Subpopulations of mandibular bone marrow mesenchymal stem cells and methods of isolation and use thereof

Abstract

The application discloses a mandibular mesenchymal stem cell subpopulation and a separation method and application thereof, and belongs to the field of biomedical technology. The stem cell subpopulation simultaneously highly expresses mandibular specific markers Barx1, Ifitm5 and Fbln2 on the cell surface, and the method comprises the following steps: washing and cutting a mandible, and sequentially digesting the mandible with protease and collagenase P; after digestion termination, centrifugation is performed to discard the supernatant; red blood cell lysis solution is added to resuspend; after red blood cell lysis termination, centrifugation is performed again to discard the supernatant; subsequently, cell culture medium is added to resuspend the precipitate, a cell population with negative CD31, CD45 and TER119 is sorted, and dead cells are removed, so that the mandibular mesenchymal stem cell subpopulation is efficiently enriched. The application further discloses application of the stem cell subpopulation in preparation of a drug or medical material for treating bone defects. The application can efficiently and rapidly separate and obtain a large amount of mandibular mesenchymal stem cells, and the stem cells have unique molecular markers and stronger osteogenic differentiation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a subpopulation of mesenchymal stem cells from jawbone bone marrow, its isolation method, and its application. Background Technology

[0002] The maxillofacial bones, as one of the most complex skeletal structures in the human body, not only participate in the formation of facial contours but also play a crucial role in protecting the digestive and respiratory systems and supporting adjacent soft tissues and teeth. Clinically, maxillofacial bone defects caused by factors such as trauma, inflammation, tumors, and congenital diseases are relatively common, often accompanied by significant functional impairment and facial disfigurement, severely affecting patients' mental health and social adaptability.

[0003] Currently, the main methods for repairing jawbone defects include autologous bone grafting, allogeneic bone grafting, and implantation of bone repair materials. Among these, autologous bone grafting has limitations such as limited donor bone volume, secondary trauma, long healing periods, and the need for multiple surgeries. Allogeneic bone grafting faces risks such as immune rejection and potential disease transmission. While alternative strategies, represented by artificial bone repair materials, have good biocompatibility and morphological adaptability, their lack of effective osteogenic induction activity often leads to slow bone integration and unsatisfactory regeneration results.

[0004] In recent years, mesenchymal stem cells (MSCs) have shown great promise in the field of bone tissue regeneration. MSCs with multi-lineage differentiation potential exist in the bone marrow of the jawbone; they can not only self-renew but also differentiate into osteoblasts, adipocytes, and chondrocytes, playing a crucial regulatory role in jawbone development, homeostasis maintenance, and injury repair. Furthermore, these cells participate in the regulation of the bone metabolic microenvironment through various mechanisms, including paracrine signaling, thus possessing unique research value in exploring new strategies for maxillofacial bone repair.

[0005] Multiple studies have shown that bone marrow mesenchymal stem cells (MSCs) derived from the jawbone differ significantly from cells from common sources such as long bones in terms of developmental origin, phenotypic markers, gene expression, and osteogenic / chondrogenic differentiation potential. During jawbone defect repair, they exhibit stronger osteogenic capacity than long bone MSCs, significantly promoting new bone formation, increasing bone density, and effectively inducing angiogenesis. This suggests the existence of a subpopulation of MSCs in the jawbone that is fundamentally different from those derived from long bones. However, to date, no method has been able to efficiently isolate and systematically identify these unique subtypes from the jawbone, nor has their essential differences been revealed at the molecular, transcriptomic, and functional levels, greatly hindering the development of precise jawbone regeneration technologies.

[0006] The maxillofacial skeletal structure is intricate and complex, and the content of mesenchymal stem cells (MSCs) in the bone marrow of the jawbone is low, making it difficult to directly obtain a sufficient number of cells for in-depth research. Existing MSC extraction techniques are all based on long bone tissues such as the femur and tibia, and the mainstream methods fall into two categories: one is to culture digested bone tissue fragments to release cells, as disclosed in publication number CN111349599A; the other is to obtain cells by acquiring and digesting long bone marrow, as described in patent publication number CN110777114A. In both methods, collagenase and dispersase are commonly used digestive enzymes. Long bones have large medullary cavities and abundant bone marrow tissue, making the above methods highly efficient for obtaining long bone MSCs.

