Mandibular skeletal stem cell subpopulation as well as sorting method and application thereof

Abstract

The invention provides a mandibular skeletal stem cell subpopulation as well as a sorting method and application thereof. Wherein the mandibular skeletal stem cell subpopulation is from a mandibular mesenchymal cell population, and the mandibular skeletal stem cell subpopulation is FOXP4 positive. The mandible bone stem cell subpopulation provided by the invention has stronger osteogenic ability, and has great potential to be clinically applied to repair of mandible bone defects; and meanwhile, the dental implant also has stronger tooth forming ability, can realize whole tooth regeneration, and provides an effective new thought for clinically treating tooth deficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of cell separation and purification, and particularly to a subpopulation of mandibular skeletal stem cells, a sorting method thereof, and an application thereof. Background Art

[0002] Hard tissue development refers to the formation and development process of hard tissues such as bones and teeth. This process involves the differentiation and functions of various cell types (such as osteoblasts, odontoblasts, etc.), and is a complex dynamic process. Skeletal stem cells (SSCs) play a key role in this process. The development of craniofacial bones and teeth is also mediated by specific skeletal stem cells. Abnormalities in their quantity and function lead to craniofacial hard tissue deformities such as mandibular dysplasia, abnormal tooth structure, and even tooth loss.

[0003] Bone tissue development malformations and bone structure defects caused by trauma usually require surgical reconstruction, but this repair and regeneration process is relatively long and accompanied by risks such as postoperative infection. At the same time, as a special hard tissue, the development process of teeth involves the interaction between epithelial cells and mesenchymal cells, and the molecular mechanism is relatively complex. Currently, clinically, only inorganic materials such as implants and porcelain crowns can be used to repair tooth morphological abnormalities or tooth loss, but their structure and function cannot be compared with natural teeth.

[0004] Forkhead box (Fox) proteins belong to the superfamily of transcription factors and are closely related to various biological processes such as cell proliferation and differentiation. FOXP4 belongs to the FOXP subfamily. Currently, no study has proven that the FOXP4 transcription factor is related to the odontogenic and osteogenic potential of skeletal stem cells. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a subpopulation of mandibular skeletal stem cells, a sorting method thereof, and an application thereof to solve the problems in the prior art.

[0006] To achieve the above object and other related objects, the present invention is obtained through the following technical solutions.

[0007] In the first aspect of the present invention, there is provided a subpopulation of mandibular skeletal stem cells, which are derived from a mandibular mesenchymal cell population, and the subpopulation of mandibular skeletal stem cells is FOXP4 positive.

[0008] Preferably, the mandibular mesenchymal cell population is derived from a human source or a mouse source.

[0009] Specifically, the Ensembl ID of the mouse-derived FOXP4 gene in this application is ENSMUSG00000023991; the Ensembl ID of the human-derived FOXP4 gene is: ENSG00000137166.

[0010] Preferably, the subpopulation of mandibular skeletal stem cells has any one or more of the following abilities (i)-(iv):

[0011] (i) The ability to differentiate into bone tissue

[0012] (ii) The ability to differentiate into cartilage tissue

[0013] (iii) The ability to differentiate into adipocytes

[0014] (iv) The ability to form teeth.

[0015] Preferably, when the mandibular mesenchymal cell population is of mouse origin, the subpopulation of mandibular skeletal stem cells is CD200 positive and ENG negative.

[0016] Preferably, when the mandibular mesenchymal cell population is of human origin, the subpopulation of mandibular skeletal stem cells is CADM1 positive and PDPN positive.

[0017] The second aspect of the present invention provides a method for sorting the subpopulation of mandibular skeletal stem cells as described above, comprising the following steps:

[0018] Surface stain the mandibular mesenchymal cell population with a combination of flow antibodies labeled with different fluorophores. After resuspending the stained mandibular mesenchymal cell population, sort it using a flow cytometer to obtain a population of mandibular skeletal stem cells that are FOXP4 positive, CD200 positive and ENG negative, or FOXP4 positive, CADM1 positive and PDPN positive.

[0019] Preferably, the population of mandibular skeletal stem cells is also CD31 negative, CD45 negative, Ter119 negative, Thy1.2 negative and 6C3 negative.

[0020] Preferably, the combination of flow antibodies includes one or more of the antibodies CADM1 antibody, PDPN antibody, CD200 antibody, ENG antibody.

[0021] Preferably, the combination of flow antibodies further includes: one or more of CD31 antibody, CD45 antibody, Ter119 antibody, Thy1.2 antibody, 6C3 antibody.

[0022] Preferably, when the mandibular mesenchymal cell population is of human origin, the combination of flow antibodies includes CADM1 antibody and PDPN antibody.

[0023] Preferably, when the mandibular mesenchymal cell population is of murine origin, the flow antibody combination includes a CD200 antibody and an ENG antibody.

[0024] The third aspect of the present invention provides a use of the mandibular skeletal stem cell subset as described above in any one or more of the following:

[0025] a) Preparing a medical material for treating tooth defects;

[0026] b) Preparing a medical material for treating bone defects.

[0027] Preferably, the medical material includes all materials of pharmaceuticals (pharmaceutical compositions), medical devices, and medical products.

[0028] Preferably, the pharmaceuticals (pharmaceutical compositions) further include a pharmaceutically acceptable carrier.

[0029] The fourth aspect of the present invention provides a medical material for treating tooth defects or bone defects, the medical material including stem cells in an effective amount of the mandibular skeletal stem cell subset as described above.

[0030] The fifth aspect of the present invention provides an antibody composition for identifying the mandibular skeletal stem cell subset as described above, the antibody composition including a CADM1 antibody and a PDPN antibody, or the antibody composition including a CD200 antibody and an ENG antibody.

[0031] Preferably, the antibody composition further includes one or more of a CD31 antibody, a CD45 antibody, a Ter119 antibody, a Thy1.2 antibody, and a 6C3 antibody.

[0032] The sixth aspect of the present invention provides a use of the antibody composition as described above in identifying the mandibular skeletal stem cell subset as described above.

[0033] The seventh aspect of the present invention provides a kit for identifying the mandibular skeletal stem cell subset as described above, including the antibody composition as described above.

[0034] The eighth aspect of the present invention provides a method for treating tooth defects or bone defects, which is to administer to a subject a medical material including stem cells in an effective amount of the mandibular skeletal stem cell subset as described above.

[0035] Preferably, the subject is a mammal, and the mammals include humans and mice.

[0036] The ninth aspect of the present invention provides a use of a murine sinus defect model for detecting the in vivo odontogenic potential of the mandibular skeletal stem cells as described above.

