Chondrogenic stem cell for promoting joint repair and use thereof

Abstract

Provided are a chondrogenic stem cell for promoting joint repair and use thereof. Specifically, disclosed is a cell preparation used for alleviating or treating osteoarthritis, comprising a Procr-positive pluripotent stem cell. The Procr-positive chondrogenic stem cell can differentiate and generate downstream chondrocytes in vivo and promote cartilage repair in osteoarthritis damage, which will be a more effective cell treatment method. In addition, Procr proteins can be used as a new target of a drug for treating osteoarthritis.

Description

A type of chondrocyte stem cell that promotes joint repair and its application Technical Field

[0001] This invention relates to the fields of biotechnology and medicine, and more specifically, to a chondrocyte stem cell that promotes joint repair and its application. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative joint disease that primarily affects the elderly. It is characterized by irreversible, progressive destruction of articular cartilage and is the most common cause of pain and disability worldwide, placing a significant burden on patients' families and society. The development of OA is related to multiple pathological factors, such as excessive load, trauma, imbalance of the inflammatory system, and impaired anti-inflammatory pathways, leading to chondrocyte senescence, decreased cell density, abnormal secretory activity, extracellular matrix degradation, and impaired articular cartilage development. The main pathological features of OA include articular cartilage erosion, synovitis, and subchondral bone degeneration. Its main symptoms include joint pain, limited mobility, and eventual disability.

[0003] Currently, there are no effective drugs for treating osteoarthritis (OA). Conservative treatment is the main approach for OA, primarily focused on pain control. Various medications, such as nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 inhibitors (COX-2 inhibitors), glucosamine, steroids, and hyaluronic acid, have been used clinically to slow the progression of OA, but they are limited to pain control and do not reverse OA; moreover, long-term use has significant side effects. Joint replacement surgery is the main treatment for advanced OA, but it has limited lifespan and a high complication rate, making it unsuitable for younger populations. Therefore, there is an urgent need for effective treatments to alleviate or reverse OA progression. Identifying and characterizing stem cell populations capable of regenerating articular cartilage in adult joints will have profound implications for OA treatment and regenerative medicine.

[0004] The maintenance of skeletal homeostasis and damage repair in adults are jointly regulated by skeletal stem cells (SSCs) located in different regions. Currently, multiple SSC populations have been identified in the bone marrow, growth plate, and periosteum. Furthermore, chondrocyte progenitor cell populations capable of differentiating into articular chondrocytes have been discovered in the superficial layer of articular cartilage.

[0005] In recent years, cell transplantation to promote articular cartilage regeneration and repair has gradually become a novel treatment for osteoarthritis. Currently, the main cell types used for articular cartilage repair include autologous chondrocytes and mesenchymal stem cells from various tissue sources. Among them, matrix-induced autologous chondrocyte implantation (MACI) is currently the only FDA-approved cell therapy for osteoarthritis. This method involves surgically obtaining autologous cartilage from the non-weight-bearing area of ​​the damaged joint, digesting it with collagenase to obtain chondrocytes, expanding them in vitro, placing them on a bioabsorbable porcine collagen membrane, and implanting them onto the surface of the damaged articular cartilage. The disadvantages of MACI are that autologous chondrocytes are prone to dedifferentiation and it cannot treat large areas of cartilage loss.

[0006] Therefore, there is an urgent need in this field to develop a novel type of cartilage stem cell that can maintain articular cartilage homeostasis and promote joint repair, providing a new strategy for the treatment of cartilage degenerative diseases. Technical issues

[0007] To address the aforementioned problems, the purpose of this invention is to provide a novel chondrocyte stem cell that maintains articular cartilage homeostasis and promotes joint repair, as well as its applications. Technical solutions

[0008] In a first aspect of the invention, a cell preparation for alleviating or treating osteoarthritis is provided, the cell preparation comprising Procr-positive pluripotent stem cells.

[0009] In one embodiment, the Procr-positive pluripotent stem cells are chondrocyte stem cells.

[0010] In one embodiment, the pluripotent stem cells are Piezol-positive cells.

[0011] In one embodiment, the pluripotent stem cells are CD105-positive cells.

[0012] In one embodiment, the pluripotent stem cells are Procr-positive and CD105-positive cells.

[0013] In one embodiment, the pluripotent stem cells are CD45-negative, Ter119-negative, and Tie2-negative cells.

[0014] In one embodiment, the pluripotent stem cells have characteristics selected from the group consisting of:

[0015] (1) Cells located on the surface of the tibial joint, articular cartilage, or meniscus;

[0016] (2) Differentiate to produce articular cartilage and chondrocytes in the deep layer of the meniscus;

[0017] (3) Promotes cartilage repair;

[0018] (4) Prevention and / or treatment of osteoarthritis.

[0019] In a second aspect of the invention, a method is provided for isolating pluripotent stem cells from adult mammalian tissue, wherein the adult mammalian tissue is cultured; and cells that are positive for the cell surface marker Procr are isolated to obtain the pluripotent stem cells.

[0020] In one embodiment, the adult mammals include: rodents (rats, mice), cynomolgus monkeys, and humans.

[0021] In one embodiment, the adult mammalian tissue is selected from joint tissue.

[0022] In one embodiment, the joint tissues include the tibia, femur, and meniscus.

[0023] In a third aspect of the invention, the use of the cell preparations as described in the second aspect of the invention is provided for the prevention and / or treatment of osteoarthritis or related diseases.

[0024] In a fourth aspect of the invention, the use of Procr protein in the preparation of a diagnostic reagent for the prevention and / or treatment of osteoarthritis is provided, the diagnostic reagent using Procr protein as a biomarker for osteoarthritis.

[0025] In one embodiment, the Procr protein refers to the protein C receptor in chondrocytes.

[0026] In a fifth aspect of the invention, the use of the Procr protein in the preparation of a medicament for treating osteoarthritis is provided, the medicament targeting the Procr protein and the medicament being able to promote the transcription or expression of the Procr protein gene.