[0007] However, the jawbone structure is delicate and complex, with a narrow medullary cavity, extremely low bone marrow content, and dense trabecular structure. Directly applying the long bone MSCs extraction protocol will result in problems such as low cell yield and significant sample heterogeneity, making it impossible to achieve efficient and high-purity acquisition of jawbone-derived MSCs.

[0008] To address the aforementioned issues, existing technologies disclose MSCs-specific extraction methods adapted to the characteristics of jawbone tissue. For example, patent CN120249195A discloses a method for high-concentration enzymatic digestion combined with flow cytometry sorting of transgenic mouse-labeled MSCs. This method involves repeatedly digesting jawbone tissue with a high-concentration complex enzyme to prepare a single-cell suspension, followed by flow cytometry sorting to obtain target stem cells. However, this technology is highly dependent on transgenic animal fluorescent labeling systems such as Foxp4-CreERT2 and tdTomato, making it unsuitable for wild-type animal and human jawbone tissue samples. Furthermore, the high-intensity, multiple-time enzymatic digestion process easily damages the biological activity and surface-specific markers of stem cells, resulting in poor cell fidelity and stability, thus limiting its applicability.

[0009] Patent CN121046307A discloses a protocol for adhering and expanding jawbone tissue blocks. This protocol involves directly adhering jawbone cancellous bone tissue blocks to a culture vessel, inducing spontaneous cell migration and expansion, and then using a limiting dilution method to obtain a mixed population of MSCs. This method requires no enzymatic digestion and does not rely on transgenic mice. However, it has significant limitations: First, adherent culture has a natural selection bias, enriching only cell subpopulations with strong migration ability and adapted to the in vitro adherent growth environment. MSC subtypes located deep within the tissue, with weaker migration ability but potentially crucial stemness maintenance functions, are easily missed, leading to the loss of important functional cell resources. Second, during in vitro adherent culture and expansion, cells are prone to adaptive changes, exhibiting shifts in proliferation characteristics, transcriptional profiles, epigenetic status, and functional phenotypes, gradually deviating from the original biological characteristics of freshly isolated MSCs from the tissue, making it difficult to accurately reflect the in vivo physiological state. Finally, this technology can only obtain mixed MSC populations and cannot achieve precise identification and separation of different functional subtypes. The experimental results obtained essentially reflect the average effect of heterogeneous cell populations, making it difficult to accurately analyze the inherent biological characteristics of each functional subpopulation of jawbone-specific MSCs and their specific roles in maintaining tissue homeostasis and regeneration repair.

[0010] In summary, traditional methods for extracting MSCs from long bones are ill-suited to the anatomical characteristics of the jawbone, which is characterized by low bone marrow content, dense trabeculae, and a fine and complex anatomical structure. Existing jawbone-derived MSC extraction techniques also have inherent limitations. Current technologies not only lack a suitable isolation scheme for jawbone tissue that efficiently extracts highly active and pure MSCs from bone tissue, but more importantly, existing techniques generally fail to recognize a specific stem cell subtype in the jawbone that differs from traditional long bone MSCs in molecular markers, transcriptomic characteristics, and differentiation potential. Furthermore, they cannot achieve precise isolation and identification of this subtype. Therefore, developing a method that enables efficient isolation of jawbone-derived MSCs, high-fidelity preservation of their original characteristics, and precise identification of specific stem cell subtypes is a core technological prerequisite for advancing maxillofacial bone regeneration research and clinical translation, and a key technical challenge that urgently needs to be addressed in this field. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a method for efficiently isolating a subpopulation of bone marrow mesenchymal stem cells with specific molecular markers (Barx1, Ifitm5 and Fbln2) from jawbone tissue, as well as the jawbone bone marrow mesenchymal stem cell subpopulation with unique molecular markers and functional characteristics obtained by the method and its applications.

[0012] In existing technologies, neither MSC extraction methods based on long bones nor publicly available jawbone MSC extraction protocols can solve the separation challenges posed by the low bone marrow content, dense trabecular bone, and intricate and complex anatomical structure of the jawbone. Furthermore, they fail to identify and separate specific MSC subtypes that may exist in the jawbone. This invention, through the synergy of a sequential enzymatic digestion protocol designed specifically for jawbone tissue characteristics and immunomagnetic bead sorting technology, achieves for the first time the efficient, highly active, and highly pure separation of primary jawbone-derived MSCs.

