Cartilage stem cell for promoting joint repair and application thereof

Abstract

The invention relates to a cartilage stem cell for promoting joint repair and application thereof. In particular, the present invention provides a cellular preparation for alleviating or treating osteoarthritis. The cell line comprises Procr-positive pluripotent stem cells. The novel cartilage stem cell provided by the invention can be differentiated in vivo to generate downstream cartilage cells, and cartilage repair in osteoarthritis injury is promoted; it is prompted that selection of Procr positive cartilage stem cells for cell treatment will be a more effective treatment method. And the Procr protein can be used as a new target of the medicine for treating osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medicine, and more specifically, to a cartilage stem cell for promoting joint repair and application thereof. Background Art

[0002] Osteoarthritis (OA) is a common degenerative joint disease that mainly occurs in the elderly. It is mainly characterized by irreversible progressive destruction of articular cartilage. It is the most common cause of pain and disability worldwide, and it brings a huge burden to the patient's family and society. The occurrence and development of OA are related to a variety of pathological factors, such as excessive load, trauma, imbalance of the inflammatory system, and impaired anti-inflammatory pathways, which lead to chondrocyte aging, decreased cell density, abnormal secretory activity, degradation of the extracellular matrix, and impaired development of articular cartilage. The main pathological characteristics of OA include articular cartilage erosion, synovitis, and subchondral bone degeneration, and its main symptoms include joint pain, limited activity, and end-stage disability.

[0003] There is currently no effective drug for the treatment of OA. Conservative treatment is currently the main treatment for OA, which is mainly limited to pain control. Various drugs, such as non-steroidal anti-inflammatory drugs, cyclooxygenase-2 inhibitors, glucosamine, steroids and hyaluronic acid have been used clinically to slow the progression of OA, but they are limited to pain control, do not have the effect of reversing OA, and have significant side effects when used for a long time. Joint replacement is the main treatment for advanced OA, but it has a limited lifespan and a high incidence of complications, making it unsuitable for young people. Therefore, there is an urgent need for the emergence of effective treatments to alleviate or reverse the progression of OA. Identifying and characterizing stem cell populations that can regenerate articular cartilage in adult joints will have far-reaching implications for OA treatment and regenerative medicine.

[0004] The maintenance of adult bone homeostasis and injury repair are jointly regulated by skeletal stem cells (SSCs) located in different regions. Currently, multiple SSC populations have been found in the bone marrow, growth plate, and periosteum. In addition, cartilage stem progenitor cell populations that can differentiate into articular chondrocytes have been found in the superficial layer of articular cartilage.

[0005] In recent years, promoting articular cartilage regeneration and repair through cell transplantation has gradually become a new type of osteoarthritis treatment. At present, the main cell types used for articular cartilage repair include autologous chondrocytes and mesenchymal stem cells from a variety of different tissue sources. Among them, matrix-induced autologous chondrocyte implantation (MACI) is the only osteoarthritis cell therapy currently approved by the FDA. This method obtains autologous cartilage from the non-load-bearing area of ​​the damaged joint through surgery, and then digests it with collagenase to obtain chondrocytes. After in vitro expansion, they are placed on a bioabsorbable porcine collagen membrane and implanted on the surface of the damaged articular cartilage. The disadvantages of MACI are that autologous chondrocytes are easy to dedifferentiate and cannot treat large areas of cartilage defects.

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

[0007] In view of the above problems, the purpose of the present invention is to provide a new type of cartilage stem cell and its application for maintaining articular cartilage homeostasis and promoting joint repair.

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

[0009] In one embodiment, the Procr-positive pluripotent stem cells are cartilage 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 cell has a property selected from the group consisting of:

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

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

[0017] (3) Promote cartilage repair;

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

[0019] In a second aspect of the present invention, a method for isolating pluripotent stem cells from adult mammalian tissue is provided, wherein the adult mammalian tissue is cultured; cells positive for the cell surface marker Procr are isolated, thereby obtaining 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 tissue includes tibia, femur and meniscus.

[0023] In the third aspect of the present invention, there is provided a use of the cell preparation according to the second aspect of the present invention for preventing and / or treating osteoarthritis or diseases related thereto.

[0024] In a fourth aspect of the present invention, provided is the use of Procr protein in the preparation of a detection reagent for preventing and / or treating osteoarthritis, wherein the detection reagent uses Procr protein as a biomarker for osteoarthritis.

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

[0026] In the fifth aspect of the present invention, there is provided the use of Procr protein in the preparation of a drug for treating osteoarthritis, wherein the drug targets the Procr protein and can 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 can increase the expression level of Procr protein in chondrocytes.

[0029] In a sixth aspect of the present invention, a pharmaceutical composition for preventing and / or treating osteoarthritis is provided, wherein the pharmaceutical composition comprises the cell preparation described in the first aspect of the present invention.

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

[0031] In one embodiment, the pharmaceutical composition further comprises a Procr protein promoter and a targeting agent, wherein the Procr protein promoter is loaded on the targeting agent, and the targeting agent targets the Procr protein.