[0027] In one embodiment, the drug targets the Procr protein in chondrocytes.

[0028] In one embodiment, the drug is able to increase the expression level of Procr protein in chondrocytes.

[0029] In a sixth aspect of the invention, a pharmaceutical composition for the prevention and / or treatment of osteoarthritis is provided, the pharmaceutical composition comprising the cell preparation described in the first aspect of the invention.

[0030] In one embodiment, the pharmaceutical composition further comprises other drugs for the prevention and / or treatment of osteoarthritis.

[0031] In one embodiment, the pharmaceutical composition further comprises a Procr protein promoter and a targeting agent, the Procr protein promoter being loaded onto the targeting agent, the targeting agent targeting the Procr protein.

[0032] In one embodiment, the targeting agent is selected from one or more of AAV virus, miRNA, and polymer micelles.

[0033] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Beneficial effects

[0034] Compared with the prior art, the present invention has the following technical effects:

[0035] The novel chondrocyte stem cells provided by this invention can differentiate into downstream chondrocytes in vivo and contribute to the repair of osteoarthritis damage. In vitro experiments demonstrated the multi-lineage differentiation capacity of Procr-positive chondrocyte stem cells, and in vivo transplantation experiments also proved their effective cartilage repair capabilities. Using Procr-positive chondrocyte stem cells for cell therapy would be a more effective treatment method. Furthermore, it suggests that the Procr protein could serve as a novel drug target for treating osteoarthritis. Attached Figure Description

[0036] Figure 1 shows a schematic diagram of the in vivo lineage tracing results of Procr-positive cells under steady-state conditions. Figure A represents the immunofluorescence image analysis of tdTomato-positive cell localization results in Procr-CreERT2;tdTomato mice of different ages 2 days after tamoxifen injection; Figure B represents the statistical analysis of the number of tdTomato-positive cells in the tibia, femur, and meniscus regions of Procr-CreERT2;tdTomato mice of different ages 2 days after tamoxifen injection; Figure C represents the number of tdTomato-positive cells in the combined tibia and femur regions of Procr-CreERT2;tdTomato mice of different ages 2 days after tamoxifen injection. The statistical analysis results of the number of tdTomato positive cells in the femur region are shown in Figures D and E, respectively. Figures D and E represent the immunofluorescence image analysis of tdTomato positive cells in tdTomato mice after lineage tracing for 4 weeks (1 month) and 8 weeks (2 months) following tamoxifen injection at 4 weeks of age. Figures F and G represent the statistical analysis results of the number of tdTomato positive cells in the tibia / femur region and the meniscus region after tamoxifen injection at 4 weeks of age and after 4 and 8 weeks of tracking, respectively.

[0037] Figure 2 shows the in vivo lineage tracing of Procr-positive cells under osteoarthritis injury and the analysis results of the effect of deleting Procr-positive cells on osteoarthritis progression. Figure A represents the experimental modeling process: 8-week-old Procr-CreERT2;tdTomato mice underwent surgery to model osteoarthritis (DMM) 2 days after tamoxifen injection, and the analysis was performed 8 weeks later. Figure B represents the working principle of the Procr-CreERT2;tdTomato;DTA mice. Figure C represents the immunofluorescence image analysis results of tdTomato-positive cells in the sham-operated group (Sham) and the osteoarthritis model group (DMM) 8 weeks after surgery in Procr-CreERT2;tdTomato;DTA mice 2 days after tamoxifen injection to model osteoarthritis, and the localization results of tdTomato-positive cells in the sham-operated group (Sham) and the osteoarthritis model group (DMM) 8 weeks later. The EH figures represent the immunofluorescence image analysis results of tdTomato positive cells in the tibia, femur, meniscus, and combined tibia / femur regions of Procr-CreERT2; tdTomato; DTA mice 8 weeks after tamoxifen injection followed by surgical osteoarthritis modeling. The results were obtained from the sham-operated group (Sham) and the osteoarthritis model group (DMM). The I figure represents the analysis of Safranin-Fixed Green staining images, where "F" represents femur and "T" represents tibia. The J figure represents the OARSI score statistics. The K figure represents the microCT osteophyte analysis image. The L figure represents the bone mass statistics of the osteophyte region analyzed by microCT.

[0038] Figure 3 shows a schematic diagram of the effect of Piezo1 gene knockout on osteoarthritis progression in Procr-positive cells. Figure A represents Procr-CreERT2;tdTomato (Control) and Procr-CreERT2;tdTomato;Piezo1. fl / flImmunofluorescence image analysis of cKO mice 8 weeks after surgical osteoarthritis modeling 2 days after tamoxifen injection; Image B represents the statistical results of the number of tdTomato positive cells in the combined tibia and femur (Tibia / Femur) region; Image C represents the analysis of Safranin-Fix-Green staining images; Image D represents the statistical results of OARSI score for arthritis; Images E and F represent images and statistical results of subchondral bone thickness; Image G represents images and statistical results of bone mass in the osteophyte region from microCT analysis; Image H represents images and statistical results of bone mass in the calcified meniscus from microCT analysis.

[0039] Figure 4 shows the results of the analysis of the effect of Piezo1 agonist on the progression of osteoarthritis. In Figure A, images of wild-type C57 mice after surgical osteoarthritis modeling followed by injection of different concentrations of Yoda1 agonist for 8 weeks are analyzed using Safranin-Fix-Green staining. Figure B represents the statistical results of the OARSI score for articular cartilage arthritis. Figures C and D represent images and statistical results of subchondral bone thickness, respectively. Figures E and F represent images and statistical results of bone mass in the osteophyte region obtained using microCT, respectively. Figures G and H represent images and statistical results of bone mass in the calcified meniscus obtained using microCT, respectively.