[0013] One of the objectives of this invention is to provide a subpopulation of jawbone bone marrow mesenchymal stem cells, wherein the cell surface of this subpopulation simultaneously highly expresses jawbone-specific markers Barx1, Ifitm5, and Fbln2.

[0014] The second objective of this invention is to provide a simple and effective method for isolating jawbone bone marrow mesenchymal stem cells. The sequential digestion step optimizes the type and time of digestive enzymes to improve the viable cell rate. Using this method, jawbone bone marrow mesenchymal stem cell populations with unique molecular markers and stronger differentiation potential can be specifically obtained.

[0015] A third objective of this invention is to provide the application of the jawbone bone marrow mesenchymal stem cell subpopulation in the preparation of drugs or medical materials for treating bone defects; wherein the bone defect is preferably a jawbone defect.

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a subpopulation of jawbone bone marrow mesenchymal stem cells, which is obtained by sequentially digesting jawbone tissue with a specific concentration of protease and collagenase P for an appropriate time, followed by immunomagnetic bead sorting to select a cell population that is negative for CD31, CD45, and TER119, and finally removing dead cells; and the cell surface of the jawbone bone marrow mesenchymal stem cell subpopulation simultaneously highly expresses jawbone-specific markers Barx1, Ifitm5, and Fbln2.

[0017] In this invention, the term "high expression" refers to a fold change (≥2) in the expression level of the target gene in the test sample relative to the expression level in the control sample.

[0018] A second aspect of this invention discloses a method for isolating a subset of jawbone bone marrow mesenchymal stem cells, wherein the cell surface of the jawbone bone marrow mesenchymal stem cell subset simultaneously highly expresses Barx1, Ifitm5, and Fbln2, and the method includes the following steps: S1. Sequential enzyme digestion: The mandible is cleaned and cut into pieces. First, it is digested with a digestive solution containing protease at 37°C for 10-40 minutes; then, it is digested with a digestive solution containing collagenase P at 37°C for 30-140 minutes. S2. Cell collection: After digestion is complete, centrifuge and discard the supernatant; resuspend in erythrocyte lysis buffer; after erythrocyte lysis is complete, centrifuge again and discard the supernatant, and collect the cell pellet; S3. Stem cell sorting: The collected cell pellet is resuspended in magnetically activated cell sorting buffer, and cell populations that are negative for CD31, CD45, and TER119 are sorted by immunomagnetic beads, and dead cells are removed to efficiently enrich jawbone bone marrow mesenchymal stem cells.

[0019] Addressing the anatomical characteristics of the jawbone—low volume and complex structure—this invention employs a sequential enzymatic digestion method to efficiently obtain a sufficient quantity of jawbone bone marrow mesenchymal stem cells. Compared to traditional PBS washing and collagenase-dispersant enzyme digestion methods, the jawbone bone marrow mesenchymal stem cells extracted using the method of this invention contain a higher number of viable cells. The jawbone involved in this invention has had soft tissue, teeth, and condylar cartilage removed; preferably, the surface muscles and fascia of the mandible have been removed, along with one incisor and three molars, and the cartilage surface layer, including the condylar cartilage.

[0020] Compared to long bones, the jawbone has a more complex structure, containing teeth and condyles. To reduce interference from stem cells in tissues such as dental pulp, periodontal ligament, and condylar cartilage, this invention removes not only soft tissues such as muscles and fascia from the bone surface, but also incisors and molars, as well as the cartilage surface layer including condylar cartilage.

[0021] In some embodiments of the present invention, the digestive solution containing protease is prepared by dissolving protease in αMEM culture medium, wherein the concentration of protease is 2-8 mg / ml, preferably 4 mg / ml; The digestion solution containing collagenase P is prepared by dissolving collagenase P in αMEM culture medium, wherein the concentration of collagenase P is 1~4 mg / ml, preferably 2 mg / ml.

[0022] In some embodiments of the present invention, in step S1, the digestion is carried out on a shaker.