[0037] Preferably, the method for constructing the mouse sinus defect model comprises the following steps: After anesthetizing the mouse to be modeled, incise the skin on the dorsal nose of the mouse, drill a hole in the nasal bone with a drill bit, and stop when a sense of falling through is felt without damaging the sinus mucosal epithelium to obtain the mouse sinus defect model.

[0038] Preferably, when actually using the mouse sinus defect model, cell spheres treated differently are transplanted into the sinus, such that the cell spheres are as close as possible to the sinus mucosal epithelium and then sutured, and the growth or differentiation of the cells is observed.

[0039] Preferably, the mouse sinus defect model can also be used to detect the osteogenic and / or chondrogenic potential of the mandibular skeletal stem cells as described above in vivo.

[0040] Preferably, the diameter of the drilled hole is 0.5 mm to 2 mm. For example, it can be 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.5 mm.

[0041] Advantages of the present invention:

[0042] 1) The present invention uses single-cell sequencing technology to discover and isolate a subpopulation of mandibular skeletal stem cells with high odontogenic and osteogenic potential from traditional mandibular skeletal stem cells. Compared with other traditional mandibular skeletal stem cells (skeletal stem cells negative for FOXP4), this subpopulation of mandibular skeletal stem cells has stronger osteogenic ability and great potential for application in the clinical repair of jaw bone defects, which is beneficial to shortening the bone repair time and significantly improving the prognosis effect.

[0043] 2) The subpopulation of mandibular skeletal stem cells provided by the technical solution of the present invention has stronger odontogenic ability compared with other traditional mandibular skeletal stem cells (skeletal stem cells negative for FOXP4), and can achieve total tooth regeneration, providing an effective new idea for the clinical treatment of tooth loss.

[0044] 3) The present invention also provides a new mouse sinus defect model, which can be used to detect the odontogenic potential of stem cells in vivo. This model utilizes the complex environment in the mouse sinus that has both epithelial cells and mesenchymal cells, thereby effectively simulating the tooth development environment and avoiding the operation of artificially mixing epithelial cells in the models of the prior art to simulate the odontogenic environment. This model can not only provide a more natural odontogenic environment to directly verify the in vivo odontogenic potential of mesenchymal stem cells and improve the accuracy of the experiment; at the same time, it can also reduce the process of co-culturing epithelial cells and mesenchymal cells when using other models, thereby simplifying the experimental operation process and improving the experimental efficiency. Description of the Drawings

[0045] Figure 1Shown is the analysis chart of the flow sorting results of mouse-derived mandibular mesenchymal cells in Example 1 of the present invention.

[0046] Figure 2 Shown is one of the analysis charts of the single-cell sequencing results of mouse-derived mandibular mesenchymal cells in Example 2 of the present invention.

[0047] Figure 3 Shown is the second analysis chart of the single-cell sequencing results of mouse-derived mandibular mesenchymal cells in Example 2 of the present invention.

[0048] Figure 4 Shown is the flow sorting result chart of mouse-derived mandibular skeletal stem cells in Example 3 of the present invention.

[0049] Figure 5 Shown is the result chart of the in vitro cell stemness verification of mouse-derived mandibular skeletal stem cells in Example 4 of the present invention.

[0050] Figure 6 Shown is the result chart of the in vivo transplantation promoting mandibular injury repair experiment of mouse-derived mandibular skeletal stem cells in Example 5 of the present invention.

[0051] Figure 7 Shown is the result chart of the in vivo transplantation-mediated tooth regeneration experiment of mouse-derived mandibular skeletal stem cells in Example 7 of the present invention.

[0052] Figure 8 Shown is one of the analysis charts of the single-cell sequencing results of human-derived mandibular periosteal mesenchymal cells in Example 8 of the present invention.

[0053] Figure 9 Shown is the second analysis chart of the single-cell sequencing results of human-derived mandibular periosteal mesenchymal cells in Example 8 of the present invention.

[0054] Figure 10 Shown is the flow sorting result chart of human-derived mandibular skeletal stem cells in Example 9 of the present invention.

[0055] Figure 11 Shown is the result chart of the in vitro cell stemness verification of human-derived mandibular skeletal stem cells in Example 10 of the present invention.

[0056] Figure 12 Shown is the result chart of the in vivo transplantation-mediated osteogenesis experiment of human-derived mandibular skeletal stem cells under the renal capsule in Example 11 of the present invention.

[0057] Figure 13 Shown is the result chart of the in vivo transplantation-mediated tooth regeneration experiment of human-derived mandibular skeletal stem cells in Example 12 of the present invention. Detailed implementation manners

[0058] The following specific embodiments illustrate the implementation manners of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0059] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation manners, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following embodiments are generally in accordance with conventional conditions or the conditions recommended by each manufacturer.

[0060] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, any value at both ends of each numerical range and any value between the two ends can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of the present technology and the description of the present invention, any methods, devices, and materials similar or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0061] In this application, CD31 is Platelet endothelial cell adhesion molecule-1; CD45 is Protein Tyrosine Phosphatase Receptor Type C; Ter119 is Lymphocyte Antigen 76; Thy1.2 is Thymocyte Antigen1.2; 6C3 is Glutamyl Aminopeptidase; CD200 is Cluster of Differentiation 200, that is, OX-2 Membrane Glycoprotein; ENG is Endoglin; CADM1 is Cell Adhesion Molecule 1; PDPN is Podoplanin.

[0062] The ENG protein / gene in this application is completely the same or equivalent to the CD105 (Cluster of Differentiation 105) protein / gene. In other words, the ENG protein / gene throughout this application is the CD105 protein / gene.

[0063] The Enpep protein / gene in the present application is completely identical or equivalent to the 6C3 protein / gene. In other words, the Enpep protein / gene throughout the present application is the 6C3 protein / gene.

[0064] Positive in the present invention means that when detected by flow cytometry (FACS), the fluorescence signal intensity detected by the flow cytometer for the target gene / protein > 10 3 threshold; negative means that the fluorescence signal intensity detected by the flow cytometer for the target gene / protein ≤ 10 3 threshold.

[0065] The subpopulation of mandibular skeletal stem cells of the present invention has high odontogenic / osteogenic potential. Therefore, the cell population of the present invention can be used as a medical material for treating tooth defects or bone defects (tooth regeneration or bone regeneration). It should be noted that the "medical material" of the present invention includes all materials classified as pharmaceuticals (pharmaceutical compositions), medical devices, and medical products according to the regulations and standards of a country or region.

[0066] The cell population that can be used as a medical material can be autologous cells, allogeneic cells, or it can also be cell lines. When problems such as infection and rejection reactions are involved, it is preferably to use cells derived from the patient prepared from the patient's tissue.

