PDGFRA < + > mesenchymal stem cells as well as identification and identification, sorting and culture methods and application thereof

Abstract

The invention is applicable to the technical field of biological medicine, and provides PDGFRA < + > mesenchymal stem cells as well as identification, sorting and culture methods and application thereof.A new dental developmental mesenchymal stem cell subset PDGFRA < + > MSCs is screened on the basis of single cell sequencing, in-vitro magnetic bead sorting and cell subset verification are performed through a marker gene, and under the serum-free and PDGFBB treatment conditions, the PDGFRA < + > mesenchymal stem cells can be obtained. The PDGFRA + MSCs with good multiplication capacity, osteogenic differentiation capacity and endothelial cell migration and lumen formation promoting capacity are obtained. The adult tooth developmental mesenchymal stem cells identified and screened by the invention can provide a more sufficient seed cell source for regeneration and transformation application based on human dental stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology and in particular to a PDGFRA + Mesenchymal stem cells and their identification, sorting, culture methods and applications. Background Art

[0002] Dental and maxillofacial diseases, cardiovascular diseases, and cancer are listed by the World Health Organization as the "three major non-communicable diseases that need to be prevented and treated". Maxillofacial trauma, tumors, infections, congenital malformations, etc. often lead to tooth loss / missing and jaw bone defects, which have a high incidence rate. They not only seriously damage the physiological functions of the oral and maxillofacial region, but also seriously affect the patient's psychological and overall health, significantly reducing the quality of life. Therefore, exploring the effective regeneration of periodontal tissues has become an important topic in the field of stomatology.

[0003] During tooth development, the formation of tooth germs originates from cranial neural crest cells. After undergoing epithelial-mesenchymal transformation, the mesenchyme differentiates into dental follicles and dental papillae under the induction of the epithelium. After the cap stage, bud stage, and bell stage, it gradually develops and differentiates into dental tissues such as dental pulp and periodontal tissues. At present, in the field of tissue engineering regeneration, guiding tissue regeneration according to the principles of organ development has become a key research direction. However, the cell composition of tooth development tissues is highly heterogeneous, and cells with different markers are also heterogeneous in function. However, a large number of current studies are based on the commonly used mesenchymal stem cells for tissue regeneration applications. The definition of dental mesenchymal stem cells lacks specificity, and there is a lack of clear research on the regulation of developmental regeneration mechanisms under complex physiological microenvironments. Therefore, screening and defining a group of dental developmental mesenchymal progenitor cells from dental developmental tissues and isolating, obtaining, and continuously culturing them has become the focus of research.

[0004] In particular, whether a specific group of dental mesenchymal stem cells with strong periodontal regeneration ability can be discovered is crucial to improving the effect of stem cells in periodontal tissue regeneration therapy, thereby providing more potential seed cells for the effective regeneration of periodontal defects in clinical practice. Summary of the invention

[0005] The purpose of the present application embodiment is to provide a PDGFRA + Mesenchymal stem cells and their identification, sorting, culture methods and applications, aiming to obtain PDGFRA with good proliferation ability, osteogenic differentiation ability, endothelial cell migration and tube formation ability + Mesenchymal stem cells provide a more sufficient source of seed cells for regenerative transformation applications based on human dental and maxillofacial stem cells.

[0006] The embodiment of the present application is implemented as follows: a PDGFRA + Methods for identifying and authenticating mesenchymal stem cells include:

[0007] Preparing a single cell suspension of tooth developmental tissue; the tooth developmental tissue includes dental follicle tissue and apical dental papilla tissue surrounding the mandibular third molar;

[0008] The single cell suspension is labeled with gel microbeads containing unique barcodes, further mixed with oil to form water-in-oil droplets, full-length cDNA is produced by reverse transcription reaction, and the emulsion is destroyed by a recovery agent, and the cDNA is cleaned by magnetic beads. After the amplified cDNA is further modified, a single cell RNA sequencing library is constructed;

[0009] Through single-cell transcriptome sequencing and bioinformatics analysis, PDGFRA, which has a common high expression of odontogenic and osteogenic protein genes, was identified. + Mesenchymal stem cells.

[0010] The present application embodiment also provides a PDGFRA + Methods for isolating mesenchymal stem cells, including:

[0011] Preparing a single cell suspension of tooth developmental tissue; the tooth developmental tissue includes dental follicle tissue and apical dental papilla tissue surrounding the mandibular third molar;

[0012] The single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, the resulting cells are combined with positive selection beads, washed, and placed in a magnetic field to sort out PDGFRA + Mesenchymal stem cells.

[0013] The present application embodiment also provides a PDGFRA + Mesenchymal stem cells, the PDGFRA + Mesenchymal stem cells are expressed by the PDGFRA + Mesenchymal stem cells are obtained by sorting.

[0014] The present application also provides a method for maintaining PDGFRA + The method for culturing mesenchymal stem cells for stable expression comprises:

[0015] The PDGFRA + Mesenchymal stem cells were cultured until the cells adhered to the wall, then the medium was changed to serum-free medium for continued culture, and characteristic gene expression was verified when the cell fusion rate reached 90%.

[0016] Preferably, PDGFBB is supplemented into the serum-free medium at a concentration of 100 ng / mL.

[0017] The present application also provides a PDGFRA+ Application of mesenchymal stem cells in the preparation of drugs for the treatment of periodontal tissue defects.

[0018] The present application also provides a drug for treating periodontal tissue defects, the drug comprising the above-mentioned PDGFRA + Mesenchymal stem cells, and pharmaceutically acceptable excipients and carriers.

[0019] The present application embodiment provides PDGFRA + Methods for identification and sorting of mesenchymal stem cells and maintenance of PDGFRA + A method for culturing mesenchymal stem cells that stably express PDGFRA. This application is based on single-cell sequencing to screen for a new subpopulation of tooth developmental mesenchymal stem cells + MSCs were sorted by magnetic beads in vitro and cell subpopulations were verified by marker genes. Under serum-free and PDGFBB treatment conditions, PDGFRA with good proliferation ability, osteogenic differentiation ability, and the ability to promote endothelial cell migration and tube formation was obtained. + MSCs. The adult tooth developmental mesenchymal stem cells identified and screened in this application will provide a more sufficient source of seed cells for the regenerative transformation application based on human dental and mandibular stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the process of preparing single cell suspension of human dental follicle tissue and apical papilla tissue and single cell sequencing provided in Example 1 of the present application;

[0021] Figure 2 This is a result analysis diagram of single-cell transcriptome sequencing provided in Example 1 of the present application;

[0022] Figure 3 This is a result analysis diagram of PDGFRA labeling of mesenchymal progenitor cells common in the dental follicle and apical dental papilla provided in Example 1 of the present application;

[0023] Figure 4 This is a verification diagram of the expression of PDGFRA in dental follicle tissue provided in Example 1 of the present application;

[0024] Figure 5 The microscopic images of DFSCs under the culture conditions of hypoxia with serum, hypoxia without serum, normoxia with serum, and normoxia without serum and the detection result image of PDGFRA expression provided in Example 2 of the present application;

[0025] Figure 6 The unsorted group and PDGFRA provided in Example 3 of the present application + Group, PDGFRA - Figure 2. Proliferation ability and cell cycle detection results of dental mesenchymal stem cells (DFSCs) in the group;

[0026] Figure 7 The unsorted group and PDGFRA provided in Example 3 of the present application + Group, PDGFRA - Figure 2 shows the results of osteogenic ability test of dental mesenchymal stem cells (DFSCs).