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

[0033] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

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

[0035] The novel cartilage stem cells provided by the present invention can differentiate into downstream chondrocytes in vivo, and also contribute to the repair of osteoarthritis damage. The multidirectional differentiation ability of Procr-positive cartilage stem cells has been demonstrated through in vitro experiments, and their effective cartilage repair ability has also been demonstrated through in vivo transplantation experiments. Selecting Procr-positive cartilage stem cells for cell therapy will be a more effective treatment method. It also suggests that Procr protein can be used as a new target for drugs to treat osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1The figure shows the results of in vivo lineage tracing of Procr-positive cells under steady-state conditions. Figure A represents the results of immunofluorescence image analysis of tdTomato-positive cell localization in Procr-CreERT2; tdTomato mice of different ages 2 days after tamoxifen injection; Figure B represents the statistical analysis results of the number of tdTomato-positive cells in the tibia (Tibia), femur (Femur) and meniscus (Meniscus) 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 (Tibia / Figure 3: Statistical analysis results of the number of tdTomato-positive cells in the combined tibia and femur (Tibia / Femur) region and in the meniscus (Meniscus) region when Procr-CreERT2;tdTomato mice were injected with tamoxifen at 4 weeks of age, and 4w represents 4 weeks of age, 12w represents 12 weeks of age, and 6m represents 6 months of age; Figure D and Figure E represent the immunofluorescence image analysis results of tdTomato-positive cell localization results 4 weeks (1 month) and 8 weeks (2 months) after lineage tracing, respectively; Figure F and Figure G represent the statistical analysis results of the number of tdTomato-positive cells in the combined tibia and femur (Tibia / Femur) region and in the meniscus (Meniscus) region when Procr-CreERT2;tdTomato mice were injected with tamoxifen at 4 weeks of age, and 4 weeks and 8 weeks of tracing, respectively.

[0037] Figure 2Shown is a schematic diagram of the in vivo lineage tracing of Procr-positive cells under osteoarthritis injury and the analysis of the effects of deleting Procr-positive cells on the progression of osteoarthritis. Figure A represents the schematic diagram of the experimental modeling process. Procr-CreERT2; tdTomato mice aged 8 weeks underwent surgery to model osteoarthritis (DMM) 2 days after tamoxifen injection, and then the analysis was performed 8 weeks later. Figure B represents the schematic diagram of the working principle of Procr-CreERT2; tdTomato; DTA mice. Figure C represents the results of immunofluorescence analysis of the localization of tdTomato-positive cells in the sham group (Sham) and the osteoarthritis model group (DMM) 8 weeks after the surgery was performed 2 days after tamoxifen injection. Figure D represents the results of immunofluorescence analysis of the localization of tdTomato-positive cells in the sham group (Sham) and the osteoarthritis model group (DMM) 8 weeks after the surgery was performed 2 days after tamoxifen injection in Procr-CreERT2; tdTomato; DTA mice underwent surgery to model osteoarthritis 8 weeks after the surgery was performed 2 days after tamoxifen injection. Figures E, H represent Procr-CreERT2; tdTomato and Procr-CreERT2; tdTomato; DTA mice, which underwent surgery 2 days after tamoxifen injection to model osteoarthritis. Immunofluorescence image analysis 8 weeks after the sham group (Sham) and osteoarthritis model group (DMM) showed the statistical results of the number of tdTomato-positive cells in the tibia (Tibia), femur (Femur), meniscus (Meniscus) and combined tibia and femur (Tibia / Femur) areas; Figure I represents the analysis of safranin fast green staining images, "F" represents femur (Femur); "T" represents tibia (Tibia); Figure J represents the statistical results of OARSI scores; Figure K represents micro CT osteophyte analysis images; Figure L represents the statistical results of bone mass in the osteophyte area by micro CT analysis.

[0038] Figure 3 The figure shows the effect of knocking out Piezo1 gene in Procr-positive cells on the progression of osteoarthritis. Figure A represents Procr-CreERT2; tdTomato (Control) and Procr-CreERT2; tdTomato; Piezo1 fl / flImmunofluorescence image analysis of cKO mice 8 weeks after osteoarthritis modeling was established 2 days after tamoxifen injection; Panel B represents the statistical results of the number of tdTomato-positive cells in the combined tibia and femur (Tibia / Femur) area by immunofluorescence image analysis; Panel C represents the image analysis of safranin fast green staining; Panel D represents the statistical results of OARSI score for articular osteoarthritis; Panels E and F represent the images of subchondral bone thickness and the statistical results of subchondral bone thickness; Panel G represents the micro CT osteophyte analysis image and the statistical results of bone mass in the osteophyte area; Panel H represents the micro CT calcified meniscus analysis image and the statistical results of bone mass in the calcified meniscus.

[0039] Figure 4 The diagram shows the results of the analysis of the effect of Piezo1 agonists on the progression of osteoarthritis. Among them, Figure A represents the analysis of the safranin fast green staining images of wild-type C57 mice injected with different concentrations of Yoda1 agonists for 8 weeks after surgical osteoarthritis modeling; Figure B represents the statistical results of OARSI scores for articular cartilage arthritis; Figures C and D represent the subchondral bone thickness images and the statistical results of subchondral bone thickness, respectively; Figures E and F represent the micro CT osteophyte analysis images and the statistical results of bone mass in the osteophyte area, respectively; Figures G and H represent the micro CT calcified meniscus analysis images and the statistical results of calcified meniscus bone mass, respectively;

