4 and FGF-9.

[0004] In summary, current technologies heavily rely on exogenous induction environments for chondrogenic differentiation of stem cells, leading to two major problems: first, the safety risks and high GMP production costs associated with the in vivo application of exogenous growth factors (such as TGF-β); and second, the cumbersome differentiation induction process and significant batch-to-batch variability. Therefore, there is currently a lack of stem cell subpopulations that possess strong endogenous chondrogenic driving forces and can spontaneously transform into chondrocytes without exogenous induction. Summary of the Invention

[0005] To address the technical problems in the prior art, this invention provides a subpopulation of synovial stem cells with spontaneous chondrogenic differentiation capacity. This subpopulation can spontaneously differentiate into chondrocytes under basic culture conditions without the addition of growth factors, providing superior seed cells for the repair of articular cartilage damage. This solves the difficulties in the prior art, such as the imprecise source of seed cells for cartilage regeneration and the cumbersome induction process. Furthermore, this invention also provides methods for isolating, identifying, and applying this subpopulation of synovial stem cells with spontaneous chondrogenic differentiation capacity.

[0006] The first aspect of the present invention provides a synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity, wherein the cell surface of the synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity simultaneously expresses CDH13 and CD90, and NOTCH3 expression is absent or the NOTCH3 expression level is below a preset threshold.

[0007] In one embodiment of the present invention, NOTCH3 expression deficiency or NOTCH3 expression level below a preset threshold refers to NOTCH3 expression negativity. NOTCH3 expression negativity means that the proportion of NOTCH3 positive cells is less than 5% in flow cytometry detection, or that its mean fluorescence intensity (MFI) is less than 10% of that of the isotype control group.

[0008] Furthermore, the synovial stem cell subpopulation, after being cultured in a medium containing only basal medium for 14-21 days, can spontaneously differentiate into functional chondrocytes without the addition of exogenous growth factors.

[0009] A second aspect of the present invention provides a method for isolating the above-mentioned synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity, comprising the following steps: S1. Fresh human synovial tissue was obtained by enzymatic digestion and separation to obtain a single-cell suspension; S2. The single-cell suspension obtained in step S1 is sorted using flow cytometry or immunomagnetic bead sorting: S3. Collect and sort the cells, which are the synovial stem cell subpopulation with the ability to spontaneously differentiate into chondrocytes.

[0010] In one embodiment of the present invention, the sorting of the single-cell suspension obtained in step S1 by flow cytometry includes the following steps: using antibodies against CDH13, CD90, and NOTCH3, according to CDH13... + / CD90 + / NOTCH3 - The strategy for sorting cells.

[0011] In one embodiment of the present invention, the antibodies used in flow cytometry sorting include FITC-labeled anti-human CDH13 antibody, PE-labeled anti-human CD90 antibody, and APC-labeled anti-human NOTCH3 antibody.

[0012] In one embodiment of the present invention, the sorting of the single-cell suspension obtained in step S1 using immunomagnetic beads includes the following steps: using positive-selective magnetic beads that bind to CDH13 and CD90, and negative-selective magnetic beads that bind to NOTCH3, to separate cells.

[0013] In one embodiment of the present invention, in step S1, the enzymatic digestion is performed to produce type I collagenase, the concentration of type I collagenase is 0.1%-0.3% (w / v), and the digestion time is 1.5-2.5 hours.

[0014] A third aspect of the present invention provides a method for using the aforementioned synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity, comprising the following steps: (1) Obtaining cell suspensions derived from human synovial tissue; (2) The cell suspension is incubated with the antibody combination; the antibody combination includes an antibody against CDH13, an antibody against CD90, and an antibody against NOTCH3; (3) The expression of cell surface markers was analyzed by flow cytometry. Cells that were positive for CDH13 and CD90 and had no NOTCH3 expression or NOTCH3 expression level below the preset threshold were identified as synovial stem cell subpopulations with spontaneous chondrogenic differentiation ability.