[0027] Figure 8 The unsorted group and PDGFRA provided in Example 3 of the present application + Group, PDGFRA - The results of the indirect co-culture of culture supernatant of dental mesenchymal stem cells (DFSCs) and endothelial cells on the migration and tube-forming ability of endothelial cells.

[0028] Fig. 9 Under the PDGFBB treatment conditions provided in Example 3 of this application, the unsorted group, PDGFRA + Group, PDGFRA - Figure 2. Proliferation ability and cell cycle detection results of dental mesenchymal stem cells (DFSCs) in the group;

[0029] Fig.10 Under the PDGFBB treatment conditions provided in Example 3 of this application, the unsorted group, PDGFRA + Group, PDGFRA - Figure 2. Detection results of osteogenic ability of dental mesenchymal stem cells (DFSCs) in the group;

[0030] Fig.11 This is a graph showing the effect of indirect co-culture of culture supernatant of dental mesenchymal stem cells (DFSCs) treated with PDGFBB and endothelial cells on the migration and tubular formation ability of endothelial cells as provided in Example 3 of the present application. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0032] The present application embodiment provides a PDGFRA + Methods for identifying and authenticating mesenchymal stem cells include:

[0033] Preparation of single cell suspensions of tooth developmental tissues;

[0034] The single cell suspension is labeled with gel microbeads containing unique barcodes, further mixed with oil to form water-in-oil droplets, full-length cDNA is produced by reverse transcription reaction, and the emulsion is destroyed by a recovery agent, and the cDNA is cleaned by magnetic beads. After the amplified cDNA is further modified, a single cell RNA sequencing library is constructed;

[0035] Through single-cell transcriptome sequencing and bioinformatics analysis, PDGFRA, which has a common high expression of odontogenic and osteogenic protein genes, was identified. + Mesenchymal stem cells.

[0036] The tooth developmental tissue includes dental follicle tissue and apical papilla tissue around the mandibular third molar that needs to be extracted due to impaction; taking dental follicle tissue as an example, the group of human dental follicle stem cells are sorted in vitro according to marker genes, cultured in vitro, and their proliferation ability, osteogenic differentiation ability, and ability to promote endothelial cell migration and lumen formation are detected. Although the present application only takes dental follicle tissue as an example, it should not be used to limit the scope of protection of the present application.

[0037] Furthermore, the present application embodiment also provides a method for in vitro magnetic bead sorting of PDGFRA based on characteristic genes. + A method for producing DFSCs comprising the following steps:

[0038] Preparing a single cell suspension of tooth developmental tissue; the tooth developmental tissue includes dental follicle tissue and apical dental papilla tissue surrounding the mandibular third molar;

[0039] The single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, and the resulting cells are combined with positive selection beads and placed in a magnetic field to separate PDGFRA + Mesenchymal stem cells.

[0040] Alternatively, the positive selection beads used for sorting can be streptavidin magnetic beads, CytoSinct TM Nanoscale cell magnetic beads, etc.

[0041] Optionally, the step of preparing a single cell suspension of tooth developmental tissue comprises:

[0042] The tooth developmental tissue was digested with 0.02% type I collagenase at 37°C for 45 min to 1 h, and then the digestion was terminated by an equal amount of 10% to 20% α-MEM medium, and then centrifuged at 1000 rpm for 10 min to obtain a cell pellet;

[0043] The tooth developmental tissue and cell precipitate were resuspended in 20% α-MEM culture medium and then inoculated into a culture device. When the cell fusion rate reached 90%, digestion and passage were performed to obtain a single cell suspension.

[0044] Optionally, the single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, the resulting cells are combined with positive selection beads, and placed in a magnetic field to sort out PDGFRA + The steps of mesenchymal stem cells include:

[0045] The single cell suspension was resuspended in PBS containing 3% to 5% serum to adjust the cell concentration to 1×10 6 100uL, add 2uL of CD140a biotinylated antibody to each system, incubate at room temperature for 10min, wash with PBS containing 3% to 5% serum, discard the supernatant after centrifugation, add 10μL of avidin positive selection beads to the obtained cells, place them in a magnetic field, stand at room temperature for 5min, and sort out PDGFRA + Mesenchymal stem cells.

[0046] The present application embodiment also provides a PDGFRA + Mesenchymal stem cells, the PDGFRA + Mesenchymal stem cells are expressed by the PDGFRA + Mesenchymal stem cells are obtained by sorting.

[0047] The present application also provides a method for maintaining the above-mentioned PDGFRA + The method for culturing mesenchymal stem cells for stable expression comprises:

[0048] The PDGFRA + Mesenchymal stem cells were cultured until the cells adhered to the wall, then the medium was changed to serum-free medium for continued culture, and characteristic gene expression was verified when the cell fusion rate reached 90%.

[0049] Optionally, the serum-free medium is an α-MEM medium without FBS; the culture conditions are: 37°C, 5% CO 2 .

[0050] Preferably, PDGFBB is supplemented into the serum-free medium at a concentration of 100 ng / mL. + Mesenchymal stem cells can enhance the migration and tube-forming ability of endothelial cells.

[0051] The following specific examples are used to describe PDGFRA. + Mesenchymal stem cells and their identification, sorting, culture methods and applications are described in detail as follows. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are all commercially available unless otherwise specified.

[0052] Example 1: PDGFRA + Methods for identifying mesenchymal stem cells, such as Figure 1 The single-cell suspension preparation and single-cell sequencing workflow of human dental follicle tissue and apical papilla tissue are shown.

[0053] (1) Acquisition of dental follicle tissue and apical papilla tissue

[0054] All tissue donors were patients of the School of Stomatology, Air Force Medical University, with approval from the Medical Ethics Committee of the Air Force Medical University Stomatological Hospital (approval number: IRB-REV-2022187). After obtaining informed consent from the patients, dental follicle tissue was collected according to the following inclusion and exclusion criteria: patients aged between 18 and 22 years, no history of pain or infection in the third molars, and healthy surrounding tissues. The dental papilla was obtained from the roots of the undeveloped third molars with open apical foramina. All patients underwent oral panoramic radiographs before surgery, and third molars with abnormal low-density shadows around them were excluded. The third molars with attached dental follicle or apical papilla tissue were immersed in 10% α-minimal essential medium (α-MEM; 12571-048, Invitrogen, USA) and immediately transferred to the Tissue Engineering Center of the Air Force Medical University Stomatological Hospital in an ice box.