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

[0041] Figure 6The figure shows the results of the analysis of the repair of articular cartilage defects by in vitro culture and differentiation of Procr-positive cells and in vivo transplantation after culture. Among them, Figure A represents the flow cytometry sorting strategy of mouse articular chondrocytes; Figure B represents the results of toluidine blue staining 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 results of chemical staining after in vitro adipogenic, osteogenic, and chondrogenic differentiation of different cell populations of mouse articular cartilage; Figure E represents the results of qPCR analysis of mRNA expression 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 in vitro, among which "control" refers to the differentiation control, cells without the addition of differentiation induction medium, and "Induced" refers to cells with the addition of adipogenic, osteogenic, or chondrogenic differentiation induction medium; Figure F represents the results of chemical staining of tissue sections of different cell populations of mouse articular cartilage transplanted under the kidney capsule for 4 weeks; Figure G represents the statistical results of the proportion of osteochondral tissue and fibrous tissue formed by different cell populations of mouse articular cartilage after 4 weeks of subrenal transplantation; Figure H represents the schematic diagram of the process of orthotopic transplantation of different cell populations of mouse articular cartilage into spheres; Figure I represents the results of safranin-fast green staining of different cell populations of mouse articular cartilage after 4 weeks of orthotopic transplantation of different cell populations of mouse articular cartilage into spheres; Figure J represents the statistical results of the proportion of cartilage and fibrous tissue formed by different cell populations of mouse articular cartilage after 4 weeks of orthotopic transplantation of different cell populations of mouse articular cartilage into spheres; Figure K represents the schematic diagram of the process of human articular cartilage tissue sampling and orthotopic transplantation; Figure L represents the statistical results of the proportion of cartilage and fibrous tissue formed by orthotopic transplantation of different cell populations of articular cartilage in young people (Young); Figure N represents the results of safranin-fast green staining of orthotopic transplantation of different cell populations of articular cartilage in old people (Old); Figure O represents the statistical results of the proportion of cartilage and fibrous tissue formed by orthotopic transplantation of different cell populations of articular cartilage in old people (Old).

[0042] the term

[0043] Protein C receptor (Procr)

[0044] Procr is a transmembrane glycoprotein that can bind to protein C (PC) and activated protein C (APC). Procr has been reported to mark a variety of 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 have hematopoietic reconstruction capabilities 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 show cell characteristics of epithelial-mesenchymal transition. Transplantation experiments have shown that Procr-positive cells can differentiate into all cell types of the mammary epithelium, so they are considered to be a new type of mammary stem cell. Other studies have also found that Procr also marks vascular endothelial stem cells, which have strong cloning ability in vitro and a high rate of vascular reconstruction in transplantation experiments. Procr-positive progenitor cells can be used as the main cell source for repairing ovarian epithelium, and a long-term culture and expansion system of ovarian epithelial cells has been established in vitro using Procr-positive cells. Further studies have found that Procr can mark a group of pancreatic islet progenitor cells. In vivo genetic lineage tracing experiments found 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 ability.

[0045] The present invention uses single-cell transcriptomics to discover that there is a group of Procr+ cells in the outer membrane of adult mouse bones and are enriched in articular cartilage.

[0046] Lineage Tracing System

[0047] The CreER lineage tracing system is a method for accurately tracing cell lineages in biological research, and is particularly suitable for controlling gene expression in time and space. By selecting different cell type-specific promoters, the expression of CreER can be restricted to target cells, thereby achieving genetic manipulation of specific cells. The CreER system is a variant of the Cre-LoxP system, which fuses the Cre recombinase and the hormone binding domain (ER) to form a controlled CreER fusion protein. In the absence of exogenous hormones (usually tamoxifen), the CreER protein will be locked in the cytoplasm and cannot enter the nucleus, so that genetic recombination does not occur. When tamoxifen is added, tamoxifen binds to the CreER protein, causing CreER to enter the nucleus and cut the sequence between the LoxP sites in the nucleus.

[0048] tdTomato fluorescent protein marker: tdTomato is a red fluorescent protein gene, which is usually designed downstream of a "LoxP-Stop-LoxP" sequence to make a tool mouse with the genotype LoxP-Stop-LoxP-tdTomato. Usually, Cre or CreER genotype mice driven by cell type-specific promoters are crossed with LoxP-Stop-LoxP-tdTomato tool mice. In the absence of Cre activation, the tdTomato gene will be inhibited by the upstream "Stop" signal and will not be transcribed and expressed. Only when CreER removes the "Stop" signal under the action of tamoxifen will the tdTomato gene be activated and begin to express red fluorescent protein, thereby achieving specific labeling of cells. This activation is permanent, which means that once the seed cells are labeled with red fluorescence, they will continue to express, allowing researchers to track the distribution, proliferation and differentiation of such cells in tissues, making it a powerful tool for tracking cell lineage dynamics in biological research.