[0067] The method of applying the medical material (pharmaceutical composition, medical device, medical product) of the present invention to an organism is not particularly limited. Depending on the application site, local transplantation by surgical means, intravenous administration, lumbar puncture administration, local injection administration, subcutaneous administration, intradermal administration, intraperitoneal administration, intramuscular administration, intracerebral administration, intraventricular administration, or intravenous administration, etc. can be considered.

[0068] The medical material of the present invention may further contain a supporting material, component, or other pharmaceutically acceptable carrier that aids in the maintenance / proliferation of cells and the administration to the affected part. As components essential for the maintenance and proliferation of cells, culture medium components such as a carbon source, nitrogen source, vitamins, minerals, salts, various cytokines, or extracellular matrix preparations such as MatrigelTM can be cited. As supporting materials and components that aid in the administration to the affected part, biodegradable polymers such as collagen, polylactic acid, hyaluronic acid, cellulose and their derivatives, and composites formed from two or more of them can be cited, as well as aqueous injection solutions such as physiological saline, culture medium, physiological buffer solutions such as PBS, and isotonic solutions containing glucose and other adjuvants (such as D-sorbitol, D-mannose, D-mannitol, sodium chloride), etc. For example, it can also be used in combination with suitable solubilizers such as alcohols (specifically ethanol, polyols (such as propylene glycol, polyethylene glycol)), nonionic surfactants (such as polysorbate 80, HCO-50), etc.

[0069] Furthermore, if necessary, it may also contain pharmaceutically acceptable organic solvents, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, mannitol, sorbitol, lactose, surfactants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, etc. that are permitted to be used as pharmaceutical additives.

[0070] The actual additives can be selected from the above additives alone or in appropriate combinations according to the form of the medical material of the present invention, but are not limited to these. For example, when used as an injectable preparation, a purified antibody can be dissolved in a solvent (such as physiological saline, buffer solution, glucose solution, etc.), and it can contain anti-adsorbents such as Tween 80, Tween 20, gelatin, etc., isotonic agents such as glycerol, D-mannitol, stabilizers such as human serum albumin, preservatives such as methyl p-hydroxybenzoate, local anesthetics such as benzyl alcohol, etc. In addition, other agents that promote tissue regeneration can also be combined.

[0071] The shape / morphology of the medical material of the present invention can be appropriately determined according to the application site and tissue. For example, in addition to ordinary cell preparations, it can also be processed into a sheet shape or disc shape for use. In addition, in addition to being used for in vivo organ / tissue regeneration, it can also be used for in vitro organ / tissue regeneration.

[0072] In the present application, the symptoms / diseases that can be the object of the medical material of the present invention include bone defects caused by trauma, surgery or diseases. The bone defects caused by diseases include bone-related diseases caused by congenital jaw dysplasia, diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, periodontal disease, oral cancer; and also include tooth defects caused by trauma, surgery or diseases. The tooth defects caused by diseases include tooth-related diseases caused by congenital tooth loss or hypoplasia, periodontal disease, dental caries, tooth trauma, osteoporosis, diabetes, oral cancer, rheumatoid arthritis, systemic lupus erythematosus, systemic malnutrition.

[0073] In the "effective amount of stem cells in the subpopulation of mandibular skeletal stem cells as described in any one of claims 1 to 3" of the present invention, the "effective amount" means an amount sufficient for preventing, treating, alleviating, or improving the symptoms or diseases of the subject. The "effective amount" can be appropriately determined according to the condition of the disease, the degree of the disease, the administration route, the number of administrations, etc. The subpopulation of mandibular skeletal stem cells of the present invention has high regenerative ability and can obtain sufficient regenerative effects with an amount less than the known common amounts of conventional mandibular mesenchymal cells and the like in the past.

[0074] Example 1 Flow cytometry was used to sort Foxp4-positive cells from mouse-derived mandibular mesenchymal cells

[0075] Specifically, it includes the following experimental steps:

[0076] 1) Construct Foxp4-CreERT2; tdTomato-traced mouse cells, and label Foxp4-positive cells with tamoxifen at embryonic day E11.5. At embryonic day E12.5, collect the mandibles of embryonic mice.

[0077] 2) Under a stereomicroscope, separate the intact mandibles of the embryonic mice obtained in step 1), remove the surrounding soft tissues with micro forceps, and cut the mandibular tissues into pieces with microsurgical scissors.

[0078] 3) Digest the mandibular tissues obtained in step 2) with a mixed enzyme solution, and perform water bath digestion at 37°C three times, 15 minutes each time. The components of the mixed enzyme solution are: 3 mg / ml type I collagenase (Worthington, LS004197), 4 mg / ml neutral protease (Sigma, 04942078001), and 1 U / ml DNase (Sigma, D4527), and the balance is HBSS buffer containing Ca 2+ and Mg 2+ of HBSS buffer.

[0079] 4) Terminate the digestion of the cell suspension obtained in step 3) with a neutralizing solution. The components of the neutralizing solution are: 2 mM EDTA, 2% FBS, and the balance is HBSS buffer without Ca2+ and Mg 2+ in HBSS buffer.

[0080] 5) Centrifuge the cell suspension obtained in step 4), remove the supernatant, resuspend the cells with the mixed antibody diluent, and incubate in the dark on ice for 30 minutes.

[0081] The mixed antibodies are: APC-CD31, APC-CD45, APC-Ter119, APC-Thy1.2, APC-6C3 (APC-CD31, APC-CD45, APC-Ter119, APC-Thy1.2, APC-6C3 are fluorescently labeled antibodies for detecting blood lineages and endothelial cells).

[0082] 6) Wash the cell suspension containing antibodies in step 5) with HBSS buffer, centrifuge to remove the supernatant, and resuspend with DAPI diluent for subsequent sorting of live and dead cells.

[0083] 7) Perform flow cytometry sorting using BD FACS AriaIII and BD FACSDiva software.

[0084] Collect cells that are negative for APC signal (Lineage negative, i.e., removing blood lineages and endothelial cells) and positive for tdT signal (i.e., Foxp4 positive) for single-cell sequencing analysis.

[0085] The flow cytometry sorting results in Example 1 are as Figure 1 shown, and it can be seen from Figure 1 that the desired target cell population has been sorted in this example. The target cell population is cells that are negative for APC signal and positive for tdT signal, i.e., cells that are negative for CD31, negative for CD45, negative for Ter119, negative for Thy1.2, negative for 6C3, and positive for Foxp4.

[0086] Example 2: Perform single-cell transcriptome sequencing on mouse-derived mandibular mesenchymal cells

[0087] In this example, further single-cell transcriptome sequencing is performed on the target cell population obtained in Example 1.