[0040] Figure 5 shows a schematic diagram of the single-cell transcriptome sequencing analysis results of long bone peritumoral cells. Figure A represents the processing flow of long bone peritumoral cells; Figure B represents the clustering results of long bone peritumoral cells in single-cell transcriptome analysis; Figure C represents the expression of marker genes for cell clustering; Figure D represents the Slingshot trajectory analysis results of peritumoral fibrous layer cells; Figure E represents the Slingshot trajectory analysis results of peritumoral cambium layer cells; and Figure F represents the expression of CD200 and CD105 in different cell populations.

[0041] Figure 6 shows the results of in vitro cultured Procr-positive cells and their subsequent in vivo transplantation for repairing articular cartilage defects. Figure A represents the flow cytometry sorting strategy for mouse articular cartilage cells; Figure B represents the toluidine blue staining results after clonal culture of different cell populations of mouse articular cartilage; Figure C represents the statistical results of the number and size of clones after clonal culture of different cell populations of mouse articular cartilage; Figure D represents the chemical staining results of in vitro adipogenic, osteogenic, and chondrogenic differentiation of different cell populations of mouse articular cartilage; Figure E represents the mRNA expression results of in vitro qPCR analysis of adipogenic differentiation-related genes (Pparg), osteogenic differentiation-related genes (Col1a1), and chondrogenic differentiation-related genes (Col2a1) of different cell populations of mouse articular cartilage. "Control" refers to the differentiation control, cells without induction culture medium, while "Induced" refers to cells with induction culture medium for adipogenic, osteogenic, or chondrogenic differentiation; Figure F represents the chemical staining results of tissue sections 4 weeks after subrenal transplantation of different cell populations of mouse articular cartilage. Figure G represents the statistical results of the proportion of osteochondral and fibrous tissue formed 4 weeks after subrenal transplantation of different cell groups of mouse articular cartilage; Figure H represents the schematic diagram of the process of orthotopic transplantation of different cell groups of mouse articular cartilage after spheroid culture; Figure I represents the Safranin-Fix-Green staining results of orthotopic transplantation of different cell groups of mouse articular cartilage 4 weeks after spheroid culture; Figure J represents the statistical results of the proportion of cartilage and fibrous tissue formed 4 weeks after orthotopic transplantation of different cell groups of mouse articular cartilage; Figure K represents the schematic diagram of the process of obtaining and orthotopic transplantation of human articular cartilage tissue; Figure L represents the statistical results of the proportion of cartilage and fibrous tissue formed by orthotopic transplantation of different cell groups of young articular cartilage; Figure N represents the Safranin-Fix-Green staining results of orthotopic transplantation of different cell groups of old articular cartilage; Figure O represents the statistical results of the proportion of cartilage and fibrous tissue formed by orthotopic transplantation of different cell groups of old articular cartilage. Embodiments of the present invention

[0042] the term

[0043] Procr is a transmembrane glycoprotein that binds to both protein C (PC) and activated protein C (APC). Procr has been reported to label various tissue-specific stem and progenitor cells. Studies have found that Procr is expressed on the surface of hematopoietic stem cells, and transplanted Procr-positive bone marrow cells exhibit hematopoietic reconstitution capacity similar to transplanted hematopoietic stem cells, indicating that Procr can specifically enrich mouse hematopoietic stem cells. Another study found that Procr-positive cells are located in the basal layer of the mammary gland and exhibit cellular characteristics of epithelial-mesenchymal transition. Transplantation experiments have demonstrated that Procr-positive cells can differentiate into all cell types of mammary epithelium, thus being considered a novel type of mammary stem cell. Other studies have also found that Procr also labels vascular endothelial stem cells, which have strong cloning ability in vitro and high vascular reconstitution rates in transplantation experiments. Procr-positive progenitor cells can serve as a major cell source for repairing ovarian epithelium, and a long-term culture and expansion system for ovarian epithelial cells has been established in vitro using Procr-positive cells. Further research has found that Procr can label a group of pancreatic islet progenitor cells. In vivo genetic lineage tracing experiments revealed that Procr+ cells can produce all cell types in adult pancreatic islets, and Procr+ cells cultured in vitro can be continuously passaged and maintain differentiation capacity.

[0044] This invention uses single-cell transcriptomics to discover a group of Procr+ cells in the periostracum of adult mice that are enriched in articular cartilage.

[0045] The CreER lineage tracing system is a method for precisely tracking cell lineages in biological research, particularly well-suited for controlling gene expression temporally and spatially. By selecting different cell type-specific promoters, CreER expression can be restricted to target cells, thereby achieving gene manipulation of specific cells. The CreER system is a variant of the Cre-LoxP system, fusing Cre recombinase and a hormone-binding domain (ER) to form a controlled CreER fusion protein. In the absence of exogenous hormones (usually tamoxifen), the CreER protein is locked in the cytoplasm, unable to enter the nucleus and thus preventing gene recombination. When tamoxifen is added, it binds to the CreER protein, causing CreER to enter the nucleus and cleave sequences located between LoxP sites.

[0046] tdTomato fluorescent protein labeling: tdTomato is a red fluorescent protein gene, typically engineered downstream of a "LoxP-Stop-LoxP" sequence to create LoxP-Stop-LoxP-tdTomato genotype mice. Cell type-specific promoter-driven Cre or CreER genotype mice are usually crossbred with LoxP-Stop-LoxP-tdTomato genotype mice. Without Cre activation, the tdTomato gene is repressed by the upstream "Stop" signal and is not transcribed or expressed. Only when the "Stop" signal is removed by CreER under the action of tamoxifen is the tdTomato gene activated, beginning to express red fluorescent protein, thus achieving cell-specific labeling. This activation is permanent, meaning that once cells are labeled with red fluorescence, they will continue to express it, allowing researchers to track the distribution, proliferation, and differentiation of these cells in tissues, making it a powerful tool for tracking cell lineage dynamics in biological research.