[0049] DTA (Diphtheria Toxin A, DTA) cell deletion mice

[0050] DTA is the cytotoxic component of diphtheria toxin, which blocks protein synthesis and causes apoptosis. When DTA is expressed in specific cells, it inhibits protein synthesis in these cells, ultimately inducing cell death. The DTA gene is usually placed downstream of a "LoxP-Stop-LoxP" region to create a tool mouse with the genotype LoxP-Stop-LoxP-DTA. In the absence of activation, the "Stop" sequence between the LoxPs prevents the expression of DTA. By placing the Cre gene under the promoter of a specific cell type, only when Cre is expressed, the Stop sequence in the DTA mouse gene is deleted, and DTA is expressed in the target cells, inducing the death of these cells. CreER (Cre recombinase activated under tamoxifen induction) is used in some experiments, which allows researchers to activate DTA at a specific time, thereby deleting target cells at a specific stage. This DTA-induced cell deletion model helps researchers gain a deeper understanding of the specific functions of cells in complex biological systems by precisely controlling cell types and time points. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solution, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, all the embodiments described are only partial embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Example 1 Using lineage tracing to confirm the distribution of Procr-positive cells and their effects on cartilage development under steady-state conditions

[0053] To analyze the localization and differentiation potential of Procr-positive cells in adult long bones, the inventors obtained Procr-CreERT2 mice from Researcher Zeng Yi of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences (this strain of mice is also available for purchase at Jaxlab, Strain #: 033052), and mated them with Loxp-Stop-Loxp-tdTomato mice to obtain Procr-CreER; tdTomato mice were used for in vivo lineage tracing experiments.

[0054] 100 mg / kg of tamoxifen was intraperitoneally injected into the above mice at 1 week, 4 weeks, 12 weeks and 6 months of age for 5 consecutive days. When tamoxifen is administered, tamoxifen binds to the estrogen receptor ER, induces the release of Cre recombinase into the nucleus to play a recombination role, cuts the Stop sequence, and makes Procr-positive cells marked to express tdTomato fluorescent protein.

[0055] The samples were collected immediately 2 days after the completion of 5 consecutive days of intraperitoneal injection of tamoxifen. At this time, the indication of tdTomato-positive cells indicates Procr-positive cells. If the samples were collected after a longer period of time, the indication of tdTomato-positive cells indicates the daughter cells produced by the downward differentiation of the Procr-positive cells marked at that time within a certain period of time, which is convenient for tracking the differentiation fate of Procr-positive cells.

[0056] Two days after the completion of 5 consecutive days of intraperitoneal injection of tamoxifen, joint tissues were collected for analysis. The joint tissues were fixed with 4% PFA for 6 hours, decalcified with 20% EDTA for 3 days, and dehydrated with 30% sucrose for 12 hours, and then embedded in OCT for frozen sections.

[0057] Joint tissue sections were collected and photographed using a fluorescence microscope. DAPI reagent binds to DNA and emits blue-purple fluorescence, which is used to mark cell nuclei; tdTomato fluorescent protein marks Procr-positive cells. Immunofluorescence images were used to analyze the location and number of tdTomato-positive cells in long bone joints.

[0058] The results are as follows Figure 1 As shown, there is no labeling on the joints of mice at 1 week of age, and tdTomato-positive cells do not appear until 4 weeks of age. Initially, cells on the surface of articular cartilage and meniscus were labeled ( Figure 1 A). These Procr-positive cells are mainly located on the tibial joint surface, but less on the femoral joint surface. The number of markers decreases with age ( Figure 1 B and 1C).

[0059] 4-week-old Procr-CreERT2; tdTomato mice were intraperitoneally injected with 100 mg / kg tamoxifen (TAM) for five consecutive days. One month after the end of TAM injection (the mice were 8 weeks old at this time), the number of tdTomato-positive cells on the tibial joint surface and meniscus increased significantly, and they differentiated into chondrocytes in the deep layer of articular cartilage and meniscus ( Figure 1 D). After two months of lineage tracing (when the mice were 12 weeks old), more chondrocytes were differentiated in the articular cartilage and deep layers of the meniscus ( Figure 1 E). Combined tibial and femoral regions ( Figure 1 F) and the meniscus area ( Figure 1 G) also supports the same result.

[0060] Example 2 Analysis of the effect of Procr-positive cells on cartilage repair in osteoarthritis using a lineage tracing system

[0061] In order to explore the role of Procr-positive cells on the joint surface in joint injury and repair, this example used the Destabilization of the medial meniscus (DMM) surgical model to construct a traumatic osteoarthritis mouse model. This model ruptures the medial meniscus ligament of the knee joint, causing direct friction between the femur and tibia after the mouse meniscus slips, thereby inducing osteoarthritis.

[0062] 8-week-old Procr-CreERT2; tdTomato mice were intraperitoneally injected with 100 mg / kg tamoxifen for 5 consecutive days. Two days after the injection, DMM surgery was performed on the right knee joint. The left knee was used as a sham control (Sham). Samples were collected and analyzed 8 weeks after modeling ( Figure 2A). Immunofluorescence images of joint tissue sections show ( Figure 2 C), the number of tdTomato-positive chondrocytes on the tibial joint surface in the DMM group increased compared with the Sham control group (2E), and a large number of tdTomato-positive chondrocytes appeared on the femoral joint surface in the DMM group ( Figure 2 F), but there were only a few tdTomato-positive chondrocytes on the femoral joint surface in the sham control group. The number of tdTomato-positive chondrocytes in the meniscus region of the DMM group also increased compared with the sham control group ( Figure 2 G), indicating that in the case of traumatic osteoarthritis, mechanical wear stimulates the accelerated differentiation of Procr-positive cells on the joint surface into chondrocytes.