[0088] Entrust a biotechnology company to perform single-cell transcriptome sequencing on the target cell population obtained in Example 1 and perform UMAP analysis on the sequencing data. The UMAP analysis results are as Figure 2As shown in the left figure. At the same time, the regulatory genes related to mesenchymal stem cells, osteogenesis, and chondrogenesis (PRRX1, MEIS2, CXCL12, COL11A1, MSX2, PAX9, ACAN, SOX9, MYOG, CD200, ENG, THY1, ENPEP) were analyzed in each cell subset, and the specific results are as Figure 2 shown in the right figure.

[0089] As can be seen from Figure 2 the left figure, the Foxp4-positive cell population obtained in Example 1 was further divided into 7 cell subsets (Cluster); as can be seen from Figure 2 the right figure, compared with the Cluster 2-Cluster 7 subsets, in the Cluster1 subset, the genes related to skeletal stem cell markers (PRRX1, MEIS2, CD200, ENG) were distributed more specifically, indicating that the Cluster1 subset is likely to be skeletal stem cells or cells with similar characteristics to skeletal stem cells.

[0090] The applicant also performed pseudotime analysis and Regulon transcriptional feature analysis of each cell subset, and the results are shown in the left and right figures of Figure 3 respectively.

[0091] As can be seen from Figure 3 the left figure, the Cluster1 cell subset is located at the uppermost reaches of the differentiation trajectories of each subset, indicating that the Cluster1 cell subset has more cell stemness; as can be seen from Figure 3 the right figure, the Regulon (Meis1) related to skeletal stem cells is specifically concentrated and distributed in the Cluster1 cell subset, indicating that the Cluster1 cell subset has the characteristics of skeletal stem cells.

[0092] In summary, the Foxp4-positive Cluster1 cell subset provided in this example has the characteristics of skeletal stem cells.

[0093] In Example 3, flow cytometry sorting was performed on mandibular skeletal stem cells derived from mice

[0094] This example is to further refine the target cell population collected by flow cytometry on the basis of Example 1, and further refine the mandibular mesenchymal cell population to the mandibular skeletal stem cell population.

[0095] Specifically, it includes the following experimental steps:

[0096] Specific steps 1)-7) are the same as those in Example 1,

[0097] Except in step 5), the color - coding scheme for the flow - cytometry antibodies is as follows: PE / Cy7 - CD200, PE / Cy5 - ENG, APC - CD31, APC - CD45, APC - Ter119, APC - Thy1.2, APC - 6C3 (APC - CD31, APC - CD45, APC - Ter119, APC - Thy1.2, APC - 6C3 are fluorescent - labeled antibodies for detecting blood lineages and endothelial cells; PE / Cy7 - CD200, PE / Cy5 - ENG are fluorescent - labeled antibodies for detecting skeletal stem - cell markers).

[0098] And in step 7), on the basis of collecting cells with negative APC signal (Lineage negative, that is, removing blood lineages and endothelial cells), positive PE / Cy7 signal, and negative PE / Cy5 signal, cells with positive tdT signal are simultaneously collected as experimental - example cells; on the basis of collecting cells with negative APC signal (Lineage negative, that is, removing blood lineages and endothelial cells), positive PE / Cy7 signal, and negative PE / Cy5 signal, cells with negative tdT signal are simultaneously collected as control - example cells for the analysis and comparison of in - vitro cell stemness.

[0099] The specific experimental results are shown in Figure 4 . From Figure 4 it can be seen that mandibular skeletal stem cells with negative APC signal, positive PE / Cy7 signal, negative PE / Cy5 signal, and positive tdT signal are selected as experimental - example cells; mandibular skeletal stem cells with negative APC signal, positive PE / Cy7 signal, negative PE / Cy5 signal, and negative tdT signal are selected as control - example cells.

[0100] Example 4 Verification of In - Vitro Cell Stemness (Mouse - Derived)

[0101] In this example, the experimental - example cells (i.e., Foxp4 - positive mandibular skeletal stem cells derived from mice) and control - example cells (i.e., Foxp4 - negative mandibular skeletal stem cells derived from mice) obtained in Example 3 are detected for their in - vitro colony - forming ability and tridirectional differentiation ability.

[0102] The specific steps of the detection method are as follows:

[0103] Detection of colony - forming ability:

[0104] 1) Resuspend the experimental - example cells and control - example cells sorted by flow cytometry in Example 3 with mesenchymal - stem - cell complete medium (α - MEM + 10% FBS, hereinafter collectively referred to as complete medium), and count them.

[0105] 2) After counting, at a density of 10 4 / cm 2Coat the single-cell suspension on a 10-cm culture dish at a certain density and culture it in a hypoxic incubator (the gas environment in the hypoxic incubator: 5% O2, 10% CO2, 85% N2).

[0106] 3) Discard half of the culture medium the next day and supplement it with half of the fresh complete culture medium.

[0107] 4) Discard all the culture medium on the third day, wash away the non-adherent cells with 1×PBS, and add fresh complete culture medium. Then change the fresh complete culture medium every two days.

[0108] 5) Perform crystal violet staining on the seventh day, observe and count the colonies formed by cloning under an inverted microscope.

[0109] See the specific experimental results in Figure 5 a). As can be seen from Figure 5 a), compared with the cells in the comparative example, the number of stained cells in the cells of the example is more, indicating that the number of clones formed by the cells of the example is higher. It shows that the cells of the example, namely the Foxp4-positive mandibular skeletal stem cells derived from mice, have stronger clone formation ability.

[0110] Detection of osteogenic differentiation ability:

[0111] 1) Culture the experimental example cells and the cells of the comparative example sorted by flow cytometry in Example 3 with the complete culture medium (α-MEM + 10% FBS) until the confluence reaches 80%.

[0112] 2) Prepare the osteogenic induction differentiation medium, and the specific components are as follows:

[0113] Osteogenic induction differentiation medium (50 mL volume)

[0114]

[0115]

[0116] 3) Discard the complete culture medium in step 1), add the osteogenic differentiation induction medium to the culture dish, and culture it in an incubator at 37°C.

[0117] 4) Change the fresh osteogenic differentiation induction medium every 3 days. Induce for 3 weeks, perform alizarin red staining experiment, and collect cells to extract RNA for qRT-PCR quantitative detection of Bglap gene and Alpl gene.

[0118] See the specific experimental results in Figure 5 b).

[0119] As can be seen from Figure 5As shown in b), compared with the cells of the comparative example, significantly more dark red was observed in the cells of the example, indicating that calcium nodules that could be stained red by alizarin red S staining agent were formed in the cells of the example, suggesting that the cells of the example were successfully differentiated into osteoblasts. Meanwhile, the expression levels of Bglap gene and Alpl gene (osteoblast markers) in the cells of the experimental example were much higher than those of the comparative example, which was consistent with the results of the alizarin red staining experiment. It shows that the cells of the example, namely the Foxp4-positive mandibular skeletal stem cells derived from mice, have stronger osteogenic ability.