[0055] a. Take out the tooth in the clean bench and place it in a culture dish. After repeatedly rinsing it with PBS, transfer it to another culture dish. Use ophthalmic scissors to separate the soft tissue around the tooth to obtain the dental follicle or the soft tissue outside the apical foramen to obtain the dental papilla.

[0056] b. Rinse the dental sac or dental papilla repeatedly with PBS until there is no obvious blood stain on the surface and then transfer it to another culture dish. Use ophthalmic scissors to cut it into pieces of about 2 to 4 mm. 3 The tissue pieces were washed 1 to 2 times with phosphate-buffered saline (PBS; P5493, Sigma-Aldrich, USA);

[0057] c. Collect the chopped dental follicle tissue or dental papilla tissue into a 1.5 mL cryopreservation tube, resuspend it in 1 ml of freezing solution (FBS:DMSO=9:1), let it stand at room temperature for 10 min, then transfer it to a gradient cooling cryopreservation box and store it in a -80°C refrigerator.

[0058] (2) Dissociation and sequencing of single cells from tooth developmental tissues

[0059] a. 0.02% collagenase type I (17018029, Gibco, USA) was used to dissociate the collected tissues into single cells;

[0060] b. Cells were barcoded by 10× gel beads and encapsulated in oil to form gel beads-cell complexes (GEMs);

[0061] c. A reverse transcription reaction is then performed to generate full-length cDNA, and a recovery agent is used to destroy the emulsion, and magnetic beads are used for cDNA cleaning, and the amplified cDNA is further modified;

[0062] d. Single-cell RNA sequencing (scRNA-seq) libraries were constructed using the Single Cell 3' Kit v3 (1000268, 10× Genomics, USA), and sequencing was performed on the IlluminaNova 6000PE150 platform (Illumina, USA);

[0063] e. Quality control was performed using Cell Ranger (version 7.0.1) software (10×Genomics, USA), and read comparison was performed by aligning the spliced ​​transcripts to the reference genome (STAR ​​aligner). Cells that did not meet the criteria were discarded: 1) the number of genes > 200, unique multiple barcode index (UMI) > 1000, and log10GenesPerUMI > 0.7; 2) UMI of mitochondrial genes < 15%, UMI of hemoglobin genes < 5%;

[0064] f. To mitigate batch effects, mutual nearest neighbor (MNN) analysis was performed; the cells were then clustered using the MNN clustering algorithm and visualized using the 2D uniform manifold approximation and projection (UMAP) algorithm.

[0065] Figure 2 This is the result analysis diagram of single-cell transcriptome sequencing; Figure 2 A is a UMAP image showing 9997 cells in the dental follicle tissue and 9,048 cells in the apical papilla tissue colored according to 9 cell populations; Figure 2 B is the integrated UMAP map of the distribution of dental follicle and dental papilla cell populations; Figure 2 C is a bar graph of the relative abundance of different cell types in dental follicle and dental papilla tissues; Figure 2 D is the composition ratio of different cell populations in dental follicle and dental papilla tissue. Figure 2It can be seen that cells were classified into 9 different cell populations by specific markers, including dental follicle stem cells (DFSCs), apical dental papilla stem cells (SCAP), endothelial cells (ECs), Schwann cells, smooth muscle cells (SMCs), T cells, B cells, plasma cells and macrophages; DFSCs and various immune cells were significantly enriched in the dental follicle, while SCAP and Schwann cells, ECs and SMCs were enriched in the dental papilla. In fact, both tissues contain significant stem cell populations (DFSCs and SCAP) as well as abundant ECs and SMCs, and in particular, the dental follicle contains a high proportion of T cells.

[0066] (3) Identification and characterization of PDGFRA-labeled dental mesenchymal progenitor cells

[0067] a. The top 100 genes of DFSCs and SCAP were analyzed and the differentially expressed genes and the intersection gene sets between the two were displayed in the form of Venn diagrams, using the OECloud tool at https: / / cloud.oebiotech.com;

[0068] b. The bubble diagram and feature diagram of genes are performed on the OECloud tool at https: / / cloud.oebiotech.com;

[0069] c. After integrated analysis of mesenchymal stem cell populations (DFSCs and SCAP), their gene expressions were sorted by gene_diff.

[0070] Figure 3 This is the result analysis of PDGFRA labeling of mesenchymal progenitor cells common in dental follicle and apical dental papilla; Figure 3 A is a Venn diagram of gene expression of dental follicle stem cells and apical papilla stem cells; Figure 3 B is a bubble chart showing the expression of genes related to tooth development in dental sac and dental papilla cell populations; Figure 3 C is a table sorting the gene expression of mesenchymal stem cell populations (DFSCs and SCAP) by gene_diff; Figure 3 D is the visualization characteristic diagram of PDGFRA in dental follicle and dental papilla cells. Figure 3 It can be seen that this application found that a total of 1,275 genes were co-expressed in these two stem cell types, DFSCs and SCAPs. The gene expression visualization results showed that PDGFRA was specifically highly expressed in these two groups of cells (gene_diff: 19.811). At the same time, these two groups of cells also expressed tooth development-related genes, such as WNT10A, MSX1, PAX9, RUNX2, FGFR1 and LTBP3.

[0071] (4) Immunofluorescence staining in dental follicle tissue to verify PDGFRA expression

[0072] a. Collect dental follicle tissue according to the inclusion and exclusion criteria described in (1) and fix it directly in 4% paraformaldehyde solution for 24 hours;

[0073] b. After removal, rinse with PBS three times, 5 minutes each time;

[0074] c. Dehydrate the tissue in 30% sucrose solution for 12 to 16 hours;

[0075] d. After dehydration, embed with OCT embedding medium, and cut 10 μm thick frozen sections of dental follicle tissue after OCT is completely solidified;

[0076] e. Before staining, wash the slides three times with PBS, permeabilize the membrane with 0.3% Triton for 15 min, wash three times with PBS, and block with goat serum for 30 min;

[0077] f. After discarding the goat serum, the primary antibodies Runx2, MSX1, and PAX9 were diluted with PDGFRA in proportion and mixed, and then incubated at 4°C in the dark for 12 to 16 hours;

[0078] g. After discarding the primary antibody, wash with PBS three times, add the secondary antibody embedded with FITC fluorescent group according to the corresponding primary antibody species, and incubate at room temperature in the dark for 1 hour.