[0063] In order to explore the role of Procr-positive cells in joint injury repair, the inventors mated to obtain Procr-CreERT2; tdTomato; DTA mice. Only when CreER enters the nucleus to play a recombination role, the Stop sequence in the DTA mouse gene is deleted, and DTA is expressed in the target Procr-positive cells, inducing the death of Procr-positive cells ( Figure 2 B). Procr-CreERT2; tdTomato; DTA mice at 8 weeks old were intraperitoneally injected with tamoxifen for 5 consecutive days to achieve Procr-positive cell deletion. Two days after the injection, an osteoarthritis model was established by DMM surgery, and the joint phenotype was analyzed 8 weeks later.

[0064] After 8 weeks of in vivo tracing, no obvious tdTomato-positive cells could be observed in the articular cartilage of Procr-CreERT2; tdTomato; DTA mice ( Figure 2 D) Proving that Procr-positive cells are efficiently deleted ( Figure 2 EH ).

[0065] After the long bone joint tissues were removed and fixed with 4% PFA for 6 hours, the overall knee joint injury was observed by MicroCT scanning, and the amount of extra osteophytes on the bone edge was analyzed.

[0066] Then, 20% EDTA was used for decalcification for 2 weeks, followed by gradient alcohol dehydration, xylene clearing, and paraffin immersion. After the joint tissue was sliced ​​in paraffin, it was stained with safranin and fast green. Safranin is a positive dye that mainly binds to glycosaminoglycans (such as chondroitin sulfate) in the cartilage matrix, making the cartilage appear red or orange-red. Fast green is a negative dye that can bind to collagen fibers and other cell components, making the bone tissue appear green. First, the paraffin sections were dewaxed and rehydrated, and the sections were immersed in hematoxylin for 3 minutes, and the surface floating color was washed off with running water for about 1 minute. After absorbing the excess water on the surface, it was immersed in 0.2% fast green dye solution for staining for 1 minute. Then, it was immersed in 1% acetic acid for 3 minutes to wash away the excess floating color. Then, it was immersed in 0.5% safranin dye solution for staining for 7 minutes. After removing the safranin dye on the surface, the sections were placed in 100% anhydrous ethanol to absorb the excess safranin dye and decolorize, and rinsed 3 times. The slices were placed in xylene for decolorization and transparency, and sealed with a neutral resin containing xylene after three times. The slices were photographed after the resin was completely dry.

[0067] The observation and scoring area needs to include the following four parts of the articular surface (articular cartilage): medial femoral condyle, lateral femoral condyle, medial tibial plateau and lateral tibial plateau. The four parts are scored separately and the sum is used to get the final score. Score description:

[0068] 0 = normal;

[0069] 0.5 = Very little degeneration. Minor loss of blue (or other cationic dye) staining (loss of proteoglycan), but no structural changes;

[0070] 1 = Very little degeneration: Minor surface wear and subsurface fibrosis is visible, with no loss of chondrocytes or cartilage matrix, and the area of ​​damage is less than 5% of the total area;

[0071] 2 = Mild degeneration: The damage is vertical and below the surface of the cartilage, but rarely extends to the deeper cartilage layers. There is some loss of the cartilage surface matrix, or localized loss of chondrocytes / proteoglycans, but there is still good collagen preservation. The area of ​​cartilage damage accounts for about 5% to 10% of the total cartilage surface;

[0072] 3 = Moderate degeneration: The lesion extends vertically down to the calcified cartilage layer with focal loss of chondrocytes / proteoglycans, involving approximately 10% to 24% of the articular cartilage;

[0073] 4 = marked degeneration: vertical fissure / lesion extending beneath the calcified cartilage, exceeding 25% to 50% of the cartilage at the articular surface, or focal area loss of chondrocytes / proteoglycan involving 25% to 50% of the thickness of the articular cartilage;

[0074] 5 = Severe degeneration: vertical cracks / destructions extending below the calcified cartilage of the joint, 50% to 75% of the cartilage surface destroyed, or focal areas of chondrocyte / proteoglycan loss, approximately 50% to 75% of the cartilage thickness destroyed;

[0075] 6 = Very severe degeneration: Vertical cracks / destruction of cartilage extending below the calcified cartilage, with greater than 75% of the articular surface destroyed.

[0076] The results showed that the OARSI score of articular cartilage after deleting Procr-positive cells ( Figure 2 I and 2J) and osteophyte formation ( Figure 2 K and 2L) 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.

[0077] Example 3 Knockout of Piezo1 gene in Procr-positive cells aggravates the progression of osteoarthritis

[0078] 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 mechanical force receptors. In order to explore the regulatory mechanism of Procr-positive cells regenerating cartilage on the joint surface, the inventors obtained Piezo1 from Dr. Zou Weiguo of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. fl / fl mice, mated with Procr-CreERT2 mice to obtain Procr-CreERT2; Piezo1 fl / fl Mouse. Procr-CreERT2; Piezo1 fl / fl Mice were intraperitoneally injected with 100 mg / kg tamoxifen for 5 consecutive days, and DMM surgery was performed 2 days after the end of the injection. The progression of osteoarthritis was analyzed 8 weeks later.