[0015] The fourth aspect of this invention provides the use of the above-mentioned synovial stem cell subpopulation in the preparation of drugs and / or medical materials for repairing articular cartilage defects.

[0016] In one embodiment of the present invention, the drug comprises the above-mentioned synovial stem cell subset and a pharmaceutically acceptable carrier; the cell concentration of the synovial stem cell subset in the pharmaceutical composition is 1 × 10⁻⁶. 5 ~5×10 6 Cells / mL.

[0017] In one embodiment of the present invention, the preparation of the medical material includes the following steps: (1) The above-mentioned synovial stem cell subpopulation was seeded onto a three-dimensional scaffold material; (2) The medical material was obtained by culturing it in a basal culture medium lacking exogenous chondrogenic factors for 14-21 days.

[0018] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: This invention provides a subpopulation of synovial stem cells with unique surface markers and the ability to autonomously differentiate into cartilage, solving the problems of inaccurate source of seed cells for cartilage regeneration and cumbersome induction steps in the prior art, and has significant innovation and practical value.

[0019] The present invention provides a subpopulation of synovial stem cells with spontaneous chondrogenic differentiation capacity, which is identified by a combination of cell surface markers (CDH13). + / CD90 + / NOTCH3 - As defined, this synovial stem cell subpopulation possesses a unique ability to spontaneously differentiate into chondrocytes. Traditional chondrocyte differentiation relies on exogenous growth factors (such as TGF-β and BMP), which are not only costly but may also cause adverse reactions such as heterotopic ossification and tumor formation. The synovial stem cell subpopulation of this invention can spontaneously differentiate without exogenous induction, significantly reducing the safety risks of clinical applications.

[0020] The synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity provided by this invention has a highly consistent spontaneous chondrogenic differentiation potential, reducing intercellular variability and ensuring predictable and efficient chondrogenic formation.

[0021] The synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity provided by this invention can provide seed cells with a stable source and easier expansion. Specifically, theoretically, all mesenchymal stem cells have the potential for chondrogenic differentiation. However, during the in vitro culture of mesenchymal stem cells, their chondrogenic differentiation capacity is reduced due to the influence of cytokines and culture conditions. The synovial stem cell subpopulation provided by this invention can spontaneously differentiate into chondrogenic cells, thus providing seed cells with a stable source and easier expansion.

[0022] The present invention provides a method for isolating synovial stem cell subsets with spontaneous chondrogenic differentiation capacity. Based on flow cytometry or immunomagnetic bead sorting technology, it can rapidly and efficiently enrich CDH13 from synovial tissue. + / CD90 + / NOTCH3 - Stem cell subpopulations. Compared to cumbersome traditional methods such as density gradient centrifugation and adherent culture, this method offers higher purity and recovery rate.

[0023] The present invention provides a method for isolating synovial stem cell subpopulations with spontaneous chondrogenic differentiation capacity. The flow cytometry or immunomagnetic bead sorting used are mature cell isolation techniques with standardized and highly reproducible operation procedures. Using this isolation method, researchers or clinicians can reliably obtain the required number of high-quality stem cell subpopulations.

[0024] The method for identifying synovial stem cell subpopulations with spontaneous chondrogenic differentiation capacity provided by this invention is simple to operate, easy to master, requires no special skills or expensive equipment, and has good repeatability and accuracy.