[0120] Detection of chondrogenic differentiation ability:

[0121] 1) The experimental example cells and comparative example cells sorted by flow cytometry in Example 3 were cultured in complete medium (α-MEM + 10% FBS) until the confluence reached 100%.

[0122] 2) Discard the complete medium in the culture plate, digest with 0.25% trypsin at 37 °C for 3 min, then add complete medium to terminate digestion, centrifuge at 1000 rpm for 3 min, and discard the supernatant.

[0123] 3) Prepare chondrogenic induction differentiation medium, and the specific components are as follows:

[0124] Chondrogenic induction differentiation medium (50 mL volume)

[0125]

[0126] 4) Add 1 ml of chondrogenic induction differentiation medium to the precipitate in step 2) to resuspend and count.

[0127] 5) The chondrogenic differentiation detection experiment was carried out in 15 ml centrifuge tubes, and the standard cell number in each tube was 2.5x10 5 cell / tube. Centrifuge at 1000 rpm for 2 minutes at high speed, resuspend with 500 μl of chondrogenic induction differentiation induction medium, and centrifuge at 1000 rpm for 2 minutes at high speed again. Then loosen the lid and culture in a 37 °C constant temperature incubator.

[0128] 6) Replace the fresh chondrogenic induction differentiation induction medium every 3 days. Induce for 4 weeks, collect chondrocyte pellets for toluidine blue staining experiment, and collect cells to extract RNA for qRT-PCR quantitative detection of Col2a1 gene and Acan gene.

[0129] The specific experimental results are shown in Figure 5 c).

[0130] From Figure 5As shown in c), compared with the cells of the comparative example, the cells of the example showed obvious blue-violet staining after staining, indicating that chondrocyte spheres were formed in the cell group of the example, while they were absent in the cell group of the comparative example. At the same time, the expression levels of Col2a1 gene and Acan gene (chondroblast markers) in the cells of the experimental example were much higher than those of the comparative example, which corroborated the results of the toluidine blue staining experiment. This shows that the cells of the example, namely the Foxp4-positive mandibular skeletal stem cells derived from mice, have stronger chondrogenic ability.

[0131] Detection of adipogenic differentiation ability

[0132] 1) The experimental example cells and comparative example cells sorted by flow cytometry in Example 3 were cultured in complete medium (α-MEM + 10% FBS) until the confluence reached 100%.

[0133] 2) Prepare an adipogenic induction differentiation medium, and the specific components are as follows:

[0134] Adipogenic induction differentiation medium (50 mL volume)

[0135]

[0136] 3) Replace the complete medium (α-MEM + 10% FBS) with the adipogenic induction differentiation medium. On the 7th day of differentiation induction, the cells were fixed and collected for oil red O staining experiment, and the cells were collected to extract RNA for qRT-PCR quantitative detection of Adipq gene.

[0137] The specific experimental results are shown in Figure 5 d).

[0138] As Figure 5 shown in d), compared with the cells of the comparative example, the cells of the example showed obvious red color, indicating that the oil red O dye combined with the lipids formed by the cells of the example to form small lipid droplets showing red color. This shows that the cells of the example were successfully differentiated into lipids. At the same time, the expression level of Adipq gene (adipocyte marker) in the cells of the experimental example was much higher than that of the comparative example, which corroborated the results of the oil red O staining experiment. This shows that the cells of the example, namely the Foxp4-positive mandibular skeletal stem cells derived from mice, have stronger adipogenic ability.

[0139] In summary, this example proves that compared with the cells of the comparative example, the cells of the example have stronger colony formation ability and stronger osteogenic, chondrogenic and adipogenic differentiation abilities. This shows that the cells of the example, namely the subpopulation of Foxp4-positive mandibular skeletal stem cells derived from mice, exhibit better colony formation and multi-directional differentiation abilities than other skeletal stem cells.

[0140] Example 5 In vivo transplantation experiment to promote mandibular injury repair (derived from mice)

[0141] In this example, the experimental cells obtained in Example 3 (i.e., Foxp4-positive mandibular skeletal stem cells derived from mice) and the control cells (i.e., Foxp4-negative mandibular skeletal stem cells derived from mice) were transplanted into a distraction osteogenesis mouse model to observe the promoting effect of cells in different groups on the injury repair of distraction osteogenesis mice.

[0142] The specific steps are as follows:

[0143] The experimental cells and control cells obtained in Example 3 were amplified and cultured in vitro. After reaching 80% confluence, they were digested and seeded into a 96-well low-attachment U-bottom plate (Corning, Cat#7007) at a cell density of 3×10 5 / well. Cell spheres were obtained 2 days after seeding for in vivo transplantation. The cell spheres were encapsulated with 0.01 ml of Matrigel (Corning, Cat#356238) and transplanted into the injury repair site of distraction osteogenesis mice.

[0144] Grouping of distraction osteogenesis mice: They were randomly divided into an experimental cell transplantation group and a control cell transplantation group. Both groups received two cell transplantations on the 2nd and 3rd days after surgery. The cell spheres were transplanted into the injury site of distraction osteogenesis mice through a syringe at the skin suture. On the 24th day after surgery, X-ray imaging examinations were performed on the mandibles of mice in each group.

[0145] The results of X-ray and immunofluorescence staining are as Figure 6 shown. It can be Figure 6 seen that compared with the control cell transplantation group, the X-ray density of the injury repair site of mice in the experimental cell transplantation group was higher. It indicates that compared with the control cells, the experimental cells of the present invention, i.e., Foxp4-positive mandibular skeletal stem cells derived from mice, have higher osteogenic potential and can significantly promote bone repair of distraction osteogenesis in mice.

[0146] Among them, the specific construction method of the distraction osteogenesis mouse model used in this example is as follows:

[0147] Wild-type mice (male, 8 - 12 weeks old) were selected to establish a distraction osteogenesis model. Analgesia was performed by intraperitoneal injection of ketoprofen before and after surgery (administered once every 12 hours and continued until 72 hours after surgery). After anesthetizing the mice, the skin was disinfected. A 1 cm incision was made on the surface of the right jawbone with a scalpel, the masseter muscle was separated to expose the mandible, and then two holes with a diameter of 0.6 mm were drilled on the bone surface at an interval of 6 mm (centered on the subsequent osteotomy site). The mandible was transected behind the third molar with a fissure drill with a diameter of 0.8 mm. The distraction device was fixed to the transected mandible of the mouse with a 0.8 mm diameter screw, and the muscle and skin were sutured layer by layer.