[0079] Immunofluorescence images of joint tissue sections showed that Piezo1 knockout mice with DMM had fewer tdTomato-positive chondrocytes ( Figure 3 A and 3B). Histomorphometric analysis showed that Piezo1 knockout mice had a significantly lower OARSI score after DMM ( Figure 3 C and 3D) and subchondral bone thickness ( Figure 3 E and 3F) showed a significant increase in osteophytes. MicroCT analysis of osteophytes ( Figure 3 G) and calcified meniscus ( Figure 3 H) The results also showed that Piezo1 knockout mice with DMM had aggravated osteoarthritis symptoms. These data indicate that Piezo1 is required for the activation of Procr-positive cells to promote articular cartilage regeneration under pathological conditions.

[0080] Example 4 Pharmacological activation of Piezo1 can reduce the progression of osteoarthritis

[0081] Yoda1 is a specific agonist of Piezo1. In order to explore whether the activation of articular chondrocyte progenitor cells Piezo1 can slow the progression of osteoarthritis. In this example, wild-type C57 mice were used for DMM surgery to construct an osteoarthritis model. After DMM surgery, 10 μl of Yoda1 was injected into the joint cavity every week, and 40 μM, 120 μM, and 360 μM injection concentrations were designed. PBS was used as the solvent control (Vehicle), and the osteoarthritis phenotype was analyzed after 8 weeks.

[0082] Histomorphometric analysis of the slices showed that the OARSI scores of the articular cartilage of the DMM mice injected with 120 μM and 360 μM Yoda1 were significantly lower than those of the DMM mice injected with Vehicle ( Figure 4 A and 4B).

[0083] The analysis of subchondral bone thickness also showed that the subchondral bone thickness of DMM mice injected with 120 μM and 360 μM Yoda1 was significantly lower than that of DMM mice injected with Vehicle ( Figure 4 C and 4D).

[0084] Micro CT analysis showed that the injection of 360 μM Yoda1 could significantly reduce the volume of osteophytes (Figures 4E and 4F), but had no effect on the volume of calcified meniscus ( Figure 4 G and 4H). These results indicate that intra-articular injection of Yoda1 to activate Piezo1 can dose-dependently slow the progression of OA.

[0085] Example 5 Single-cell transcriptomic analysis of long bone pericytes

[0086] To characterize the stem and progenitor cell activity of Procr-positive cells in vivo, the inventors attempted to identify more cell surface protein markers in order to further enrich and isolate these cells from articular cartilage. To this end, Prrx1-Cre; tdTomato; Col2.3-GFP mice were purchased and hybridized from Jax lab. Prrx1 is a transcription factor widely expressed in the limb bud mesenchyme, so genetic lineage tracing using Prrx1-Cre; tdTomato will mark cells of all skeletal lineages in the long bones of the limbs, including periosteum cells. Col2.3-GFP mice are transgenic mice 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.

[0087] DAPI was obtained by flow cytometry from the periosteum of 8-week-old Prrx1-Cre; tdTomato; Col2.3-GFP mice - CD45 - Ter119 - Tie2 - tdTomato + GFP - of cells ( Figure 5 A). DAPI is a fluorescent dye used to mark cell nuclei. - This means that all stained dead cells are excluded, because DAPI usually does not penetrate the membrane of living cells, but will stain apoptotic or dead cells; CD45 is a commonly used leukocyte antigen used to mark blood cells. - Indicates that blood cells were excluded to avoid interference with the periosteal cell population of interest in the experiment; Ter119 is a specific erythroid marker. - Indicates that all red blood cells or red blood cell progenitor cells are excluded; Tie2 is a receptor tyrosine kinase that marks endothelial cells and is mainly expressed in vascular endothelial cells. Tie2 - Indicates that endothelial cells are excluded; tdTomato + Refers to skeletal stem cells and their derivatives marked by the Prrx1-Cre system; GFP expression is driven by the Col2.3 promoter, which is specifically expressed in differentiated osteoblasts. GFP - This means that all Col2.3-expressing osteoblasts were excluded to obtain immature or undifferentiated cell populations. Subsequently, 3' droplet-based scRNA-seq library construction and sequencing analysis were performed by 10X Genomics ( Figure 5 A).

[0088] Cluster analysis divided the patients into 9 subgroups ( Figure 5 B) The clusters were annotated by their highly expressed genes ( Figure 5C). It includes three stromal cell subpopulations (0-2) of the periosteum fibrous layer and four subpopulations (3-6) of the periosteum cambium layer. Among them, cell 0 highly expresses Fst and Gdf10, cell 1 highly expresses Pi16 and Ly6a, and cell 2 highly expresses Pdgfra and Ccl1, which are defined as stromal cell 1, stromal cell 2, and stromal cell 3, respectively. Cell 3 highly expresses Postn and Alpl, which is an osteoblast progenitor cell; cell 4 highly expresses Acan and Col2a1, which is a chondrocyte; cell 5 highly expresses Fmod and Tnmd, which is a tendon cell; cell 6 highly expresses Abi3bp and Thbs2, which is a perichondrial cell. Cell 7 highly expresses Acta2 and Myh11, which is a smooth muscle cell population. Cell 8 expresses Prg4 and Procr, which is defined as the surface cell of articular cartilage.

[0089] Slingshot is a method for analyzing the differentiation trajectory of single-cell transcriptome data. It infers the developmental path of cells by identifying the continuous states of cells. Slingshot differentiation trajectory analysis shows that there is a hierarchical differentiation relationship between the three subpopulations of the periosteum fiber layer ( Figure 5 D). In the periosteum cambium, perichondrial cells No. 6 can differentiate into osteoblast progenitor cells No. 3, growth plate chondrocytes No. 4, and tenocytes No. 5 ( Figure 5 E).