[0025] The synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity provided by this invention can be combined with biological scaffold materials to prepare medical materials for repairing cartilage defects. Furthermore, drugs that can promote chondrogenic differentiation of these cells can be screened to develop novel cartilage repair drugs. Transplanting these cells into the joint cavity can promote cartilage regeneration without additional induction. Moreover, when constructing tissue-engineered cartilage in vitro, cartilage tissue with good mechanical properties and biocompatibility can be obtained without adding exogenous growth factors, simplifying the tissue engineering process and reducing production costs. Attached Figure Description

[0026] Figure 1 A graph showing the UMAP dimensionality reduction clustering results of synovial cells; Figure 2 It consists of a subset of synovial fibroblasts and a subset of synovial stem cells (CDH13) capable of spontaneous chondrogenic differentiation. + / CD90 + / NOTCH3 - (Subgroup) distribution; Figure 3 A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - Subgroup gene expression characteristics; Figure 4 A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - Subgroup) cell function enrichment analysis; Figure 5 A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - (Subgroup) Flow sorting strategy diagram; Figure 6A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - Subgroup) CCK-8 assay and cell scratch assay; among which Figure 6 a is the CCK-8 proliferation curve. Figure 6 b is the migration diagram from the scratch test. Figure 6 c is a statistical graph of the scratch test; Figure 7 A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - Results of the three-lineage differentiation experiment (subgroup); among which, Figure 7 a represents adipogenic differentiation 200X. Figure 7 b represents osteogenic differentiation at 200X. Figure 7 c represents 400X chondrogenic differentiation; Figure 8 A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - Staining results of 14-day culture of subgroup (chondrocytes) chondrocytes (100X); among which Figure 8 a is a diagram of safranin-fix-green staining. Figure 8 b is the Alcian Blue staining pattern; Figure 9 A subset of synovial stem cells (CDH13) with spontaneous chondrogenic differentiation capacity. + / CD90 + / NOTCH3 - Subgroup) 7-day immunofluorescence results (200X); among which Figure 9 a is the COL2A1 staining diagram. Figure 9 b is the ACAN staining pattern. Figure 9 c is the SOX9-stained image. Detailed Implementation

[0027] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0028] This invention, based on single-cell RNA sequencing combined with bioinformatics analysis, isolated a subset of synovial stem cells (CDH13) within synovial cells that possess spontaneous chondrogenic differentiation capacity and play a crucial role in chondrogenic tissue regeneration. + / CD90 + / NOTCH3 - The study identified a subpopulation of cells and clarified the isolation method for this subpopulation. It also verified that the subpopulation could spontaneously differentiate into cartilage under normal basal culture conditions without the addition of exogenous inducing factors (including TGF-β, BMP-2, bFGF, etc.). This provides a factor-free cell therapy for clinical autologous cartilage repair. Furthermore, the cells are derived from normal synovium, eliminating the risk of tumorigenesis. Extraction is relatively simple and standardized, avoiding the safety and cost issues of exogenous factors. This facilitates large-scale preparation and clinical translation, and has broad clinical application prospects in cartilage repair and regeneration.

[0029] The synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity provided by this invention was obtained by the following isolation method: primary cells were obtained from synovial tissue of healthy individuals, and CDH13 was obtained by flow cytometry or immunomagnetic bead sorting. + / CD90 + / NOTCH3 - Synovial stem cell subpopulations were obtained. These subpopulations were directly seeded into conventional culture medium without any pretreatment or induction, and their gradual transformation into a chondrocyte phenotype was observed, producing a chondrocyte-like extracellular matrix rich in proteoglycans and type II collagen. Specifically, after a period of culture, Alcian blue staining showed a positive result (blue mucopolysaccharide matrix deposition), and immunofluorescence detection of the chondrocyte marker protein COL2A1 (type II collagen) and transcription factor SOX9 also showed positive expression, confirming that this subpopulation of cells had differentiated into a chondrocyte phenotype.

[0030] All cells described in this invention are derived from normal human (non-pathological tissue) synovium. After sorting, they are directly cultured without any gene modification or chemical pre-induction, demonstrating a safe and convenient application prospect.

[0031] The present invention will be described in detail below through specific embodiments.

[0032] Example 1 This embodiment provides a subpopulation of synovial stem cells with spontaneous chondrogenic differentiation ability. The cell surface of this subpopulation of synovial stem cells with spontaneous chondrogenic differentiation ability simultaneously expresses CDH13 and CD90, and NOTCH3 expression is absent or the NOTCH3 expression level is lower than a preset threshold.