[0079] h. After discarding the secondary antibody, wash three times with PBS, stain the cell nucleus with DAPI containing mounting medium, and mount the slides.

[0080] i. Fluorescence images were captured using a CLSM (Nikon, Japan) confocal microscope and analyzed using NIS-Elements Viewer 4.50 software.

[0081] Figure 4 A is the immunofluorescence staining of RUNX2, MSX1 or PAX9 co-localized with PDGFRA in dental follicle tissue, scale bar = 200 μm. Figure 4 As shown in A, immunofluorescence staining confirmed the expression of PDGFRA in tooth developmental progenitor cells marked by RUNX2, MSX1, and PAX9.

[0082] (5) Flow cytometry verification of PDGFRA expression in dental follicle tissue

[0083] a. Collect the dental follicle tissue fragments according to the method described in (1), transfer them to a 15 mL centrifuge tube, add type I collagenase to immerse the tissue fragments, and digest them in a 37°C incubator in the dark for 45 min to 1 h, shaking every 5 to 10 min to ensure that the tissue is fully digested;

[0084] b. Add an equal amount of 10% α-MEM to terminate digestion;

[0085] c. Centrifuge at 1000r / min for 5min and discard the supernatant;

[0086] d. Use the piston end of a 10 mL syringe to squeeze and grind the remaining tissue cell pellet, pass it through a 100 μm metal filter to decompose it into a single cell suspension, and collect it in a 15 mL centrifuge tube;

[0087] e. Centrifuge at 800 rpm for 5 min and discard the supernatant;

[0088] f. Add 1 mL of α-MEM medium containing 10% serum to resuspend and count;

[0089] g. The cells were stained with anti-human PDGFRA-PE antibody (5 μL / test, sc-398206PE, Santa Cruz Biotechnology, USA) and incubated at 4°C in the dark for 1 hour before being analyzed using a flow cytometer (CytoFLEX, Beckman, USA).

[0090] Figure 4 B is the flow cytometry analysis of PDGFRA-positive cells in isolated dental follicle tissue cells and cultured dental stem cells (DFSCs). Figure 4 B As shown in the left figure, PDGFRA in dental follicle tissue + The proportions of cells were basically consistent with the theoretical predictions based on bioinformatics analysis.

[0091] (6) Flow cytometry analysis of cultured dental follicle stem cells

[0092] a. Collect dental follicle cells according to the method described in (5) a to c, add 1 mL of α-MEM medium (containing 10% FBS, 100 U / mL penicillin and streptomycin) and resuspend;

[0093] b. Inoculate the resuspended cells in a 25cm 2 Add 4 mL of culture medium to the culture bottle and place it in CO 2 Culture in a 37°C incubator with a saturation of 5% to allow tissue blocks to adhere to the wall, and change the medium after 7 days;

[0094] c. Subculture when the cell fusion rate in the culture flask reaches more than 90%;

[0095] d. Discard the original culture medium and rinse twice with PBS;

[0096] e. Add 2 mL of 2.5% trypsin and digest in a 37°C incubator for 1 to 2 minutes;

[0097] f. Observe under an inverted microscope. When the cells shrink into a round shape, add an equal amount of culture medium to stop digestion, and mix with a pipette to obtain a single cell suspension.

[0098] g. Centrifuge at 800 r / min for 5 min;

[0099] h. Discard the supernatant, add culture medium and repeatedly pipette to resuspend the cells, and count;

[0100] i. The cells were stained with anti-human PDGFRA-PE antibody (5 μL / test, sc-398206PE, Santa Cruz Biotechnology, USA) and incubated at 4°C in the dark for 1 hour before analysis using a flow cytometer (CytoFLEX, Beckman, USA).

[0101] The results are as follows Figure 4 B As shown in the right figure, PDGFRA + DFSCs were still detectable, but the proportion of PDGFRA-positive cells was less than 5%.

[0102] (7) Immunofluorescence staining of cultured dental follicle stem cells to verify PDGFRA expression

[0103] a. Obtain DFSC (P1) cell resuspension according to method (6) a to h, and add 1×10 4 / well density was inoculated in a 24-well plate with cell slides pre-placed;

[0104] b. When the cell confluence rate reaches 80%, wash twice with PBS and then fix with 4% PFA at room temperature for 30 minutes;

[0105] c. The fixed cells were permeabilized with 0.5% Triton X-100 (X100PC, Sigma-Aldrich, USA) for 15 min;

[0106] d. The cells were blocked with 5% bovine serum albumin (BSA; 0881066-CF, MP Biomedicals, USA) for 30 minutes at room temperature;

[0107] e. Use the primary antibody PDGFRA (sc-398206PE, Santa Cruz Biotechnology, USA; dilution 1:100) embedded with PE fluorescent group for staining and incubate at 4°C for 12-16h;

[0108] f. Discard the primary antibody, wash three times with PBS, add 10 μL of DAPI containing mounting medium on the slide, use a pre-bent 1mL syringe needle and tweezers to take out the cell slide and turn it upside down on it to stain the nucleus (ab104139, Abcam, UK);

[0109] g. Cell fluorescence images were collected under a fluorescence microscope (Olympus, Japan).

[0110] Figure 4 C is a positive immunofluorescence staining image of PDGFRA in cultured dental mesenchymal stem cells (DFSCs), scale bar = 100 μm. Figure 4 As shown in C, PDGFRA is also expressed on the cell membrane surface of subcultured DFSCs. In summary, PDGFRA can mark the common odontogenic progenitor cells in DFSCs and SCAPs.

[0111] Example 2 Maintaining the expression of PDGFRA in dental mesenchymal stem cells under serum-free culture conditions

[0112] In the verification process of Example 1, it was found that compared with the proportion of PDGFRA-positive cells in the tissue flow cytometry results, the expression of PDGFRA in DFSCs after subculture was reduced. In order to maintain the expression of PDGFRA in dental mesenchymal stem cells, the following experiments were performed:

[0113] a. Dental mesenchymal stem cells (DFSCs) were subcultured and cultured at 1×10 5 The cells were inoculated at a density of / well in a 6-well plate. The hypoxia group with serum, the hypoxia group without serum, the normoxic group with serum, and the normoxic group without serum were set up respectively. The hypoxia group was placed in 3% O 2 , 5% CO 2 The normoxic group was placed in a 20% O 2 , 5% CO 2 The serum group was cultured in α-MEM medium containing 10% FBS, and the serum-free group was cultured in α-MEM medium without FBS.