[0047] DTA is the cytotoxic component of diphtheria toxin; it inhibits protein synthesis, leading to apoptosis. When DTA is expressed in specific cells, it suppresses protein synthesis in those cells, ultimately causing cell death. The DTA gene is typically placed downstream of a "LoxP-Stop-LoxP" region to create tool mice with the genotype LoxP-Stop-LoxP-DTA. In the absence of activation, the "Stop" sequence between LoxPs prevents DTA expression. By placing the Cre gene under a promoter specific to a particular cell type, DTA is expressed in target cells only when the Stop sequence in the DTA mouse gene is deleted, inducing cell death. Some experiments use CreER (Cre recombinase activated under tamoxifen induction), allowing researchers to activate DTA at specific times, thereby deleting target cells at specific stages. This DTA-induced cell deletion model, by precisely controlling cell type and time point, helps researchers gain a deeper understanding of the specific functions of cells in complex biological systems.

[0048] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Example 1: Using lineage tracers to confirm the distribution of Procr-positive cells and their effects on chondrogenesis under steady-state conditions.

[0050] To analyze the localization and differentiation potential of Procr-positive cells in adult long bones, the inventors obtained Procr-CreERT2 mice (this strain of mice is also available for purchase from Jax lab, Strain #: 033052) from Researcher Zeng Yi at the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, and crossed them with Loxp-Stop-Loxp-tdTomato mice to obtain Procr-CreER; tdTomato mice were used for in vivo lineage tracing experiments.

[0051] Mice aged 1 week, 4 weeks, 12 weeks, and 6 months were intraperitoneally injected with 100 mg / kg of tamoxifen for 5 consecutive days. Upon administration of tamoxifen, it binds to the estrogen receptor ER, inducing the release of Cre recombinase into the nucleus to exert its recombination effect, cleaving the Stop sequence, thereby labeling Procr-positive cells to express tdTomato fluorescent protein.

[0052] Two days after completing five consecutive days of intraperitoneal injections of tamoxifen, samples were collected immediately. At this time, the indicator of tdTomato positive cells was Procr positive cells. If samples were collected after a longer period, the indicator of tdTomato positive cells would represent the progeny cells produced by the downward differentiation of the then-labeled Procr positive cells over a certain period of time, which would facilitate the tracking of the differentiation fate of Procr positive cells.

[0053] Two days after completing five consecutive days of intraperitoneal injections of tamoxifen, joint tissue was harvested for analysis. The joint tissue was sequentially fixed with 4% PFA for 6 hours, decalcified with 20% EDTA for 3 days, and dehydrated with 30% sucrose for 12 hours before being embedded in OCT for frozen sectioning.

[0054] Joint tissue sections were photographed using a fluorescence microscope. DAPI reagent bound to DNA and emitted blue-violet fluorescence to label cell nuclei; tdTomato fluorescent protein labeled Procr-positive cells. The immunofluorescence images were analyzed to determine the location and number of tdTomato-positive cells in long bone joints.

[0055] The results are shown in Figure 1. No markers were found on the joints of mice at 1 week of age, and tdTomato positive cells appeared only at 4 weeks of age. Initial labeling was performed on surface cells of the articular cartilage and meniscus (Figure 1A). These Procr-labeled cells were mainly located on the tibial side of the joint surface, and less so on the femoral side. The number of markers decreased with increasing age (Figures 1B and 1C).

[0056] Four-week-old Procr-CreERT2; tdTomato mice were intraperitoneally injected with 100 mg / kg tamoxifen (TAM) for five consecutive days. After one month of lineage tracing following the TAM injection (when the mice were 8 weeks old), a significant increase in the number of tdTomato-positive cells was observed on the tibial articular surface and in the meniscus, with these cells differentiating into articular cartilage and deep meniscus chondrocytes (Fig. 1D). After two months of lineage tracing (when the mice were 12 weeks old), even more articular cartilage and deep meniscus chondrocytes differentiated (Fig. 1E). Statistical results from the combined tibial and femoral regions (Fig. 1F) and the meniscus region (Fig. 1G) also supported the same findings.

[0057] Example 2: Analysis of the effect of Procr-positive cells on cartilage repair in osteoarthritis using a lineage tracing system.

[0058] To investigate the role of Procr-positive cells on the joint surface in joint injury repair, this study established a mouse model of traumatic osteoarthritis using a surgical model of medial meniscus instability (DMM). This model induces osteoarthritis in mice by rupturing the medial meniscus ligament of the knee joint, causing direct friction between the femur and tibia after meniscus slippage.

[0059] Eight-week-old Procr-CreERT2;tdTomato mice were intraperitoneally injected with 100 mg / kg tamoxifen for five consecutive days. Two days after the injection, right knee DMM surgery was performed, with the left knee serving as a sham control. Tissue samples were collected for analysis eight weeks after modeling (Figure 2A). Immunofluorescence images of joint tissue sections (Figure 2C) showed an increase in tdTomato-positive chondrocytes on the tibial joint surface in the DMM group compared to the Sham control group (Figure 2E). Simultaneously, a large number of tdTomato-positive chondrocytes were observed on the femoral joint surface in the DMM group (Figure 2F), while the Sham control group showed only a few tdTomato-positive chondrocytes on the femoral joint surface. The number of tdTomato-positive chondrocytes in the meniscus region of the DMM group was also increased compared to the Sham control group (Figure 2G), indicating that in traumatic osteoarthritis, mechanical wear stimulates the accelerated differentiation of Procr-positive cells into chondrocytes on the joint surface.

[0060] To investigate the role of Procr-positive cells in joint injury repair, the inventors bred Procr-CreERT2;tdTomato;DTA mice. Only when CreER enters the nucleus and exerts its recombination effect is the Stop sequence deleted from the DTA mouse gene, leading to DTA expression in target Procr-positive cells and inducing their death (Figure 2B). Procr-positive cell deletion was achieved by intraperitoneal injection of tamoxifen into 8-week-old Procr-CreERT2;tdTomato;DTA mice for 5 consecutive days. Two days after the injection, an osteoarthritis model was established via DMM surgery, and joint phenotypes were analyzed after 8 weeks.