[0033] (I) The method for isolating and culturing synovial stem cell subsets with spontaneous chondrogenic differentiation ability provided in this embodiment is as follows: 1. Synovial Tissue Acquisition and Primary Cell Isolation: Synovial tissue from healthy individuals discarded after knee or hip joint surgery was collected. Under aseptic conditions, the tissue was rinsed with PBS to remove blood and then minced. The synovial tissue was digested with 0.2% type I collagenase for 2 hours. The digestion product was filtered through a 70µm cell filter and centrifuged to collect a single-cell suspension. Cells were seeded in culture flasks, and DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies) was added. The flasks were incubated at 37°C and 5% CO2 for 24 hours. The medium was then replaced to remove non-adherent cells, yielding primary synovial stromal cells.

[0034] 2. Single-cell RNA sequencing and bioinformatics analysis: Single-cell RNA sequencing was performed on primary synovial cells (10x Genomics platform, target capture 5000-10000 cells). Data preprocessing and cluster analysis were performed using the Seurat software package (resolution 0.5-1.0). UMAP dimensionality reduction showed that the synovial cells were divided into multiple clusters. Figure 1 Further subpopulation identification revealed a subpopulation of synovial fibroblasts with high CDH13 expression, overlapping with CD90 positivity, but with low NOTCH3 expression. Figure 2 This subset of synovial stem cells (CDH13) possesses the ability to spontaneously differentiate into chondrocytes. + / CD90 + / NOTCH3 - Subgroup gene expression characteristics showed upregulation of cartilage-related genes such as COL2A1, SOX9, and ACAN, and downregulation of fibrosis genes ( Figure 3 GO functional enrichment analysis confirmed that this subgroup was enriched in the chondrogenesis and extracellular matrix assembly pathways. Figure 4 ).

[0035] 3. Flow Cytometry Sorting of CDH13-Positive / CD90-Positive / NOTCH3-Negative Cell Subpopulations: Target subpopulations were sorted from primary synovial cells using flow cytometry. The specific steps were as follows: Synovial cells in logarithmic growth phase (P0 generation) were digested into single cells and resuspended in flow cytometry working solution. FITC-labeled anti-human CD90 antibody (BioLegend, catalog number 328107), PE-labeled anti-human CDH13 antibody (R&D Systems, catalog number FAB3264P), and APC-labeled anti-human NOTCH3 antibody (BioLegend, catalog number 345409) were added, respectively, along with Zombie NIR dye (BioLegend, catalog number 423106) for viability staining. The cells were incubated at the manufacturer's instructions for 30 minutes at 4°C in the dark. Cells were analyzed and sorted using a flow cytometer (BD FACSAria III): Dead cells and debris were first excluded (dead cells were excluded using forward scattering / side scattering and Zombie staining). Forward scattering (FSC): H Gain 20, A Gain 1, Path 1; Side scattering (SSC): Voltage 400V, H Gain 1, A Gain 1, Path 1; CD90-FITC channel: Voltage 400V, H Gain 1, A Gain 1, Path 1; CDH13-PE channel: Voltage 550V, H Gain 1, A Gain 1, Path 1; NOTCH3-APC channel: Voltage 500V, H Gain 1, A Gain 1, Path 3; Zombie channel: Voltage 400V, H Gain 1, A Gain 1, Path 3; Compensation matrix: CDH13-PE to CD90-FITC 140%, NOTCH3-APC to Zombie 76%; A dual-parameter gate was set in the surviving single-cell population for CDH13 positivity (>80% positive cell percentage, MFI>50% control group) and CD90 positivity (>80% positive cell percentage, MFI>50% control group), and then NOTCH3 positive cells (<5% positive cell percentage, MFI<10% control group) were excluded based on this. The sorting strategy is as follows: Figure 5 As shown. CDH13 was finally collected. + / CD90 + / NOTCH3 - The cell subpopulation, namely the synovial stem cell subpopulation with spontaneous chondrogenic differentiation ability described in this embodiment, can be immediately used for subsequent culture and experimental analysis after sorting.