[0114] b. After 48 hours of culture, the cell fusion rate reached 90%, and cell photos were collected under a light microscope;

[0115] c. Wash twice with PBS, add 1 mL of MIsZOL reagent (MI00617, MisZOL Biotechnology, China) to each well to extract total cellular RNA;

[0116] d. 5× PrimeScript RT MasterMix (RR036A-1, Takara, Japan) was used for cDNA synthesis, and reverse transcription was performed on a PCR amplifier (EasyCycler, Germany);

[0117] e.qPCR using TB Premix Ex Taq (RR420A, Takara, Japan) was used, and gene expression detection was performed using a real-time system (Bio-Rad, USA);

[0118] The primer sequences (5'-3') are as follows:

[0119]

[0120] f. CFX Manager software (Bio-Rad, USA) was used for result analysis.

[0121] Figure 5 Figure 2 is the expression of PDGFRA in DFSCs under serum-free culture conditions, where Figure 5 A is the light microscopic image of dental mesenchymal stem cells (DFSCs) cultured under hypoxia with serum, hypoxia without serum, normoxia with serum, and normoxia without serum, scale bar = 250 μm; Figure 5 B is a statistical chart of PDGFRA expression under four culture conditions detected by qRT-PCR. Figure 5 As shown, under light microscopy, the cell growth status under hypoxic and normoxic culture conditions was basically the same, while the cell fusion rate in the serum group was higher than that in the serum-free group. The qRT-PCR results indicated that hypoxic culture conditions had no significant effect on the expression of PDGFRA in dental mesenchymal stem cells, while PDGFRA expression increased under serum-free culture conditions, and there was no significant difference in the expression of PDGFRA between hypoxic and normoxic conditions.

[0122] Example 3 PDGFRA + Detection of characteristics of dental mesenchymal stem cells

[0123] (1) Magnetic bead separation of PDGFRA + Dental mesenchymal stem cells

[0124] a. Dental follicle stem cells (P1) after passage are used to isolate PDGFRA + Dental mesenchymal stem cells.

[0125] b. Digest with 0.02% collagenase type I at 37°C for 1 h, terminate digestion with an equal amount of 10% α-MEM, and centrifuge at 1000 rpm for 10 min to obtain a precipitate; resuspend the tissue and cell precipitate in 20% α-MEM and inoculate into 25 cm 2 When the cell fusion rate in the culture flask reaches about 90%, the cells are digested and passaged; the cell suspension obtained by digestion is resuspended in PBS containing 3% serum to adjust the cell concentration to 1×10 6100ul, add 2uL of CD140a biotinylated antibody (13-1401-80, Thermofisher, USA) to each system, incubate at room temperature for 10min, wash once with PBS containing 3% serum, and discard the supernatant after centrifugation.

[0126] c. Mix cells with 10uL MagniSort TM Streptavidin positive selection beads (MSPB-6003-74, Thermofisher, USA) were bound.

[0127] d. After washing with PBS separation buffer containing 3% FBS, the cells were placed in a magnetic field and allowed to stand at room temperature for 5 minutes, after which they were poured out to obtain negative cells; the cells obtained in the flow tube were positive cells.

[0128] (2) Detection of DFSCs clone formation ability

[0129] Take the unsorted group of P2 generation in the logarithmic growth phase, PDGFRA + , PDGFRA - DFSCs were grouped at 1×10 3 The cells were inoculated at a density of 1000 cells / well in a 6-well plate and shaken to evenly distribute the cells. After the cells adhered to the wall, the culture medium was replaced every 3 days. After 7 to 12 days of culture, when the diameter of each clone colony was about 1 cm and they were not connected to each other, they were stained with crystal violet staining solution. The specific steps are as follows:

[0130] a. Wash with PBS 2-3 times;

[0131] b. Add 1 mL of 4% paraformaldehyde for fixation and discard after fixation for 30 minutes;

[0132] c. Wash twice with PBS;

[0133] d. Add 1 mL of crystal violet stain, incubate for 20 min, and then discard;

[0134] e. Wash twice with PBS and take pictures under light microscope;

[0135] f. Image J was used to measure and analyze the clone diameter.

[0136] Figure 6 A and Figure 6 B The left figure shows the results and statistical graphs of three groups of clone formation experiments (scale bar = 250 μm); Figure 6 A and Figure 6 As shown in the left figure of B, the three groups of DFSCs obtained after sorting have the ability to form clones and good cell proliferation ability. After being seeded at low density, the cells grow in colony-like manner, but the clone-forming ability of the three groups of DFSCs does not show obvious differences.

[0137] (3) EdU detection of DFSCs proliferation ability

[0138] The proliferation capacity of DFSCs was determined using the EdU cell proliferation detection kit. The sterilized cell slides were placed in the bottom of a 24-well plate and moistened with 10% α-MEM medium at a rate of 1 × 10 cells / well. 4 The cells were inoculated at a density of 100 cells per well and their proliferation capacity was detected after 48 h.

[0139] The specific steps are as follows:

[0140] a. Dilute the EdU solution with complete culture medium (a-MEM culture medium, 100U / mL penicillin and streptomycin) to adjust the EdU concentration to 10-20μM;

[0141] b. Add 200 μL of diluted EdU working solution to each well and incubate for 48 hours;

[0142] c. Remove the EdU working solution and wash twice with PBS, 3 to 5 minutes each time;

[0143] d. Add 200 μL 4% paraformaldehyde to each well and fix at room temperature for 30 minutes;

[0144] e. Remove the paraformaldehyde and add 200 μL of 2 mg / mL glycine solution (ddH 2 O preparation), neutralize the residual fixative, and incubate at room temperature for 5 min;

[0145] f. Remove the glycine solution and add 400 μL of 3% BSA (prepared in PBS) to each well and wash twice, each time for 3 to 5 minutes;

[0146] g. Remove the washing solution, add 400 μL of 0.5% TritonX-100 (prepared in PBS) permeabilization agent to each well, and incubate at room temperature for 10 min;

[0147] h. Remove the permeation-enhancing solution and add 400 μL 3% BSA washing solution to each well and wash twice;

[0148] i. Remove the washing solution and add 200 μL Click-iT reaction mixture staining solution (prepared according to the instructions) to each well, and incubate at room temperature in the dark for 30 minutes;

[0149] j. Remove the staining solution, add 200 μL 3% BSA washing solution to each well and wash twice, retaining the last washing solution;

[0150] k. Pipette 10 μL of DAPI containing mounting medium onto the slide, pick up the slide with tweezers, and place the slide with the cell surface facing down on the DAPI, being gentle to avoid bubbles;

[0151] l. Observe under an inverted fluorescence microscope.

[0152] Figure 6 AThe following figure and Figure 6 B right figure shows the results of three groups of EdU experiments (scale bar = 50μm) and statistical chart results. Figure 6 AThe following figure and Figure 6 As shown in the right figure of B, the results of the EdU proliferation experiment showed that the proliferation status of DFSCs in the three groups was good, with no statistical difference.