[0061] After 8 weeks of in vivo tracing, no obvious tdTomato positive cells were observed in the articular cartilage of Procr-CreERT2; tdTomato; DTA mice (Fig. 2D), proving that Procr positive cells were efficiently deleted (Fig. 2E-H).

[0062] After removing long bone joint tissue and fixing it with 4% PFA for 6 hours, the overall knee joint injury was observed by MicroCT scan, and the amount of bone spurs at the bone edge was analyzed.

[0063] Subsequently, the joint tissue was decalcified with 20% EDTA for 2 weeks, followed by gradient alcohol dehydration, xylene clearing, and paraffin soaking. After paraffin sectioning, the sections were stained with safranin and fast green. Safranin is a positive dye that primarily binds to glycosaminoglycans (such as chondroitin sulfate) in the cartilage matrix, giving the cartilage a red or orange-red color. Fast green is a negative dye that binds to collagen fibers and other cellular components, giving bone tissue a green color. The paraffin sections were first dewaxed and rehydrated, then immersed in hematoxylin for staining for 3 minutes, followed by rinsing with running water to remove surface stain for about 1 minute. After removing excess surface moisture, the sections were immersed in 0.2% fast green staining solution for 1 minute. Then, they were immersed in 1% acetic acid for 3 minutes to wash away excess stain. Finally, they were immersed in 0.5% safranin staining solution for 7 minutes. After removing the surface safranin stain, the sections were placed in 100% anhydrous ethanol to remove excess safranin stain and decolorize, and rinsed 3 times. The slides were placed in xylene for decolorization and transparency, and after three rounds, they were mounted with neutral resin containing xylene. After the resin dried completely, the slides were photographed.

[0064] The observation and scoring area must include the following four articular surfaces (articular cartilage): the medial femoral condyle, the lateral femoral condyle, the medial tibial plateau, and the lateral tibial plateau. Scores are calculated separately for each of the four areas, and the sum of these scores yields the final score. Score description:

[0065] 0 = Normal;

[0066] 0.5 = Very small degradation. Staining with blue (or other cationic dyes) results in a small loss of proteoglycans, but no structural change.

[0067] 1 = Very small degeneration: small surface wear and fibrosis under the cartilage surface are visible, without loss of chondrocytes or cartilage matrix, and the damaged area is less than 5% of the total area;

[0068] 2 = Mild degeneration: The damage extends vertically to below the cartilage surface, but rarely extends to deeper cartilage layers. There is some loss of the cartilage surface matrix, or localized loss of chondrocytes / proteoglycans, but good collagen preservation is still maintained. The damaged area accounts for about 5% to 10% of the total cartilage surface.

[0069] 3 = Moderate degeneration: The damage extends vertically downwards to the calcified cartilage layer, with localized loss of chondrocytes / proteoglycans, affecting approximately 10% to 24% of the articular cartilage;

[0070] 4 = Significant degeneration: Vertical fissures / damage extend beneath the calcified cartilage, exceeding 25% to 50% of the cartilage on the joint surface, or localized loss of chondrocytes / proteoglycans, affecting 25% to 50% of the articular cartilage thickness;

[0071] 5 = Severe degeneration: Vertical cracks / damage extend below the calcified articular cartilage, 50% to 75% of the cartilage surface is damaged, or localized areas of chondrocytes / proteoglycans are lost, and approximately 50% to 75% of the cartilage thickness is damaged;

[0072] 6 = Very severe degeneration: Vertical cracks / damage to the cartilage extend beneath the calcified cartilage, with more than 75% of the joint surface damaged.

[0073] The results showed that the OARSI score (Fig. 2I and 2J) and osteophyte formation (Fig. 2K and 2L) of articular cartilage after deletion of Procr-positive cells were significantly higher than those in the Procr-CreERT2; tdTomato mouse DMM surgery group. These results indicate that in vivo deletion of Procr-positive cells significantly aggravates osteoarthritis symptoms.

[0074] Example 3: Knocking out the Piezo1 gene in Procr-positive cells exacerbates the progression of osteoarthritis.

[0075] Mechanical force may play a key regulatory role in the activation of Procr-positive cells and the regeneration of articular cartilage. In recent years, Piezo1 ion channels have been shown to be an important class of mechanoreceptors. To investigate the regulatory mechanism of cartilage regeneration by Procr-positive cells on the articular surface, the inventors obtained Piezo1 from Professor Weiguo Zou of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. fl / flMice, obtained by mating with Procr-CreERT2 mice; Piezo1 fl / fl Mice. Introduced Procr-CreERT2; Piezo1 fl / fl Mice were intraperitoneally injected with 100 mg / kg tamoxifen for 5 consecutive days. Two days after the injection, DMM surgery was performed, and the progression of osteoarthritis was analyzed 8 weeks later.

[0076] Immunofluorescence images of joint tissue sections showed a reduction in tdTomato-positive chondrocytes in Piezo1 knockout mice undergoing DMM (Fig. 3A and 3B). Histomorphological analysis revealed significantly increased OARSI scores (Fig. 3C and 3D) and subchondral bone thickness (Fig. 3E and 3F) in Piezo1 knockout mice undergoing DMM. MicroCT analysis of osteophytes (Fig. 3G) and calcified menisci (Fig. 3H) also indicated that osteoarthritis symptoms were exacerbated in Piezo1 knockout mice undergoing DMM. These data suggest that, under pathological conditions, Piezo1 is essential for the activation of Procr-positive cells to promote articular cartilage regeneration.

[0077] Example 4: Pharmacological activation of Piezo1 can reduce the progression of osteoarthritis.

[0078] Yoda1 is a specific agonist of Piezo1. To investigate whether activation of Piezo1 in articular chondrocyte progenitor cells can slow the progression of osteoarthritis, this study used wild-type C57 mice to establish an osteoarthritis model through deep vein dysplasia (DMM) surgery. Following DMM surgery, 10 μl of Yoda1 was injected into the joint cavity weekly, with injection concentrations of 40 μM, 120 μM, and 360 μM designed. PBS was used as a solvent control (vehicle). The osteoarthritis phenotype was analyzed after 8 weeks.