[0036] 4. Culture and morphological observation of sorted cells: The synovial stem cell subpopulation (CDH13) with spontaneous chondrogenic differentiation ability obtained by sorting was cultured and observed. + / CD90+ / NOTCH3 - Subpopulations were inoculated into 6-well plates or culture flasks at an initial density of approximately 5 × 10⁻⁶. 4 cells / cm 2 The culture medium was DMEM / F12 + 10% FBS (without any added growth factors). Cell culture conditions were 37℃ and 5% CO2. Initially, this subpopulation of cells exhibited a long, spindle-shaped or polygonal fibroblast-like morphology after adhesion, similar to typical synovial stromal cells. Cell proliferation and small colony formation were observed on day 3 of culture. With prolonged culture (approximately 7–10 days), local cell density increased, and morphological changes gradually occurred after confluence: some cells gradually became enlarged and rounded from their fibrous appearance, with thickened cytoplasm, exhibiting chondrocyte-like morphological characteristics. Macroscopic observation revealed translucent extracellular matrix deposition in certain areas of the cell layer within the culture dish. No exogenous inducing factors were added throughout the entire culture process; the morphological transformation of the cells occurred spontaneously under the basal culture conditions.

[0037] (II) Verification of cell proliferation and migration ability of the synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity provided in this embodiment: 1. Proliferation capacity assay (CCK-8 assay): To evaluate the proliferation capacity of the synovial stem cell subset (CDH13) obtained in step (I). + / CD90 + / NOTCH3 - The proliferation characteristics of the synovial stem cell subpopulation (CDH13) were assessed using CCK-8 assay on days 1, 2, and 3 of culture. Appropriate amounts of CCK-8 working solution were added to each well at each time point, and the cells were incubated at 37°C for 2 hours. The absorbance was then measured at 450 nm. The results showed that the synovial stem cell subpopulation (CDH13) obtained in this embodiment exhibited [positive cell proliferation activity]. + / CD90 + / NOTCH3 - The subpopulation exhibits good proliferative capacity, with OD450 values ​​increasing over time. This result demonstrates the strong proliferative capacity of the subpopulation, allowing for the acquisition of sufficient numbers for subsequent differentiation and application (see [link to relevant documentation]). Figure 6 a).

[0038] 2. Migration Assay: The scratch assay was used to verify cell migration ability, CDH13 + Cells showed a migration rate of 90% after 12 hours and 100% after 24 hours, demonstrating the efficacy of CDH13. + The cells exhibit significant migration ( Figure 6 b, 6c).

[0039] (III) Verification of Multi-directional Differentiation Potential The synovial stem cell subset (CDH13) obtained in step (I) was induced using classical induction conditions. + / CD90 + / NOTCH3 - Subgroups were subjected to adipogenic and osteogenic differentiation experiments to verify their multi-directional differentiation ability.

[0040] Adipogenic induction: Cells were seeded in 24-well plates. After confluence, the medium was replaced with adipogenic induction medium (containing 0.5 mM IBMX, 1 µM dexamethasone, 10 µg / mL insulin, 100 µM pibapyron, etc.) and cultured for 2 weeks, changing the medium every 3 days. After induction, cells were fixed with 4% paraformaldehyde, and lipid droplet formation was observed by OilRedO staining. The results showed that a small number of red lipid droplets appeared in the cells, indicating that their ability to differentiate into adipocytes was weak (see...). Figure 7 a). Osteogenic induction: After the cells reached confluence, the medium was replaced with osteogenic induction medium (containing 10 mM β-glycerophosphate, 50 µg / mL ascorbic acid, 0.1 µM dexamethasone, etc.) and cultured for 3–4 weeks. Alizarin red staining was used to detect mineralized nodules; the results showed no obvious red calcium deposits in the cell layer, indicating that they were unlikely to differentiate into osteoblasts (see...). Figure 7 b). The above results confirm that the synovial stem cell subpopulation (CDH13) obtained in this embodiment is... + / CD90 + / NOTCH3 - The CDH13-positive synovial cell subset has weak adipogenic and osteogenic differentiation capabilities.