[0090] CD200 is a previously reported marker of skeletal stem cells, while CD105 is a marker protein for bone, cartilage, and matrix progenitor cells. In the single-cell transcriptome data analysis, the expression levels of the genes of interest were extracted and normalized. Violin plots were used to display the expression distribution of target genes in different cell populations. It was found that CD200 was highly expressed in the periosteum cambium (3-6) and smooth muscle cell population 7, and CD105 was highly expressed in the surface cells of articular cartilage (8). Figure 5 F). Therefore, the inventors chose to test whether CD105 could be used to further purify Procr-positive chondrogenic progenitor cells from articular cartilage.

[0091] Example 6 Procr + CD105 + Cells differentiate into cartilage in vitro and can be transplanted in situ to repair cartilage in vivo

[0092] Obtaining mouse articular cartilage Procr by flow cytometry - 、Procr + CD105 - and Procr + CD105 + cell( Figure 6A). Add growth medium to a 6-well sterile culture dish in advance and balance it in a hypoxic incubator (5% CO2, 5% O2) to make the culture medium have the same oxygen content and temperature as the incubator. Add 200 target cell suspensions collected after flow cytometry sorting to the culture dish, mix well, and return to the incubator for in vitro CFU-F clone formation experiment. After 7 days of in vitro culture, use toluidine blue to stain the cell clones and count the number of clones formed. After digestion, count the clones and calculate the average clone size ( Figure 6 B).

[0093] With Proc - Compared with cells, Procr + The number of cell-formed clones increased significantly, and the size of the clones also increased significantly ( Figure 6 C). Among them, Procr + CD105 + The cells formed the largest number of clones and the largest clone size, suggesting that they have the strongest in vitro cloning ability.

[0094] The inventors further investigated the effect of Procr + CD105 + The cells were subjected to an in vitro tri-differentiation experiment. Growth medium was added to a 10 cm sterile culture dish in advance and balanced in a hypoxic incubator (5% CO2, 5% O2) to ensure that the oxygen content and temperature of the culture medium were the same as those of the incubator. The target cell suspension collected after flow cytometry sorting was added to the culture dish and mixed well, then returned to the incubator, with the medium changed every 5 days. When the cells grew to 70% full, the culture medium was aspirated and washed once with DPBS. After adding 5 mL of TrypLE digestion enzyme, the cells were placed at 37°C for 7 min to digest them, and then resuspended by centrifugation and counted.

[0095] (1) For osteogenic differentiation, 1x10 4 The cells were placed in a 24-well plate. When the cells grew to 80%-90%, the growth medium was removed and osteogenic differentiation medium was added. The medium was changed every 3 days. After 21 days of differentiation, 10% formalin was added for fixation for 30 min, 0.2% alizarin red was added for staining for 10 min, and the floating color was washed off with ddH2O, and the cells were photographed under a microscope.

[0096] (2) For adipogenesis differentiation, 1x10 4The cells were placed in a 24-well plate. When the cells grew to 80%-90%, the growth medium was removed and adipogenic differentiation medium was added. The medium was changed every 3 days. After 7 days of differentiation, 10% formalin was added for fixation for 30 min and washed once with 60% isopropanol. 60% oil red was added for staining for 10 min, and the floating color was washed off with ddH2O, and the cells were photographed under a microscope.

[0097] (3) For chondrogenesis, 2.5x10 5 Cells were inoculated into a 15 mL centrifuge tube and centrifuged at 1000 rpm for 3 min until the tube bottom was filled with chondrogenic differentiation medium for differentiation culture. The medium was changed every 3 days. After 28 days of differentiation, 4% PFA was added for fixation for 30 min and 30% sucrose was used for dehydration. After embedding, the cells were cut on ice and stained with toluidine blue for 10 min. PBS was used to wash away the floating color and the microscope was used for photography.

[0098] The results showed that Procr + CD105 + The cells have the ability to differentiate into adipogenic, osteogenic and chondrogenic cells in vitro ( Figure 6 D).

[0099] In addition, the differentiated cells were collected in 500 μL TRIzol, the RNA of the cells was extracted, and the expression of differentiation-related genes was detected by qPCR experiments. The expression of Pparγ (adipocyte marker gene), Col1a1 (osteoblast marker gene), and Col2a1 (chondrocyte marker gene) increased significantly after differentiation, further verifying the above conclusion ( Figure 6 E).

[0100] The primer sequences used are as follows:

[0101] Pparγ-F: 5'- GAAAGACAACGGACAAATCACC-3' (SEQ ID NO: 1);

[0102] Pparγ-R: 5'- GGGGGTGATATGTTTGAACTTG-3' (SEQ ID NO: 2);

[0103] Col1a1-F: 5'-CATGTTCAGCTTTGTGGACCT-3' (SEQ ID NO: 3);

[0104] Col1a1-R: 5'-GCAGCTGACTTCAGGGATGT-3' (SEQ ID NO: 4);

[0105] Col2a1-F: 5'-CCAGGATGCCCGAAATTA-3' (SEQ ID NO: 5);

[0106] Col2a1-R: 5'-GAGGTCCTCTGGGTCCTATGAT-3' (SEQ ID NO: 6);

[0107] β-Actin-F: 5'-GCTCTTTTCCAGCCTTCCTT-3' (SEQ ID NO: 7);

[0108] β-Actin-R: 5'-CTTCTGCATCCTGTCAGCAA-3' (SEQ ID NO: 8).