[0079] Histological analysis of the sections showed that the OARSI scores of articular cartilage in DMM mice injected with 120 μM and 360 μM Yoda1 were significantly lower than those in DMM mice injected with Vehicle (Figures 4A and 4B).

[0080] Analysis of subchondral bone thickness also showed that the subchondral bone thickness of DMM group mice injected with 120 μM and 360 μM Yoda1 was significantly lower than that of DMM group mice injected with Vehicle (Figures 4C and 4D).

[0081] Micro-CT analysis showed that injection of 360 μM Yoda1 significantly reduced osteophyte volume (Figs. 4E and 4F), but had no effect on the volume of calcified meniscus bone (Figs. 4G and 4H). These results indicate that intra-articular injection of Yoda1 to activate Piezo1 can dose-dependently slow the progression of osteoarthritis.

[0082] Example 5: Single-cell transcriptomic analysis of periosteal cells in long bones

[0083] To characterize the stem and progenitor cell activity of Procr-positive cells in vivo, the inventors sought to identify more cell surface protein markers for further enrichment and isolation of these cells from articular cartilage. For this purpose, Prrx1-Cre;tdTomato;Col2.3-GFP mice were purchased from Jax Labs and hybridized. Prrx1 is a transcription factor widely expressed in limb bud mesenchyme; therefore, genetic lineage tracing using Prrx1-Cre;tdTomato would label cells from all skeletal lineages in the long bones of the limbs, including periosteal cells. The Col2.3-GFP mouse is a transgenic mouse in which the expression of green fluorescent protein (GFP) is controlled by the Col2.3 promoter. The Col2.3 promoter is specifically expressed in osteoblasts, which allows osteoblasts to be labeled with green fluorescent protein.

[0084] DAPI was obtained from the periosteal membrane of 8-week-old Prrx1-Cre;tdTomato;Col2.3-GFP mice by flow cytometry sorting. - CD45 - Ter119 - Tie2 - tdTomato + GFP - The cells (Figure 5A). DAPI is a fluorescent dye used to label the cell nucleus. - This means that all stained dead cells are excluded, because DAPI typically does not penetrate the membranes of living cells but will stain apoptotic or dead cells; CD45 is a commonly used leukocyte antigen used to label blood cells. - This indicates that blood cells were excluded to avoid interfering with the periosteal cell population of interest in the experiment; Ter119 is a specific erythroid marker. - This indicates that all erythrocytes or erythrocyte progenitor cells have been excluded; Tie2 is a receptor tyrosine kinase that marks endothelial cells and is primarily expressed in vascular endothelial cells. - This indicates that endothelial cells have been excluded; tdTomato + This refers to skeletal stem cells and their derivatives labeled with the Prrx1-Cre system; GFP expression is driven by the Col2.3 promoter, which is specifically expressed in differentiated osteoblasts. -This indicates that all osteoblasts expressing Col2.3 were excluded to obtain an immature or undifferentiated cell population. Subsequently, 3' droplet-based scRNA-seq library construction and sequencing analysis were performed using 10X Genomics (Figure 5A).

[0085] Cluster analysis divided the cells into nine subgroups (Figure 5B), and the subgroups were annotated by the genes highly expressed in each subgroup (Figure 5C). These included three stromal cell subgroups (0-2) of the fibrous layer and four subgroups (3-6) of the camellia layer. Subgroup 0 highly expressed Fst and Gdf10; subgroup 1 highly expressed Pi16 and Ly6a; subgroup 2 highly expressed Pdgfra and Ccl1, and were defined as stromal cells 1, 2, and 3, respectively. Subgroup 3 highly expressed Postn and Alpl, and were osteoblastic progenitors; subgroup 4 highly expressed Acan and Col2a1, and were chondrocytes; subgroup 5 highly expressed Fmod and Tnmd, and were tendon cells; subgroup 6 highly expressed Abi3bp and Thbs2, and were perichondrial cells; subgroup 7 highly expressed Acta2 and Myh11, and were smooth muscle cells; and subgroup 8 expressed Prg4 and Procr, and was defined as surface cells of articular cartilage.

[0086] Slingshot is a method for analyzing differentiation trajectories in single-cell transcriptome data. It infers cellular developmental pathways by identifying continuous cellular states. Slingshot differentiation trajectory analysis revealed a hierarchical differentiation relationship among the three subpopulations of the periosteal fibrous layer (Figure 5D). In the periosteal cambium, perichondrial cells (type 6) differentiate into osteoblastic progenitor cells (type 3), growth plate chondrocytes (type 4), and tendon cells (type 5) (Figure 5E).

[0087] CD200 is a previously reported marker for skeletal stem cells, while CD105 is a marker protein for bone, cartilage, and stromal progenitor cells. In single-cell transcriptome data analysis, the expression levels of genes of interest were extracted and normalized. A violin plot was used to illustrate the expression distribution of target genes in different cell populations. High expression of CD200 was found in the periosteal cambium (3-6) and smooth muscle cell population 7, while high expression of CD105 was found in articular cartilage surface cells (population 8) (Figure 5F). Therefore, the inventors chose CD105 to test its potential use for further purification of Procr-positive chondrocyte progenitor cells from articular cartilage.

[0088] Example 6 Procr + CD105 + Cells differentiate into cartilage in vitro, and in situ transplantation in vivo can repair cartilage.