[0041] (iv) Verification of spontaneous chondrogenic differentiation The synovial stem cell subpopulation (CDH13) obtained in step (I) of this embodiment was cultured without the addition of any inducing agent. + / CD90 + / NOTCH3 - The synovial stem cell subpopulation (CDH13) obtained in step (I) of this embodiment was used to observe its cartilage differentiation. + / CD90 + / NOTCH3 - Subpopulations were cultured in DMEM / F12 + 10% FBS for approximately 2 weeks. As the cells fused and cultured further, a significant increase in extracellular matrix was gradually observed. On day 14, cartilage-related staining and molecular marker detection were performed: First, culture wells were fixed with 4% paraformaldehyde, and acidic mucopolysaccharide staining (Alcian Blue staining, pH 1.0) was used to assess the production of glycosaminoglycan sulfate (GAG) in the cartilage matrix. The results showed that the cell cultures of this invention exhibited significant Alcian Blue positive staining, with the surrounding matrix stained blue, demonstrating abundant proteoglycan / mucopolysaccharide matrix deposition (see...). Figure 7c). In chondrogenic differentiation culture, cells were simultaneously cultured in vitro as chondrocytes (without chondrogenic inducing factors). On day 14, the chondrocytes were paraffin-embedded and stained with safranin and fast green (see [link to relevant documentation]). Figure 8 a) The results showed that red staining (Safranin-Fibrin Green positive) appeared in the matrix region surrounding the cells, indicating that these cells successfully generated a cartilage-like matrix rich in glycosaminoglycans (GAGs). Alcian blue staining (see Alcian blue staining) Figure 8 (b) The extracellular matrix is ​​clearly visible as a deep blue color, further demonstrating that these cells can spontaneously generate a matrix with a cartilage phenotype in the absence of inducing factors.

[0042] Furthermore, regarding the synovial stem cell subpopulation (CDH13) obtained in step (I) of this embodiment... + / CD90 + / NOTCH3 - Immunofluorescence detection of chondrocyte-specific markers was performed on the subpopulation cultures: staining was performed using anti-human Collagen II (COL2A1) monoclonal antibody, anti-ACAN antibody, and anti-SOX9 antibody (corresponding to chondrocyte-specific type II collagen, cartilage matrix glycosaminoglycans, and key transcription factors for cartilage differentiation, respectively). Results showed that significant positive signals (red fluorescence) for Collagen II and SOX9 were observed in the cell cultures of this invention, and ACAN signal was also positive (green fluorescence). These positive cells were mostly round or oval, clustered in areas rich in matrix deposition (see...). Figure 9 ).

[0043] The above results confirm that the synovial stem cell subpopulation (CDH13) of this invention... + / CD90 + / NOTCH3 - The subpopulation had partially transdifferentiated into chondrocytes without induction, and its products contained typical chondrocytes and cartilage matrix. This is a unique characteristic reported to date in synovial stem cells.

[0044] As can be seen from the above, the synovial stem cell subpopulation (CDH13) obtained by the present invention... + / CD90 + / NOTCH3 - This subpopulation exhibits typical MSC biological characteristics (adhesive growth and multi-lineage differentiation ability), and more significantly, it can initiate cartilage differentiation without exogenous stimulation. This discovery provides a novel cellular material for cartilage tissue engineering and articular cartilage repair.