[0109] In order to further explore its differentiation potential in vivo, the inventors also cultured and expanded 2.5x10 5 Mouse Procr - 、Procr + CD105 - and Procr + CD105 + Transplanted under the renal capsule. Movat staining was performed on the sections 4 weeks after transplantation ( Figure 6 F) Identify the type of tissue it is produced from by color and morphology. It is mainly used to distinguish the different components of connective tissue, including bone, cartilage and fiber. Among them, bone tissue is dense and yellow, with obvious bone morphology, cartilage tissue is blue, and fibrous tissue is sparse and light yellow.

[0110] The results showed that Procr - and Procr + CD105 - Cells tend to produce fibrous tissue, and Procr + CD105 + The cells showed an enhanced ability to generate bone and cartilage tissue ( Figure 6 G).

[0111] To further detect Procr + CD105 + The cartilage repair capacity of cells was 2×10 5 Mouse Procr in 3D spheroid culture in low-adhesion plates in vitro - 、Procr + CD105 - and Procr + CD105 + The cells were transplanted orthotopically into the joints ( Figure 6H). A 0.5 mm-diameter drill was used to drill a 0.5 mm-deep microhole on the femoral joint surface of the knee joint of 8-week-old wild-type C57 mice. The 3D spheroid cultured spheroids were then wrapped with Matrigel and transplanted into the hole. After 2 weeks, the joint sections were collected and stained with safranin fast green to detect their chondrogenic differentiation ability. Safranin is a positive dye that mainly binds to glycosaminoglycans (such as chondroitin sulfate) in the cartilage matrix, making the cartilage appear red or orange-red; the in situ chondrogenic ability was evaluated by quantitatively evaluating the proportion of cartilage stained with safranin after transplantation into the cartilage defect.

[0112] The results showed that: - and Procr + CD105 - Compared with cells, the results of safranin fast green staining showed that Procr + CD105 + Cells have stronger in situ chondrogenic ability ( Figure 6 I and 6J).

[0113] In order to detect the transformation significance of Procr-positive cells in human joint tissues, the applicant digested and sorted the total cells and Procr-positive (PROCR + )cell.

[0114] Subsequently, the cells were expanded in vitro, cultured into spheres, and orthotopically transplanted into the knee joint defect of immunodeficient NSG mice ( Figure 6 K).

[0115] Results showed that PROCR from young biopsy samples was significantly higher than that from total human chondrocytes. + The cells were able to efficiently differentiate into chondrocytes and repair joint defects in NSG mice ( Figure 6 L and 6M). More importantly, PROCR + The cells also showed superior efficiency in repairing articular cartilage defects, indicating that PROCR + The cells could serve as a source of next-generation stem or progenitor cells for treating joint diseases.

[0116] discuss

[0117] Procr + The most notable feature of cells is mechanical sensitivity. + The cells are mainly distributed in the tibial articular cartilage which bears greater mechanical load. + The number of cells peaked at 4 weeks and then gradually decreased with age, which correlated with the mice's physical activity level.

[0118] The only FDA-approved cell therapy for the treatment of full-thickness cartilage defects is MACI. This therapy isolates chondrocytes from articular cartilage, cultures 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 that primary chondrocytes have a limited proliferation capacity, and can only repair small joint defects. In contrast, stem cells or progenitor cells are considered to be an ideal source of chondrocytes due to their unlimited proliferation capacity. Therefore, Proc + Chondroprogenitor cells appear to be a better cell source than mixed articular chondrocytes for use in cell therapies such as MACI to repair larger joint defects. Another limitation of MACI is that the proliferation capacity of articular chondrocytes is impaired during aging, so it cannot be used in patients over 55 years old. In contrast, we show that Procr + Cells can effectively repair joint defects in immunodeficient mice, further indicating that Procr + Piezo1 agonist Yoda1 significantly alleviated osteoarthritis symptoms, suggesting that Procr + In vivo activation of chondroprogenitor cells may be another effective approach to prevent or delay the progression of OA.

[0119] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which 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 technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A cell preparation for alleviating or treating osteoarthritis, characterized in that: The cell preparation comprises 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 a property selected from the group consisting of: (1) Cells located on the tibial joint surface, articular cartilage surface or meniscus surface; (2) Differentiation to produce chondrocytes in the deep layer of articular cartilage and meniscus; (3) Promote cartilage repair; (4) Prevention and / or treatment of osteoarthritis.

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

5. The method according to claim 4, characterized in that: The adult mammalian tissue is selected from joint tissue.

6. The use of the cell preparation according to claim 1, characterized in that: For preventing and / or treating osteoarthritis or its related diseases.

7. Use of Procr protein in the preparation of a drug 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 preventing and / or treating osteoarthritis, characterized in that: The pharmaceutical composition comprises the cell preparation according to claim 1.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition also contains other drugs for preventing and / or treating 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 on the targeting agent, and the targeting agent targets the Procr protein.

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