[0153] (4) Cell cycle gene detection

[0154] a. The three groups of cells were divided into 1×10 5 / well density was inoculated in a 6-well plate, and total cellular RNA was extracted when the cell fusion rate reached 90%;

[0155] b. The same as steps c to f of Example 2, wherein the primer sequences (5'-3') are as follows:

[0156]

[0157]

[0158] Figure 6 C is a statistical diagram of the expression of three groups of cell cycle-related genes detected by qRT-PCR. Figure 6 As shown in C, the cell cycle status of the three groups was basically the same, with no statistical difference.

[0159] (5) Detection of osteogenic differentiation ability of three groups of DFSCs

[0160] a. Preparation of osteogenic induction solution: weigh 0.765 g of sodium β-glycerophosphate into a 15 mL centrifuge tube, pipette 10 mL of α-MEM medium (containing 7% to 10% FBS by volume, 100 U / mL penicillin and streptomycin) into the centrifuge tube, mix well to fully dissolve the sodium β-glycerophosphate, filter with a 0.22 μm filter, add the filtrate, 0.417 mL of VC (30 mg / mL) and 50 μL of dexamethasone (0.02 mg / mL) prepared in advance into 240 mL of α-MEM medium, and store at 4°C in the dark;

[0161] b. DFSCs were seeded into 6-well culture plates at a seeding density of 1×10 5 100 cells / well, add 2 mL of 10% α-MEM medium to each well, and place in a cell culture incubator for culture; when the cells grow and fuse to 70% to 80%, replace the osteogenic differentiation medium, and replace the osteogenic induction medium every 3 days. After continuous culture for 14 days, perform alizarin red staining;

[0162] c. Washing and fixation: remove the original culture medium, wash twice with PBS, and add 1 mL of 4% paraformaldehyde fixative to each well for 30 minutes at room temperature;

[0163] d. Washing: remove the fixative and wash with ddH 2 O wash 2 times;

[0164] e. Staining: Add 1 mL of 1% Alizarin Red staining solution to each well and stain for 10 to 20 minutes at room temperature;

[0165] f. Washing: Remove the staining solution and wash with ddH 2 Wash thoroughly until the staining solution is completely washed away;

[0166] g. Observe the formation of calcium nodules under an inverted optical microscope.

[0167] Figure 7 A. Figure 7 B shows the ARS staining results and statistical graphs of the three groups after 14 days of osteogenic induction, scale bar = 100 μm. Figure 7 A. Figure 7 As shown in the left figure of B, the three groups of DFSCs were stained with Alizarin red after 14 days of osteogenic induction culture. Dark red mineralized nodules were visible under an inverted optical microscope, and there was no statistical difference in osteogenic ability.

[0168] (6) Detection of ALP activity in the supernatant of osteogenic induction fluid

[0169] a. Collect the culture medium of each group of cells after 14 days of osteogenic induction culture, and centrifuge at 1000-1500 rpm / min for 10 minutes;

[0170] b. Take the supernatant for determination;

[0171] c. ALP activity detection kit (A059-2) provided by Nanjing Jiancheng Bioengineering Research Institute was used to detect ALP activity according to the manufacturer's instructions. The absorbance of each well was measured at 520 nm.

[0172] Figure 7 C is a statistical chart showing the results of ALP activity detection in the culture supernatant of the three groups after 14 days of osteogenic induction. Figure 7 As shown in C, there was no statistical difference in the ALP activity in the culture supernatant of the three groups of DFSCs after 14 days of osteogenic induction.

[0173] (7) Osteogenesis induction gene detection

[0174] a. After discarding the culture medium, wash twice with PBS;

[0175] b. Perform qRT-PCR detection according to the method described in experimental steps c to f of Example 2.

[0176] The primer sequences are as follows:

[0177]

[0178] Figure 7 D is the statistical graph of osteogenic-related gene expression in three groups of cells detected by qRT-PCR after 14 days of osteogenic induction. Figure 7 As shown in D, there was no statistical difference in the expression of osteogenic-related genes in the three groups of DFSCs after 14 days of osteogenic induction.

[0179] (8) Detection of osteogenic protein expression

[0180] 1) Protein extraction after osteogenic induction

[0181] a. After 14 days of osteogenic induction, the cells in the 6-well plate were rinsed twice with pre-cooled PBS;

[0182] b. Aspirate PBS, add 100uL of pre-cooled lysis buffer and lyse on ice for 10min (protease and phosphatase inhibitors are pre-added in the lysis buffer);

[0183] c. Use a cell scraper to scrape the adherent cells and gently transfer the cells and lysate to a pre-cooled EP centrifuge tube;

[0184] d. Ultrasonic disruption on ice for 10 s, 3 cycles;

[0185] e. Centrifuge at 12000 rpm for 15 min at 4°C;

[0186] f. Gently aspirate the supernatant, transfer it to a newly pre-cooled centrifuge tube and place it on ice. This is the protein sample and discard the precipitate.

[0187] 2) Protein quantification

[0188] a. Add BSA standard to the 96-well plate according to Table 1 for standard curve determination; take 2 μL of sample and 18 μL of saline and add them to the 96-well plate for sample protein concentration detection. Set three replicates for both standard and sample;

[0189] b. Mix solution A and solution B in the BCA kit at a ratio of 50:1 and add 180 μL to each well;

[0190] c. Place the well plate in a 37°C incubator and incubate for 30 minutes, then detect the absorbance at 562 nm and calculate the sample concentration based on the absorbance and the concentration of the BSA standard.

[0191] d. Finally, dilute the sample protein concentration to 1 μg / μL by adding loading buffer and physiological saline, boil it in boiling water for 10 minutes, and store it in a -80℃ refrigerator.

[0192] Table 1 Standard curve drawing

[0193]

[0194] 3) Detection of protein expression by western blotting

[0195] a. Load the prepared protein samples at a loading volume of 20 μL per well;

[0196] b. Electrophoresis at a constant voltage of 80 V for 30 min, then switch to a constant voltage of 120 V to electrophoresis to a suitable position;

[0197] c. Separate the electrophoresis gel and place it together with the PVDF membrane in the transfer device. The transfer condition is 200mA constant current transfer on ice for 150min;

[0198] d. Block the transferred membrane with 5% BSA solution on a shaker at room temperature for 1 to 2 hours, then select the corresponding primary antibody (COL1, OPN, RUNX2, OSX, GAPDH) and incubate at 4°C for 12 to 16 hours;

[0199] e. After the primary antibody incubation, wash the membrane three times in PBST, 10 min each time;

[0200] f. Incubate the washed membrane in the corresponding secondary antibody for 1 to 2 hours, then wash the membrane three times with PBST, each time for 10 minutes;

[0201] g. Pour the luminescent solution on the washed membrane and observe the corresponding bands in the chemiluminescence instrument.