[0045] In summary, the synovial stem cell subpopulation (CDH13) with spontaneous chondrogenic differentiation capacity provided by this invention... + / CD90+ / NOTCH3 - (Subgroup), compared with existing technologies, has the following outstanding features; (1) Novelty of the sorting markers: Traditional identification of synovial stem cell subpopulations often uses combinations of known mesenchymal markers. For example, existing techniques identify key MFAP5-positive chondrocyte regeneration cells in the synovium through double positivity for CD34 and THY1. In contrast, this invention is the first to use CDH13 for the sorting of synovial stem cell subpopulations and introduces NOTCH3 negativity as a screening criterion. CDH13 (T-cadherin) has never played a role in the selection of synovial cells before, but this invention finds that it can effectively identify cell populations with chondrocyte differentiation potential. Equally important, the NOTCH3 negativity criterion is based on the fact that some perivascular cells in the synovium also express THY1 and CDH13 positivity. NOTCH3 negativity can exclude the influence of perivascular cells on differentiation.

[0046] (2) Breakthrough in Cellular Functional Characteristics: In vitro chondrogenic differentiation of MSCs from any source (including bone marrow, adipose tissue, synovium, etc.) typically depends on specific induction media and factors, such as TGF-β3 and BMP-2. Even the recently identified MFAP5-positive synovial stem cell subpopulation requires chondrogenic induction media for full differentiation in practical applications (existing literature and patents do not report its ability to spontaneously chondrogenicize under factor-free conditions). In contrast, the synovial stem cell subpopulation (CDH13) of this invention possesses spontaneous chondrogenic differentiation capability. + / CD90 + / NOTCH3 - The subgroup has achieved true "spontaneous chondrogenesis": it can secrete cartilage matrix and express chondrocyte markers without any external stimulation. This is a major functional improvement, simplifying cartilage tissue engineering from a "two-step" process (first inducing cells and then forming cartilage) to a "one-step" process.

[0047] (3) Comparison of cell source and safety: Bone marrow MSCs are limited in clinical application due to the pain during the sampling process and the risk of heterotopic ossification; adipose MSCs are relatively easy to obtain but have limited chondrogenic ability and require in vitro induction to improve the effect. Synovial MSCs have received attention in recent years, and their chondrogenic potential is considered superior to that of bone marrow. Existing synovial MSC products (such as the aforementioned gMSC1) do not select for chondrogenic subpopulations and require long-term culture to form chondrogenic tissue. The cells of this invention are directly obtained from normal human synovium, avoiding the influence of inflammation or abnormal signals that may be caused by using diseased tissue; at the same time, the target subpopulation cells are screened using precisely defined surface markers, unlike unsorted MSCs which are mixed with other stromal cells, which helps to improve efficacy and controllability. More importantly, the cells of this invention can be used under conditions without exogenous factors, which reduces the variability and risk of exogenous substance residues during cell processing. From the perspective of clinical translation, the technical solution of this invention uses patient-derived cells and simple sorting and culture steps, with a clear technical path, safety and feasibility, which is significantly better than the existing complex stem cell induction differentiation schemes.

[0048] (4) Regarding the research on synovial stem cell subsets for cartilage repair, existing technologies have identified the MFAP5⁺ subset through single-cell sequencing and identified it using CD34. + / THY1 + This invention distinguishes cells with high chondrogenic potential by using markers for sorting. In contrast, this invention differs in concept and technique: First, the discovery of the MFAP5 subset relies on the regenerative environment of a cartilage injury model, while this invention directly identifies cells with chondrogenic differentiation capacity in normal synovium. Second, the MFAP5 subset requires dual markers CD34 and THY1 for indirect identification, as MFAP5 cannot be used as a surface marker for sorting. This invention uses the key surface markers CDH13 and CD90, with NOTCH3 as a negative selector, making the markers more targeted. Third, and crucially, the MFAP5 subset has not been reported to spontaneously differentiate into chondrocytes without induction, while the cells in this invention clearly demonstrate spontaneous chondrogenic differentiation, a unique functional characteristic. Therefore, this invention differs fundamentally from existing technologies in both marker selection and functional implementation.