[0148] The traction operation steps are as follows: The first 3 days after surgery are for the device to stabilize, followed by a 7-day traction period at a rate of 0.2 mm every 12 hours, and then a 14-day injury repair period.

[0149] The traction device was fabricated by computer-aided design (Unigraphics NX10.0 software) and 3D printing technology (Bosheng Mold Factory, resolution 20 μm).

[0150] Construction of a mouse sinus defect model in Example 6

[0151] This example provides a mouse sinus defect model and a specific construction method thereof.

[0152] Wild-type mice (male, 8 - 12 weeks old) were selected to establish a mouse sinus defect model. Before and after surgery, ketoprofen was administered intraperitoneally for analgesia (once every 12 hours, lasting until 72 hours after surgery). After the selected mice were anesthetized, the skin on the nasal dorsum was shaved and disinfected with 75% ethanol. The skin was incised with an 11-blade, and a drill bit with a diameter of 0.8 mm was used to drill holes in the nasal bone until a sense of falling through was felt, then stopped without damaging the sinus mucosal epithelium. The incision was sutured with 6-0 sutures, thus obtaining a mouse sinus defect model for detecting the odontogenic and / or osteogenic potential of stem cells in vivo.

[0153] Example 7 In vivo transplantation-mediated tooth regeneration experiment (mouse-derived)

[0154] The experimental cells obtained in Example 3 (i.e., Foxp4-positive mandibular skeletal stem cells derived from mice) and the control cells (i.e., Foxp4-negative mandibular skeletal stem cells derived from mice) were taken and transplanted into the mouse sinus defect model provided in Example 6 to observe the tooth regeneration of cells in different groups in the recipient mice.

[0155] The specific experimental steps are as follows:

[0156] The experimental cells and control cells obtained in Example 3 were amplified and cultured in vitro. After reaching 80% confluence, they were digested and seeded into a low-attachment U-bottom 96-well plate (Corning, Cat#7007) at a cell density of 3×10 5 / well. Cell spheres were obtained 2 days after seeding for in vivo transplantation. The cell spheres were encapsulated with 0.05 ml of Matrigel (Corning, Cat#356238) and transplanted into the sinuses of the mouse sinus defect model, and during the transplantation process, the transplanted cell spheres were made to adhere as closely as possible to the sinus mucosal epithelium.

[0157] Grouping of mice with sinus defects: They were randomly divided into an experimental cell transplantation group and a control cell transplantation group. At 3 weeks after surgery, the craniofacial bones including the sinuses of mice in each group were harvested for histological section safranin-fast green staining and immunofluorescence staining experiments.

[0158] The results of safranin-fast green staining and immunofluorescence staining are as Figure 7 shown.

[0159] As Figure 7 shown in Figure 7 a) and

[0160] As Figure 7 shown in Figure 7 c) - d) and Figure 7 e), the results of safranin-fast green staining show that, compared with the normal tooth tissue structure, the experimental cell transplantation group of the present application can regenerate tooth structures in the mouse sinuses, and has complete dentin (den), odontoblasts (od), enamel (ena), and ameloblasts (ame), which are similar to the tooth structures of normal teeth. And the results of immunofluorescence staining show that only the mesenchymal cell part of the regenerated tooth structure in the experimental cell transplantation group in the mouse sinuses has red fluorescence, that is, tdT signal positive, indicating again that it is derived from the experimental cells provided in Example 3 of the present application. Figure 7 d) shows the magenta structure pointed by the black arrow). And the results of immunofluorescence staining show that the mesenchymal cell part of the regenerated bone tissue and cartilage tissue has red fluorescence, that is, tdT signal positive, indicating again that it is derived from the experimental cells provided in Example 3 of the present application.

[0161] In summary, the experimental cells provided by the present invention, namely mouse-derived Foxp4-positive mandibular skeletal stem cells, have higher in vivo odontogenic and osteogenic potential; and the odontogenic and osteogenic potential can be verified in vivo through the mouse sinus defect model provided in Example 6 of the present invention.

[0162] Example 8 performs single-cell transcriptome sequencing on human-derived mandibular periosteal mesenchymal cells

[0163] In this example, the single-cell transcriptome sequencing data of human mandibular periosteal mesenchymal cells in the open-source database (GEO: GSE289393) was analyzed, and UMAP analysis was performed on the sequencing data. The results of the UMAP analysis are as Figure 8 shown in the left figure. At the same time, the situation of genes related to human skeletal stem cells, mesenchymal cells, smooth muscle cells, and lymphocytes (CADM1, PDPN, CD164, NT5E, MCAM, LEPR, CDKN3, MKI167, SGCA, CD84) in each cell subset was analyzed. The specific results are as Figure 8 shown in the right figure.

[0164] AsFigure 8 As can be seen from the left figure, the cell population was further divided into 6 cell subpopulations (Clusters); Figure 8 As can be seen from the right figure, compared with the Cluster1-Cluster3, Cluster4, and Cluster5 subpopulations, in the Cluster4 subpopulation, the genes related to cranial neural crest-derived stem cells (PDPN and CADM1) were most specifically distributed, and at the same time, it also expressed genes related to human bone stem cell markers (CD164 and NT5E), indicating that the Cluster4 subpopulation may be bone stem cells or cells with similar characteristics to bone stem cells.

[0165] The applicant also performed Regulon transcriptional feature analysis on each cell subpopulation, and the results are as Figure 9 shown. As can be seen from Figure 9 it, the Regulons related to bone stem cells (SOX9 and TWIST1) were concentrated in the Cluster4 cell subpopulation, and at the same time, it was found that the mouse mandibular bone stem cell marker FOXP4 also had specific distribution therein.

[0166] This indicates that the Cluster4 cell subpopulation has the characteristics of bone stem cells, and at the same time indicates that the FOXP4 gene is very likely to be able to serve as a marker for mandibular bone stem cells of human origin.

[0167] Example 9 Flow sorting of mandibular bone stem cells of human origin

[0168] Discarded mandibular bone chips intercepted during the operation of patients undergoing mandibular osteotomy clinically were collected. The periosteum of the bone chips was scraped with a surgical blade and cut into small bone chips, and then the periosteum and the small bone chips were enzymatically digested respectively to obtain periosteal and endosteal cells simultaneously.

[0169] The operation of making a single-cell suspension by enzymatic digestion of the cells was the same as steps 3)-7) in Example 1.