[0202] Figure 7 E is the result of western blot detection of osteogenic protein expression in three groups of cells after 14 days of osteogenic induction. Figure 7 As shown in E, there was no significant difference in the expression of osteogenic proteins among the three groups of cells after osteogenic induction culture.

[0203] (9) Endothelial cell migration ability detection

[0204] a. Human umbilical vein endothelial cells (HUVECs) were obtained from a cell cryopreservation bank from iCell Biotech (Shanghai, China);

[0205] b. Endothelial cell culture medium (1001, ScienCell, USA) was prepared by adding 25 mL FBS, 5 mL endothelial cell growth factor and 5 mL penicillin / streptomycin solution to 500 mL ECM culture medium and storing at 4 °C in the dark;

[0206] c. Cell recovery: After thawing the frozen cells in 37℃ water, transfer them to a 15ml centrifuge tube and add 2mL PBS. Centrifuge at 800rpm for 5min at room temperature and inoculate them in a dish containing 10mL ECM. Culture them in a cell culture incubator at 37℃ and 5% carbon dioxide concentration. Change the culture medium every 3 days.

[0207] When the cells reached 80-90% confluency, they were subcultured into 6-well plates using 0.25% trypsin solution (15050057, Gibco, USA) at a density of 5×10 cells per well. 5 1 ml of the culture supernatant of the three groups of DFSCs filtered through a 0.22 μm filter and 1 ml of ECM were added to each well. When the cell fusion rate reached 90%, a sterile pipette tip was used to make a scratch;

[0208] e. Take photos at 0h, 12h, and 24h to record the scratch healing status.

[0209] Figure 8 A is the unsorted group, PDGFRA + Group, PDGFRA - Light microscopic images of endothelial cell scratch assay in group III, scale bar = 500 μm; Figure 8 B is the unsorted group, PDGFRA + Group, PDGFRA - The migration rate statistics of three groups of scratch tests are shown in Figure 2. Figure 8 A. Figure 8 As shown in B, there was no significant statistical difference in the effects of the three groups of DFSCs and ECs in indirect co-culture on the migration ability of endothelial cells.

[0210] (10) Detection of endothelial cell lumen formation ability

[0211] a. Take out Matrigel (082703, Shanghai Nova Medical Technology, China) in advance and place it on ice to melt in a 4°C refrigerator;

[0212] b. Use a pre-cooled pipette tip to spread Matrigel on μ-Slides 15Well 3D (81506, ibidi, Germany), 10 μL per well, and place flat in a 37°C incubator to solidify for more than 2 hours;

[0213] c. Endothelial cells were cultured at 1×10 4 / well were seeded on μ-Slides plates, and the supernatants of the DFSCs culture media collected from the three groups were filtered through a 0.22 μm filter, mixed with fresh serum-free endothelial cell culture media at a ratio of 1:1 and resuspended;

[0214] d. After 4 to 5 hours, the formation of tubular structures was observed and photographed under an inverted microscope (Leica, Germany);

[0215] e. The number of network structures was quantified using ImageJ software (NIH, USA).

[0216] Figure 8 C is the unsorted group, PDGFRA + Group, PDGFRA - Light microscopic image of the lumen results of group 3, scale bar = 500 μm; Figure 8 D is the unsorted group, PDGFRA + Group, PDGFRA - Statistical analysis of endothelial cell lumen formation parameters in three groups. Figure 8 C. Figure 8 D shows that there is no significant statistical difference in the effect of the three groups of DFSCs and ECs in the indirect co-culture conditions on the tube-forming ability of endothelial cells.

[0217] (11) Evaluation of H-type blood vessel formation ratio

[0218] a. After forming tubular structures on microplates (μ-Slides), fix with 4% PFA for 30 minutes;

[0219] b. Wash slowly 3 times with PBS on a shaker;

[0220] c. Block with 5% BSA for 30 min at room temperature;

[0221] d. Primary antibodies against CD31 (FAB3628G, R&D Systems, USA; dilution ratio 1:100) and EMCN (DF13357, Affinity, China; dilution ratio 1:100) were incubated overnight at 4°C in the dark;

[0222] e. After discarding the primary antibody, stain with the corresponding fluorescent secondary antibody at 37°C for 1 hour;

[0223] f. Discard the secondary antibody and stain with DAPI for 10 min;

[0224] g. Fluorescence images were captured using a confocal laser scanning microscope (CLSM) (Nikon, Japan);

[0225] h. The percentage of CD31 and EMCN double-positive vessels was analyzed using ImageJ software (NIH, USA).

[0226] Figure 8 E is the unsorted group, PDGFRA + Group, PDGFRA -Immunofluorescence staining of CD31 and EMCN for endothelial cell lumen formation in group 3; Figure 8 F is the unsorted group, PDGFRA + Group, PDGFRA - Endothelial cells CD31 + EMCN + Statistical graph of lumen formation percentage. Figure 8 E. Figure 8 F shows that under the indirect co-culture conditions of DFSCs and ECs in the three groups, CD31 + EMCN + There was no statistically significant difference in the angiogenesis ratio.

[0227] (12) Detection of proliferation capacity of three groups of DFSCs after PDGFBB treatment

[0228] a. After the cells were subcultured and adhered to the culture medium, PDGFBB (HY-P7055, MCE, China) was added to the DFSCs culture medium at a concentration of 100 ng / mL, and the corresponding PDGFBB was added each time the medium was changed;

[0229] b. The clone formation ability, proliferation ability and cell cycle status of the three groups of DFSCs were detected according to the steps of Example 3(2) to (4).

[0230] Fig. 9 Under the condition of PDGFBB treatment, the unsorted group and PDGFRA + Group, PDGFRA - The proliferation ability and cell cycle detection results of dental mesenchymal stem cells (DFSCs) in the group were shown in Figure 2. Fig. 9 A and Fig. 9 B is the unsorted group, PDGFRA + Group, PDGFRA - Group III clone formation experiment (scale bar = 250 μm) and EdU experiment (scale bar = 50 μm) results and statistical graphs; Fig. 9 C is a statistical chart showing the expression of three groups of cell cycle-related genes detected by qRT-PCR. Fig. 9 It can be seen that PDGFBB treatment promoted PDGFRA + Enhancement of DFSCs proliferation capacity.

[0231] (13) Detection of osteogenic capacity of three groups of DFSCs after PDGFBB treatment

[0232] a. After the cells were subcultured and adhered to the culture medium, PDGFBB (HY-P7055, MCE, China) was added to the DFSCs culture medium at a concentration of 100 ng / mL, and the corresponding PDGFBB was added each time the medium was changed;

[0233] b. The osteogenic differentiation ability of the three groups of DFSCs was detected according to the steps of Example 3(5).