[0023] In some embodiments of the present invention, in step S2, after adding red blood cell lysis buffer and resuspending, the mixture is allowed to stand for 5 to 20 minutes, preferably 10 minutes, and then centrifuged.

[0024] In some embodiments of the present invention, step S3, the immunomagnetic bead sorting specifically includes: The cell pellet collected in step S2 was resuspended in magnetically activated cell sorting buffer, and incubated with a primary antibody conjugated with magnetic microbeads. The primary antibody was CD31 antibody, CD45 antibody and TER119 antibody. After primary antibody incubation, magnetic separation was performed, and the negative separation solution was collected. Cells were collected by centrifugation of the negative sorting solution, and the cell pellet was washed with magnetically activated cell sorting buffer. After removing the supernatant, the cell pellet was resuspended, and dead cell removal beads were added for magnetic labeling and incubation. Magnetic separation was performed again, the negative sorting solution was collected, centrifuged, and the cell pellet was the jawbone bone marrow mesenchymal stem cells.

[0025] In some embodiments of the present invention, after adding a primary antibody coupled with magnetic microbeads, the mixture is incubated at 4°C for 0.5 to 2 hours, preferably 1 hour. After adding dead cells to remove the microbeads, incubate at 4°C in the dark for 5-30 minutes, preferably 15 minutes.

[0026] The third aspect of this invention discloses the application of a subset of jawbone bone marrow mesenchymal stem cells in the preparation of drugs or medical materials for treating bone defects.

[0027] In some embodiments of the present invention, the bone defect is a jawbone defect.

[0028] This invention, through comparative studies, reveals for the first time that bone marrow-derived mesenchymal stem cells (OMSCs) obtained from mouse jawbone using the method of this invention exhibit significant differences in cell population composition, gene expression lineage, and differentiation potential compared to bone marrow-derived mesenchymal stem / stromal cells (BMSCs) isolated using existing techniques. OMSCs represent a novel cell population with unique biological characteristics. First, single-cell transcriptome analysis shows that the distribution regions of OMSCs and BMSCs in the tSNE dimensionality reduction space almost do not overlap, indicating that they are cell subpopulations with distinctly different transcriptome characteristics. Furthermore, differentially expressed gene analysis of OMSCs and BMSCs reveals that significantly upregulated (DEGs) genes in OMSCs are mainly enriched in osteogenic differentiation and other related biological processes. The expression levels of key osteogenic genes (such as Ibsp, Spp1, and Dlx5) and chondrogenic genes (such as Sox9) are significantly higher in OMSCs than in BMSCs, suggesting a stronger osteogenic differentiation potential. This invention also identifies for the first time OMSCs-specific genes, including Barx1, Ifitm5, and Fbln2, which are almost not expressed in BMSCs. Using the method of this invention, a subpopulation of jawbone bone marrow mesenchymal stem cells with unique molecular markers and stronger osteogenic differentiation potential can be obtained.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention, for the first time, addresses the anatomical characteristics of the jawbone, such as its narrow medullary cavity, dense trabeculae, and abundant fibrous components, by establishing a method involving sequential digestion by protease and collagenase P, combined with CD31. - / CD45 - / TER119 - This invention relates to a separation method consisting of immunomagnetic bead sorting and dead cell removal steps. It effectively solves the core technical bottleneck of existing technologies, which suffer from extremely low cell yield and poor cell activity due to the inability to fully dissect dense trabeculae of the jawbone. This method enables the stable and efficient acquisition of large quantities of high-purity, highly active jawbone bone marrow-derived mesenchymal stem cells.

[0030] Building upon this foundation, this invention, for the first time, isolated and identified a subpopulation of osteogenic bone marrow mesenchymal stem cells (OMSCs) from jawbone bone marrow that simultaneously highly expresses Barx1, Ifitm5, and Fbln2 on their cell surface. Single-cell transcriptome sequencing revealed that the distribution of this subpopulation in the tSNE dimensionality-reduced space almost completely overlapped with that of long bone BMSCs, indicating that the two are cell populations with distinctly different transcriptome characteristics. Furthermore, the expression of the aforementioned three marker genes was extremely low in long bone BMSCs, suggesting that this represents a novel stem cell subtype specific to jawbone tissue. This achievement fills a long-standing gap in the field's understanding of jawbone-specific MSC subtypes, providing novel cellular resources and molecular targets for research on craniofacial development and regeneration.