[0170] Except that in step 5), the cell suspension after enzymatic digestion was centrifuged, the supernatant was removed, and the cells were resuspended with a mixed antibody diluent and incubated in the dark on ice for 30 minutes. Among them, the mixed antibodies for incubating human periosteal cells were: APC-CD31, APC-CD45, APC-CD235a, PerCP710-PDPN, biotin-CADM1; then the secondary antibody was incubated: PE / Cy7 Streptavidin antibody was used and incubated in the dark on ice for 30 minutes;

[0171] And in step 7), on the basis of collecting APC signal-negative (Lineage-negative, that is, removing blood lineage and endothelial cells), cells that are positive for PerCP710 signal and positive for PE / Cy7 signal are collected simultaneously as experimental example cells; on the basis of collecting APC signal-negative (Lineage-negative, that is, removing blood lineage and endothelial cells), cells that are negative for PerCP710 signal and negative for PE / Cy7 signal are collected simultaneously as control example cells, which are used for RT-qPCR gene detection and in vitro cell stemness analysis and comparison.

[0172] The specific experimental results are shown in Figure 10 a). As can be seen from Figure 10 a), stem cells that are negative for APC signal, positive for PerCP710 signal and positive for PE / Cy7 signal are selected as experimental example cells; stem cells that are negative for APC signal, positive for PerCP710 signal and negative for PE / Cy7 signal are selected as control example cells.

[0173] The specific RT-qPCR detection results of the FOXP4 gene expression of the experimental example cells and control example cells in this example are shown in Figure 10 b). As can be seen from Figure 10 b), the FOXP4 gene expression level of the experimental example cells is significantly higher than that of the control example cells, indicating that the FOXP4 gene has specific expression in the human-derived mandibular skeletal stem cells sorted in the present invention.

[0174] Example 10 In vitro cell stemness verification (human-derived)

[0175] In this example, the experimental example cells (that is, human-derived Foxp4-positive mandibular skeletal stem cells) and control example cells (that is, human-derived Foxp4-negative mandibular skeletal stem cells) obtained in Example 9 are detected for in vitro colony formation ability and tridirectional differentiation ability.

[0176] The specific steps of the detection method are the same as those of the in vitro cell stemness verification experiment of mouse-derived in Example 4. The specific results are shown in Figure 11 .

[0177] As can be seen from Figure 11 a), compared with the control example cells, the number of stained cells in the experimental example cells is more, indicating that the colony formation number of the experimental example cells is higher. It shows that the experimental example cells, that is, human-derived Foxp4-positive mandibular skeletal stem cells, have stronger colony formation ability.

[0178] As can be seen from Figure 11As shown in b), compared with the cells of the comparative example, the cells of the example showed obvious dark red, indicating that calcium nodules that could be stained red by alizarin red S staining agent were formed in the cells of the example, suggesting that the cells of the example were successfully differentiated into osteoblasts. At the same time, the expression levels of Bglap gene and Alpl gene (osteoblast markers) in the cells of the experimental example were much higher than those of the comparative example, which corroborated the results of the alizarin red staining experiment. It shows that the cells of the example, namely human-derived Foxp4-positive mandibular skeletal stem cells, have stronger osteogenic ability.

[0179] As can be seen from Figure 11 c), compared with the cells of the comparative example, the cells of the example showed obvious blue-violet staining after staining, indicating that chondrocyte spheres were formed in the cell group of the example, while they were not present in the cell group of the comparative example. At the same time, the expression levels of Col2a1 gene and Acan gene (chondrocyte markers) in the cells of the experimental example were much higher than those of the comparative example, which corroborated the results of the toluidine blue staining experiment. It shows that the cells of the example, namely human-derived Foxp4-positive mandibular skeletal stem cells, have stronger chondrogenic ability.

[0180] As can be seen from Figure 11 d), compared with the cells of the comparative example, the cells of the example showed obvious red, indicating that oil red O dye combined with the lipids formed by the cells of the example to form small lipid droplets showing red. It shows that the cells of the example were successfully differentiated into lipids. At the same time, the expression level of Adipq gene (adipocyte marker) in the cells of the experimental example was much higher than that of the comparative example, which corroborated the results of the oil red O staining experiment. It shows that the cells of the example, namely human-derived Foxp4-positive mandibular skeletal stem cells, have stronger adipogenic ability.

[0181] In summary, this example proves that compared with the cells of the comparative example, the cells of the example have stronger colony formation ability and stronger osteogenic, chondrogenic and adipogenic differentiation abilities. It shows that the cells of the example, namely the human-derived Foxp4-positive mandibular skeletal stem cell subset, exhibit superior colony formation and multi-directional differentiation abilities compared to other skeletal stem cells.

[0182] Example 11 In Vivo Osteogenic Experiment by Renal Subcapsular Transplantation (Human-derived)

[0183] Take the cells of the experimental example 9 obtained in the experimental example 9 (namely human-derived Foxp4-positive mandibular skeletal stem cells) and the cells of the comparative example (namely human-derived Foxp4-negative mandibular skeletal stem cells), and transplant them under the renal capsule of NCG immunodeficient mice to observe the new bone formation of cells in different groups under the renal capsule of the recipient NCG mice.

[0184] The specific steps are as follows:

[0185] The experimental example cells and control example cells obtained in Experimental Example 9 were amplified and cultured in vitro. After reaching 80% confluence, they were digested and seeded into a low-attachment U-bottom 96-well plate (Corning, Cat#7007) at a cell density of 3×10 5 / well. Cell spheres could be obtained for in vivo transplantation 2 days after seeding. The cell spheres were wrapped with 0.01 ml of Matrigel (Corning, Cat#356238) and awaited transplantation.

[0186] Grouping of recipient NCG mice: They were randomly divided into 2 groups, namely the experimental example cell transplantation group and the control example cell transplantation group. Then, the experimental example cell spheres and control example cell spheres wrapped in Matrigel were correspondingly implanted under the renal capsule of recipient mice using a capillary tube. The incision of the renal capsule was sealed with an electrocautery knife, and the skin incision was sutured. The recipient mice were euthanized at the 4th week after surgery. The right kidneys of each group of mice were taken, fixed, dehydrated, embedded in paraffin, sectioned, and stained with Movat’s Pentachrome for histological observation.

[0187] The experimental results of Movat’s Pentachrome histological staining are as Figure 12 shown. As Figure 12 can be seen, in the control example cell transplantation group, most of the tissue was regenerated fibrous tissue, with rare regenerated bone tissue and basically no regenerated cartilage tissue. In the experimental example cell transplantation group, compared with the control example cell transplantation group, the proportion of regenerated bone tissue / cartilage tissue formed under the renal capsule of the experimental example cell transplantation group mice was significantly higher (the yellow / green tissue shown in the figure).

[0188] It is shown that the experimental example cells provided by the present invention, namely human-derived Foxp4-positive mandibular skeletal stem cells, have higher osteogenic potential under the renal capsule.