[0089] Procr, a type of articular cartilage from mice, was obtained by flow cytometry. -Procr + CD105 - and Procr + CD105 + Cells (Figure 6A). Growth medium was pre-added to sterile 6-well plates and equilibrated in a hypoxic incubator (5% CO2, 5% O2) to ensure the oxygen content and temperature of the culture medium were the same as those in the incubator. A suspension of 200 target cells collected after flow cytometry sorting was added to the culture dish, mixed thoroughly, and returned to the incubator for in vitro CFU-F colony formation assay. After 7 days of in vitro culture, toluidine blue was used to stain the cell clones, and the number of clones formed was counted. The clones were digested and counted, and the average clone size was calculated (Figure 6B).

[0090] With Procr - Compared to cells, Procr + The number of clones formed by the cells increased significantly, and the clone size also increased significantly (Figure 6C). Among them, Procr + CD105 + The cell formed the most clones, and the clones were also the largest, suggesting that it has the strongest in vitro cloning ability.

[0091] The inventors further improved the articular cartilage Procr + CD105 + Cells were used for in vitro three-lineage differentiation experiments. Growth medium was added to 10 cm sterile culture dishes beforehand, and the dishes were equilibrated in a hypoxic incubator (5% CO2, 5% O2) to ensure the oxygen content and temperature of the culture medium were the same as those in the incubator. The target cell suspension collected after flow cytometry sorting was added to the culture dishes, mixed thoroughly, and then returned to the incubator. The medium was changed every 5 days. When the cells reached 70% confluence, the medium was aspirated, and the cells were washed once with DPBS. 5 mL of TrypLE digestive enzyme was added, and the cells were incubated at 37°C for 7 min to digest them. After centrifugation and resuspending, the cells were counted.

[0092] (1) For osteogenic differentiation, inoculate 1x10 4 Cells were transferred to 24-well plates and, when they reached 80%–90% confluence, the growth medium was removed, and osteogenic differentiation medium was added. The medium was changed every 3 days. After 21 days of differentiation, the cells were fixed in 10% formalin for 30 min, stained with 0.2% alizarin red for 10 min, washed with ddH2O to remove excess stain, and photographed under a microscope.

[0093] (2) For adipogenesis differentiation, inoculate with 1x10 4Cells were transferred to 24-well plates and, when they reached 80%–90% confluence, the growth medium was removed, and adipogenic differentiation medium was added. The medium was changed every 3 days. After 7 days of differentiation, the cells were fixed in 10% formalin for 30 min and washed once with 60% isopropanol. The cells were then stained with 60% Oil Red for 10 min, washed with ddH2O to remove excess stain, and photographed under a microscope.

[0094] (3) For chondrogenesis, 2.5 x 10 5 One cell line was seeded into a 15 mL centrifuge tube, centrifuged at 1000 rpm for 3 min until the bottom of the tube was reached, and chondrogenic differentiation medium was added for differentiation culture. The medium was changed every 3 days. After 28 days of differentiation, the cells were fixed with 4% PFA for 30 min and dehydrated with 30% sucrose. After embedding, the cells were ice-cut, stained with toluidine blue for 10 min, washed with PBS to remove excess stain, and photographed under a microscope.

[0095] The results showed that Procr + CD105 + The cells have the ability to differentiate into adipocytes, osteoblasts, and chondrocytes in vitro (Figure 6D).

[0096] Furthermore, differentiated cells were collected in 500 μL TRIzol, and RNA was extracted from the cells. The expression of differentiation-related genes was detected by qPCR. The expression of Pparγ (adipocyte marker gene), Col1a1 (osteoblast marker gene), and Col2a1 (chondrocyte marker gene) was significantly increased after differentiation, further validating the above conclusions (Figure 6E).

[0097] The primer sequences used are as follows:

[0098] Pparγ-F: 5'- GAAAGACAACGGACAAATCACC-3' (SEQ ID NO: 1);

[0099] Pparγ-R: 5'- GGGGGTGATATGTTTGAACTTG-3' (SEQ ID NO: 2);

[0100] Col1a1-F: 5'-CATGTTCAGCTTTGTGGACCT-3' (SEQ ID NO: 3);

[0101] Col1a1-R: 5'-GCAGCTGACTTCAGGGATGT-3' (SEQ ID NO: 4);

[0102] Col2a1-F: 5'- CCAGGATGCCCGAAATTA-3' (SEQ ID NO: 5);

[0103] Col2a1-R: 5'-GAGGTCCTCTGGGTCCTATGAT-3' (SEQ ID NO: 6);

[0104] β-Actin-F: 5'-GCTCTTTTCCAGCCTTCCTT-3' (SEQ ID NO: 7);

[0105] β-Actin-R: 5'-CTTCTGCATCCTGTCAGCAA-3' (SEQ ID NO: 8).

[0106] To further explore its differentiation potential in vivo, the inventors also cultured and expanded 2.5x10⁻¹⁰ μL of the sample. 5 Procr mice - Procr + CD105 - and Procr + CD105 + The tissue was transplanted under the renal capsule. Four weeks after transplantation, sections were stained with Movat (Figure 6F) to identify the resulting tissue type by color and morphology. This staining is primarily used to differentiate the different components of connective tissue, including bone, cartilage, and fiber. Dense bone tissue is yellow with clearly visible bone morphology, cartilage tissue is blue, and sparse fibrous tissue is pale yellow.

[0107] The results showed that Procr - and Procr + CD105 - Cells tend to produce fibrous tissue, while Procr + CD105 + The cells showed a stronger ability to produce bone and cartilage tissue (Fig. 6G).

[0108] To further detect Procr + CD105 + The chondrogenic repair capacity of cells will be 2×10 5 A mouse Procr cultured in an in vitro hypoabsorbent plate in 3D spheroidization. - Procr + CD105 - and Procr + CD105 +In situ joint transplantation experiments were performed using cells (Figure 6H). Micropores 0.5 mm deep were drilled into the femoral joint surface of the knee joint of 8-week-old wild-type C57 mice using a 0.5 mm drill bit. Cells cultured in 3D spheroids were then encapsulated in Matrigel and transplanted into the pores. Joint sections were collected two weeks later for safranin-fast green staining to assess their chondrogenesis capacity. Safranin is a positive dye that primarily binds to glycosaminoglycans (such as chondroitin sulfate) in the cartilage matrix, giving the cartilage a red or orange-red color. The in situ chondrogenesis capacity was assessed by quantitatively measuring the proportion of cartilage stained with safranin after transplantation to cartilage defects.