[0031] The OMSCs subsets obtained in this invention naturally overexpress key osteogenic genes (Ibsp, Spp1, Dlx5) and key chondrogenic genes (Sox9), possessing excellent osteogenic differentiation potential and showing significant advantages in colony formation and stemness maintenance. They can be used as high-quality seed cells for the repair and treatment of maxillofacial bone defects, overcoming the clinical challenges of limited supply and lack of bioactivity of traditional autologous bone transplantation and artificial bone materials.

[0032] The jawbone bone marrow mesenchymal stem cells obtained using the method of this invention were identified by flow cytometry and single-cell transcriptome sequencing as stably and highly expressing Barx1, Ifitm5, and Fbln2, demonstrating the reliability of the method and the characteristic stability of the obtained cell subpopulations.

[0033] This method is simple to operate and has significant isolation and culture efficiency. It is not only applicable to the isolation and culture of wild-type mouse jawbone bone marrow mesenchymal stem cells, but can also be extended to the isolation and culture of transgenic mouse and human jawbone bone marrow mesenchymal stem cells. Attached Figure Description

[0034] Appendix Figure 1 This is a flowchart of the primary isolation and culture process of mouse jawbone bone marrow mesenchymal stem cells in Example 1.

[0035] Appendix Figure 2This is a colony map of mesenchymal stem cells in the bone marrow of a mouse jawbone.

[0036] Appendix Figure 3 The images show the results of the three-lineage differentiation identification of mouse jawbone bone marrow mesenchymal stem cells. From left to right, they are the staining results of osteogenic differentiation, adipogenic differentiation, and chondrogenic differentiation.

[0037] Appendix Figure 4 UMAP diagram of mouse jawbone bone marrow cell populations; Figure 4 The English translations in the Chinese text are as follows: Cxcl12-abundant reticular cell (CAR): Cxcl12-rich reticular cell; Chondrocyte: chondroblast; Chondrocyte progenitor: chondroblast progenitor; Early mesenchymal progenitors (EMP): early mesenchymal progenitor; Late mesenchymal progenitors (LMP): late mesenchymal progenitor; Matureosteoblast: mature osteoblast; Neurological cell: nerve cell; Osteoblast: osteoblast; Osteoprogenitor: osteoblast progenitor; Preosteoblast: preosteoblast; Smooth muscle cell (SMC): smooth muscle cell.

[0038] Appendix Figure 5 UMAP diagram showing the expression of specific markers for bone marrow mesenchymal stem cells in mouse jawbone.

[0039] Appendix Figure 6 The figure shows the cell counting results of mouse jawbone bone marrow mesenchymal stem cells isolated by different methods.

[0040] Appendix Figure 7 Cell colony diagrams of mouse jawbone bone marrow mesenchymal stem cells isolated by different methods.

[0041] Appendix Figure 8 The graph shows the percentage of Gli1-positive cells in mouse jawbone bone marrow mesenchymal stem cells obtained by the method of this invention; the horizontal axis FL2-A::PE-A represents the intensity of PE fluorescence signal detected by the FL2 channel.

[0042] Appendix Figure 9 The images show fluorescence and bright-field images of mouse jawbone bone marrow mesenchymal stem cells obtained by the method of this invention after culture, with the left image being the fluorescence image and the right image being the bright-field image.

[0043] Appendix Figure 10This figure shows the expression of osteogenic and chondrogenic genes in third-generation long bone marrow mesenchymal stem cells and mouse jawbone marrow mesenchymal stem cells induced by osteogenic factors.

[0044] Appendix Figure 11 tSNE clustering diagram of jawbone MSCs and long bone MSCs.

[0045] Appendix Figure 12 Bubble chart of GO enrichment analysis of upregulated DEGs in jawbone MSCs compared to long bone MSCs; Figure 12 The corresponding Chinese names for the English terms are as follows: angiogenesis, Ossification, Osteoblast differentiation, Skeletal system development, Bloodvessel development, Positive regulation of osteoblast differentiation, Cartilage development, Bonemineralization, Chondrocyte differentiation, Gene Number, P-value, and Rich Factor.