[0189] Among them, the specific construction method of the mouse renal capsule transplantation model used in this example is as follows:

[0190] An immunodeficient NCG mouse (male, 8 - 12 weeks old) was used as the recipient mouse to establish a renal capsule transplantation model. Analgesia was performed by intraperitoneal injection of ketoprofen before and after surgery (administered once every 12 hours until 72 hours after surgery). The NCG mouse was anesthetized and disinfected. A 1 cm incision was made on the right back with a scalpel, and the right kidney was taken out. After making a small incision at the lower pole of the kidney, a mouse renal capsule transplantation model was obtained.

[0191] Example 12 In Vivo Transplantation-Mediated Tooth Regeneration Experiment (Human Origin)

[0192] Take the experimental example cells obtained in Example 9 (i.e., human-derived Foxp4-positive mandibular skeletal stem cells) and the comparative example cells (i.e., human-derived Foxp4-negative mandibular skeletal stem cells), and transplant them into a mouse sinus defect model to observe the tooth regeneration of cells in different groups in the recipient mice.

[0193] Among them, the specific construction method of the mouse sinus defect model is basically the same as that in Example 6, except that immunodeficient NCG mice need to be used to avoid immune rejection of the transplanted human-derived cells by the recipient mice.

[0194] The specific experimental steps are as follows:

[0195] Amplify and culture the experimental example cells and comparative example cells obtained in Example 9 in vitro. After reaching 80% confluence, digest them and inoculate them into a 96-well plate (Corning, Cat#7007) with a low-attachment U-bottom at a cell density of 3×10 5 / well. Cell spheres are obtained 2 days after inoculation for in vivo transplantation. Wrap the cell spheres with 0.05 ml of Matrigel (Corning, Cat#356238) and transplant them into the sinus of the mouse sinus defect model described above in this application. During the transplantation process, make the transplanted cell spheres adhere as closely as possible to the sinus mucosal epithelium.

[0196] Grouping of sinus defect mice: Randomly divide them into an experimental example cell transplantation group and a control example cell transplantation group. Collect the craniofacial bones including the sinuses of the mice in each group 3 weeks after the operation, and perform histological section safranin-fast green staining and immunofluorescence staining experiments.

[0197] The results of safranin-fast green staining and immunofluorescence staining are as Figure 13 shown.

[0198] As can be seen from Figure 13 a and Figure 13 b, compared with the normal tooth tissue structure, the experimental example cell transplantation group of this application can regenerate tooth structures in the mouse sinus, and has complete dentin (den), odontoblasts (od), enamel (ena), and ameloblasts (ame), which are similar to the tooth structure of normal teeth. And the results of immunofluorescence staining show that the mesenchymal cell part of the regenerated tooth structure in the experimental example cell transplantation group in the mouse sinus has red fluorescence, that is, positive for human-Vimentin signal, indicating that it is derived from the experimental example cells provided in Example 9 of this application.

[0199] As can be seen from Figure 13 c and Figure 13 d, the results of safranin-fast green staining show that, compared with the control example cell transplantation group, the experimental example cell transplantation group of this application can regenerate bone tissue in the mouse sinus (specifically Figure 13The blue striped structure (indicated by the black arrow in c), while in the control example cell transplantation group, no blue striped structure was seen and no obvious bone tissue formation occurred.

[0200] In summary, the experimental example cells provided by the present invention, namely human-derived Foxp4-positive mandibular skeletal stem cells, have higher in vivo odontogenic and osteogenic potential; and their odontogenic and osteogenic potential can be verified in vivo through the mouse sinus defect model provided by the present invention.

[0201] The above embodiments merely illustrate the principles and effects of the present invention, rather than limiting the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A subpopulation of mandibular skeletal stem cells, derived from a population of mandibular mesenchymal cells, wherein the subpopulation of mandibular skeletal stem cells is FOXP4 positive.

2. The subpopulation of mandibular skeletal stem cells according to claim 1, wherein The population of mandibular mesenchymal cells is of human origin or mouse origin; and / or, the subpopulation of mandibular skeletal stem cells has any one or more of the following abilities (i) to (iv): (i) The ability to differentiate into bone tissue (ii) The ability to differentiate into cartilage tissue (iii) The ability to differentiate into adipocytes (iv) The ability to form teeth.

3. The subpopulation of mandibular skeletal stem cells according to claim 2, wherein, When the population of mandibular mesenchymal cells is of mouse origin, the subpopulation of mandibular skeletal stem cells is CD200 positive and ENG negative; and / or, when the population of mandibular mesenchymal cells is of human origin, the subpopulation of mandibular skeletal stem cells is CADM1 positive and PDPN positive.

4. A method for sorting the subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3, characterized in that, Comprising the following steps: Surface staining of the population of mandibular mesenchymal cells with a combination of flow antibodies labeled with different fluorophores, after resuspending the stained population of mandibular mesenchymal cells, sorting with a flow cytometer to obtain a population of mandibular skeletal stem cells that are FOXP4 positive, CD200 positive and ENG negative, or FOXP4 positive, CADM1 positive and PDPN positive.

5. The sorting method according to claim 4, characterized in that, The population of mandibular skeletal stem cells is also CD31 negative, CD45 negative, Ter119 negative, Thy1.2 negative and 6C3 negative; and / or, the combination of flow antibodies includes one or more of CADM1 antibody, PDPN antibody, CD200 antibody, ENG antibody; preferably, the combination of flow antibodies further includes: one or more of CD31 antibody, CD45 antibody, Ter119 antibody, Thy1.2 antibody, 6C3 antibody.

6. The sorting method according to claim 5, characterized in that When the population of mandibular mesenchymal cells is of human origin, the combination of flow antibodies includes CADM1 antibody and PDPN antibody; and / or, when the population of mandibular mesenchymal cells is of mouse origin, the combination of flow antibodies includes CD200 antibody and ENG antibody.

7. Use of a subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3 in any one or more of the following: a) Preparation of a medical material for treating tooth defects; b) Preparation of a medical material for treating bone defects.

8. A medical material for treating tooth defects or bone defects, the medical material comprising a stem cell in an effective amount from a subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3.

9. An antibody composition for identifying the subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3, characterized in that, The antibody composition includes CADM1 antibody and PDPN antibody, or, the antibody composition includes CD200 antibody and ENG antibody; Preferably, the antibody composition further includes: one or more of CD31 antibody, CD45 antibody, Ter119 antibody, Thy1.2 antibody, 6C3 antibody.

10. Use of an antibody composition according to claim 9 in identifying a subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3.

11. A kit for identifying the subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3, characterized in that, Comprising an antibody composition according to claim 9.

12. A method for treating tooth defect or bone defect, characterized in that, Is to administer to a subject a medical material comprising a stem cell in an effective amount from a subpopulation of mandibular skeletal stem cells according to any one of claims 1 to 3.

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