[0109] The results show that: compared with Procr - and Procr + CD105 - Compared to cells, Safranin-Fix Green staining results showed Procr + CD105 + The cells have a stronger ability to form chondrocytes in situ (Figs. 6I and 6J).

[0110] To investigate the significance of Procr-positive cell transformation in human joint tissue, the applicant obtained total cells and Procr-positive cells (PROCR) from articular cartilage biopsy tissues from different age groups (29-68 years). + )cell.

[0111] Subsequently, these cells were cultured and expanded in vitro, then cultured into spheres, and then transplanted in situ into the knee joint defects of immunodeficient NSG mice (Figure 6K).

[0112] The results showed that, compared with total human chondrocytes, PROCR from young biopsy samples... + The cells were able to effectively differentiate into chondrocytes, thereby repairing joint defects in NSG mice (Figs. 6L and 6M). More importantly, PROCR from aged cartilage biopsy samples... + The cells also showed superior efficiency in repairing articular cartilage defects, indicating that PROCR + Cells can serve as a source of next-generation stem cells or progenitor cells for the treatment of joint diseases.

[0113] discuss

[0114] Procr + The most prominent characteristic of cells is mechanosensitivity, Procr + The cells are mainly distributed in the articular cartilage of the tibia, which bears a large mechanical load. + The number of cells peaked at 4 weeks and then gradually decreased with age, which was correlated with the mice's physical activity level.

[0115] The only FDA-approved cell therapy for treating full-thickness cartilage defects is MACI. This therapy isolates chondrocytes from articular cartilage, culturees them in a 3D porcine collagen membrane, and then autologously implants them into the joint surface to promote cartilage regeneration. One limitation of MACI is the limited proliferative capacity of primary chondrocytes; it can only repair small joint defects. Conversely, stem cells or progenitor cells are considered ideal sources of chondrocytes due to their unlimited proliferative capacity. Therefore, Procr... + Chondrocyte progenitor cells appear to be a better cell source than a mixture of articular chondrocytes, making them suitable for cell therapies like MACI to repair large-area joint defects. Another limitation of MACI is that the proliferative capacity of articular chondrocytes is impaired during aging, thus it cannot be used in patients over 55 years of age. In contrast, we present Procr cells from human biopsy samples from both young and older individuals. + Cells can effectively repair joint defects in immunodeficient mice, further demonstrating that Procr + Cells are an ideal cell source and may extend the age limit for MAI. Intra-articular administration of the Piezo1 agonist Yoda1 significantly reduced osteoarthritis symptoms, suggesting that Procr... + In vivo activation of chondrocyte progenitor cells may be another effective way to prevent or delay the progression of osteoarthritis (OA).

[0116] The preferred 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 the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention. Industrial applicability

[0117] This invention provides a novel type of cartilage stem cell that maintains articular cartilage homeostasis and promotes joint repair, which can be used to alleviate or treat osteoarthritis and has industrial applicability. Sequence List Free Content

[0118] The primer sequences are as follows:

[0119] Pparγ-F: 5'- GAAAGACAACGGACAAATCACC-3' (SEQ ID NO: 1);

[0120] Pparγ-R: 5′- GGGGGTGATATGTTTGAACTTG-3′ (SEQ ID NO:2);

[0121] Col1a1-F: 5′- CATGTTCAGCTTTGTGGACCT-3′ (SEQ ID NO:3);

[0122] Col1a1-R: 5′-GCAGCTGACTTCAGGGATGT-3′ (SEQ ID NO:4);

[0123] Col2a1-F: 5′-CCAGGATGCCCGAAAATTA-3′ (SEQ ID NO:5);

[0124] Col2a1-R: 5′- GAGGTCCTCTGGGTCCTATGAT-3′ (SEQ ID NO:6);

[0125] β-Actin-F: 5′-GCTCTTTTCCAGCCTTCCTT-3′ (SEQ ID NO:7);

[0126] β-Actin-R: 5′-CTTCTGCATCCTGTCAGCAA-3′ (SEQ ID NO:8).

Claims

1. A cell preparation for use in alleviating or treating osteoarthritis, characterized in that, The cell preparation contains Procr-positive pluripotent stem cells.

2. The cell preparation according to claim 1, characterized in that, The pluripotent stem cells are CD105-positive cells.

3. The cell preparation according to claim 1, characterized in that, The pluripotent stem cells have characteristics selected from the following group: (1) Cells located on the surface of the tibial joint, articular cartilage, or meniscus; (2) Differentiate to produce articular cartilage and chondrocytes in the deep layer of the meniscus; (3) Promotes cartilage repair; (4) Prevention and / or treatment of osteoarthritis.

4. A method for isolating pluripotent stem cells from adult mammalian tissues, characterized in that, The adult mammalian tissue was cultured; cells that were positive for the cell surface marker Procr were isolated to obtain the pluripotent stem cells.

5. The method according to claim 4, characterized in that, The adult mammalian tissues were selected from joint tissues.

6. The application of the cell preparation according to claim 1, characterized in that, Used for the prevention and / or treatment of osteoarthritis or related diseases.

7. The application of Procr protein in the preparation of drugs for treating osteoarthritis, characterized in that, The drug targets the Procr protein and can promote the transcription or expression of the Procr protein gene.

8. A pharmaceutical composition for the prevention and / or treatment of osteoarthritis, characterized in that, The pharmaceutical composition comprises the cell preparation of claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition may also contain other medications for the prevention and / or treatment of osteoarthritis.

10. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further comprises a Procr protein promoter and a targeting agent, wherein the Procr protein promoter is loaded onto the targeting agent, and the targeting agent targets the Procr protein.

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