[0049] In summary, the synovial stem cell subpopulation (CDH13) of the present invention possesses the ability to spontaneously differentiate into chondrocytes. + / CD90 + / NOTCH3 - The subgroup has significant advantages over existing technologies: it obtains cartilage progenitor cells with higher purity through a novel combination of biomarkers and achieves cartilage differentiation without external factors, which will greatly advance the development of cartilage regeneration therapy.

[0050] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. A subpopulation of synovial stem cells with spontaneous chondrogenic differentiation capacity, characterized in that, The synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity expresses both CDH13 and CD90 on its cell surface, and NOTCH3 expression is absent or the NOTCH3 expression level is below a preset threshold.

2. A method for isolating a subset of synovial stem cells with spontaneous chondrogenic differentiation capacity as described in claim 1, characterized in that, Includes the following steps: S1. Fresh human synovial tissue was obtained by enzymatic digestion and separation to obtain a single-cell suspension; S2. The single-cell suspension obtained in step S1 is sorted using flow cytometry or immunomagnetic bead sorting: S3. Collect and sort the cells, which are the synovial stem cell subpopulation with the ability to spontaneously differentiate into chondrocytes.

3. The method for obtaining a synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity according to claim 2, characterized in that, The sorting of the single-cell suspension obtained in step S1 using flow cytometry includes the following steps: using antibodies against CDH13, CD90, and NOTCH3, according to CDH13... + / CD90 + / NOTCH3 - The strategy for sorting cells.

4. The method for obtaining a synovial stem cell subpopulation with spontaneous chondrogenic differentiation capacity according to claim 3, characterized in that, The antibodies used in flow cytometry sorting included FITC-labeled anti-human CDH13 antibody, PE-labeled anti-human CD90 antibody, and APC-labeled anti-human NOTCH3 antibody.

5. The method for isolating a subset of synovial stem cells with spontaneous chondrogenic differentiation capacity according to claim 2, characterized in that, The sorting of the single-cell suspension obtained in step S1 using immunomagnetic beads includes the following steps: using positive-selective magnetic beads that bind to CDH13 and CD90, and negative-selective magnetic beads that bind to NOTCH3, to separate cells.

6. The method for isolating a subset of synovial stem cells with spontaneous chondrogenic differentiation capacity according to claim 2, characterized in that, In step S1, the enzymatic digestion produces type I collagenase, with a concentration of 0.1%-0.3% (w / v) and a digestion time of 1.5-2.5 hours.

7. A method for identifying a subset of synovial stem cells with spontaneous chondrogenic differentiation capacity as described in claim 1, characterized in that, Includes the following steps: (1) Obtaining cell suspensions derived from human synovial tissue; (2) The cell suspension is incubated with the antibody combination; the antibody combination includes an antibody against CDH13, an antibody against CD90, and an antibody against NOTCH3; (3) The expression of cell surface markers was analyzed by flow cytometry. Cells that were positive for CDH13 and CD90 and had no NOTCH3 expression or NOTCH3 expression level below the preset threshold were identified as synovial stem cell subpopulations with spontaneous chondrogenic differentiation ability.

8. The use of the synovial stem cell subpopulation of claim 1 in the preparation of drugs and / or medical materials for repairing articular cartilage defects.

9. The application according to claim 8, characterized in that, The drug comprises the synovial stem cell subset as described in claim 1, and a pharmaceutically acceptable carrier; the cell concentration of the synovial stem cell subset in the pharmaceutical composition is 1 × 10⁻⁶. 5 ~5×10 6 Cells / mL.

10. The application according to claim 8, characterized in that, The preparation of the medical material includes the following steps: (1) Seedling the synovial stem cell subpopulation described in claim 1 onto a three-dimensional scaffold material; (2) The medical material was obtained by culturing it in a basal culture medium lacking exogenous chondrogenic factors for 14-21 days.

Images