[0234] Fig.10 Under the condition of PDGFBB treatment, the unsorted group and PDGFRA + Group, PDGFRA - Figure 2 shows the results of the osteogenic ability test of dental mesenchymal stem cells (DFSCs). Fig.10 A and Fig.10 B shows the ARS staining results and statistical graphs of the three groups after 14 days of osteogenic induction, scale bar = 100 μm; Fig.10 C is a statistical graph showing the results of ALP activity detection in the culture supernatant of the three groups after 14 days of osteogenic induction; Fig.10 D is the statistical graph of osteogenic-related gene expression in three groups of cells detected by qRT-PCR after 14 days of osteogenic induction; Fig.10 E is the result of western blot detection of osteogenic protein expression in three groups of cells after 14 days of osteogenic induction. Fig.10 It can be seen that PDGFBB treatment promoted PDGFRA + Enhancement of the osteogenic capacity of DFSCs.

[0235] (14) Effects of the supernatants of three groups of DFSCs treated with PDGFBB on endothelial cells

[0236] a. PDGFBB (HY-P7055, MCE, China) was added to the DFSCs culture medium at a concentration of 100 ng / mL. After 48 hours, the conditioned medium was collected, filtered through a 0.22 μm filter, and stored at -80°C;

[0237] b. Prepared according to the experimental steps of Example 3 (10) a to c, wherein the above culture supernatant was mixed with fresh serum-free endothelial cell culture medium in a ratio of 1:1 and HUVECs were resuspended;

[0238] c. Pictures of the scratched area were taken at 0, 12, and 24 h using an inverted microscope (Leica, Germany) and quantified using ImageJ software (NIH, USA).

[0239] Fig.11 The figure shows the effect of indirect co-culture of endothelial cells with the culture supernatant of dental mesenchymal stem cells (DFSCs) treated with PDGFBB on the migration and tube formation ability of endothelial cells; Fig.11 A is the light microscopic image of the three groups of endothelial cell scratch experiments, scale bar = 500 μm; Fig.11 B is the statistical result of the migration rate of three groups of scratch experiments; Fig.11 C is the light microscopic image of the three-component lumen results, scale bar = 500 μm; Fig.11 D is the statistical diagram of the analysis of the lumen formation parameters of the three groups of endothelial cells; Fig.11 E is the immunofluorescence staining of CD31 and EMCN for endothelial cell lumen formation in the three groups, scale bar = 200 μm; Fig.11 F is the CD31 of three groups of endothelial cells + EMCN + Statistical graph of lumen formation percentage. Fig.11 It can be seen that PDGFRA after PDGFBB treatment + DFSCs can enhance the migration and tube-forming ability of endothelial cells.

[0240] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

[0241] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A PDGFRA + A method for identifying and authenticating mesenchymal stem cells, characterized in that: include: Preparation of single cell suspensions of tooth developmental tissues; The tooth developmental tissue includes dental follicle tissue and apical dental papilla tissue surrounding the mandibular third molar; The single cell suspension is labeled with gel microbeads containing unique barcodes, further mixed with oil to form water-in-oil droplets, full-length cDNA is produced by reverse transcription reaction, and the emulsion is destroyed by a recovery agent, and the cDNA is cleaned by magnetic beads. After the amplified cDNA is further modified, a single cell RNA sequencing library is constructed; Through single-cell transcriptome sequencing and bioinformatics analysis, PDGFRA, which has a common high expression of odontogenic and osteogenic protein genes, was identified. + Mesenchymal stem cells.

2. A PDGFRA + A method for sorting mesenchymal stem cells, characterized in that: include: Preparation of single cell suspensions of tooth developmental tissues; The tooth developmental tissue includes dental follicle tissue and apical dental papilla tissue surrounding the mandibular third molar; The single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, and the resulting cells are combined with positive selection beads and placed in a magnetic field to separate PDGFRA + Mesenchymal stem cells.

3. The PDGFRA according to claim 2 + A method for sorting mesenchymal stem cells, characterized in that: The step of preparing a single cell suspension of tooth developmental tissue comprises: The tooth developmental tissue was digested with 0.02% type I collagenase at 37°C for 1 h, and then digested with an equal amount of 10% to 20% α-MEM medium. The cell pellet was obtained by centrifugation at 1000 rpm for 10 min. The tooth developmental tissue and cell sediment were resuspended in 10% to 20% α-MEM culture medium, and then inoculated into a culture device. When the cell fusion rate reached 90%, digestion and passage were performed to obtain a single cell suspension.

4. The PDGFRA according to claim 2 + A method for sorting mesenchymal stem cells, characterized in that: The single cell suspension is incubated with a biotin-labeled CD140a monoclonal antibody, the resulting cells are combined with positive selection beads, and placed in a magnetic field to separate PDGFRA + The steps of mesenchymal stem cells include: The single cell suspension was resuspended in PBS containing 3% to 5% serum to adjust the cell concentration to 1×10 6 100uL, add 2uL of CD140a biotinylated antibody to each system, incubate at room temperature for 10min, wash with PBS containing 3% to 5% serum, discard the supernatant after centrifugation, add 10μL of avidin positive selection beads to the obtained cells, place them in a magnetic field, stand at room temperature for 5min, and sort out PDGFRA + Mesenchymal stem cells.

5. A PDGFRA + Mesenchymal stem cells, characterized in that The PDGFRA + Mesenchymal stem cells are PDGFRA according to any one of claims 2 to 4 + Mesenchymal stem cells are obtained by sorting.

6. A method to maintain PDGFRA + A method for culturing mesenchymal stem cells for stable expression, characterized in that: include: The PDGFRA of claim 5 was cultured in α-MEM medium containing 10% serum. + Mesenchymal stem cells were cultured until the cells adhered to the wall, then the medium was changed to serum-free medium for continued culture, and characteristic gene expression was verified when the cell fusion rate reached 90%.

7. The method for maintaining PDGFRA according to claim 6. + A method for culturing mesenchymal stem cells for stable expression, characterized in that: The serum-free culture medium is an α-MEM culture medium without FBS; the culture conditions are: 37° C., 5% CO 2 .

8. The method for maintaining PDGFRA according to claim 6 or 7. + A method for culturing mesenchymal stem cells for stable expression, characterized in that: PDGFBB was supplemented into the serum-free medium at a concentration of 100 ng / mL.

9. The PDGFRA according to claim 5 + Application of mesenchymal stem cells in the preparation of drugs for the treatment of periodontal tissue defects.

10. A drug for treating periodontal tissue defects, characterized in that: The drug comprises the PDGFRA according to claim 5 + Mesenchymal stem cells, and pharmaceutically acceptable excipients and carriers.

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