[0046] Appendix Figure 13 This image shows a comparison of the expression distribution of Barx1, Ifitm5, and Fbln2 genes in jawbone MSCs and long bone MSCs. Figure 13 The corresponding Chinese names for the English terms are as follows: UMAP_1, UMAP_2: the first and second principal components after dimensionality reduction by Unified Manifold Approximation and Projection (UMAP).

[0047] Figure 14 This figure shows the differential expression levels of Barx1, Fbln2, and Ifitm5 genes in the mouse mandible and long bone tissues.

[0048] Figure 15 The image shows a comparison of the t-SNE dimensionality reduction distribution of Experiment Example 7. The left image shows the t-SNE clustering distribution of cell subpopulations in the unselected group, and the right image shows the t-SNE distribution of target marker-positive cells in the selected group. Figure 15 The corresponding Chinese names for the English terms are: tSNE_1: t-SNE dimension 1, tSNE_2: t-SNE dimension 2. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific examples. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0050] The experimental reagents, instruments, and manufacturers used in this invention are as follows: PBS: Brand Gibco, Product No. C10010500BT; Protease: Supplied by Roche, catalog number 10165921001; Collagenase P: Supplied by Roche, catalog number 11213857001; αMEM: Brand Gibco, Product No. C12571500BT; Pancreatic enzyme: Brand Gibco, Product No. 25200056; Bovine serum albumin: Brand BioFroxx, Product No. 4240GR100; Red Blood Cell Lysis Solution (10×): Supplied by Miltenyi Biotec, Germany, catalog number 130-094-183; Dead Cell Removal Kit: Supplied by Miltenyi Biotec, Germany, catalog number 130-090-101; Anti-Ter-119 MicoBeads (mice): Supplied by Miltenyi Biotec, Germany, catalog number 130-049-901; CD45 magnetic beads for mice (CD45 MicroBeads): Supplied by Miltenyi Biotec, Germany, catalog number 130-052-301; CD31 magnetic beads for mice (CD31 MicroBeads): Supplied by Miltenyi Biotec, Germany, catalog number 130-097-418; OctoMACS™ Separator: Supplied by Miltenyi Biotec, Germany, item number 130-042-109; Magnetic sorting columns (MS Columns): Supplied by Miltenyi Biotec GmbH, Germany, item number 130-042-201.

[0051] Example 1 This embodiment discloses the method for primary cell isolation and culture of mouse jawbone bone marrow mesenchymal stem cells according to the present invention, the process of which is attached. Figure 1 As shown, the details are as follows: Step 1. Take a fresh mouse mandible, place it in PBS, and transport or store it on ice.

[0052] Step 2. In a clean bench, using microsurgical scissors and microsurgical forceps under a microscope, remove the surface muscle and fascia, remove one incisor and three molars, and remove the cartilage surface layer, including the condylar cartilage. During this process, the tissue should be immersed in PBS to keep it moist. The processed mandible is then minced and transferred to a 15ml centrifuge tube.

[0053] Step 3. Dissolve the protease in αMEM medium to prepare digestion solution 1 with a protease concentration of 4 mg / ml. Add an appropriate amount of digestion solution 1 to a centrifuge tube containing mandibular bone fragments to completely submerge the bone particles; then place the centrifuge tube in a shaker at 37°C and 100 rpm for 20 minutes to ensure complete enzymatic digestion of the tissue.

[0054] Step 4. After digestion, attach a 70μm cell filter to a 50mL centrifuge tube and filter the digestion solution into the tube. Add an equal volume of complete culture medium (αMEM medium + 10% fetal bovine serum + 1% penicillin / streptomycin) to terminate the digestion reaction, and temporarily store the filtrate at 4℃.

[0055] Step 5. Dissolve collagenase P in αMEM medium to prepare digestion solution 2 with a collagenase P concentration of 2 mg / ml. Add digestion solution 2 to the remaining tissue fragments from step 4, and continue digestion at 37°C and 100 rpm for 70 min.

[0056] Step 6. After digestion, filter the digestion solution from step 5 into the filtrate obtained in step 4 using the same 70μm filter screen; then wash the remaining tissue with complete culture medium and filter it into the above filtrate to terminate digestion.

[0057] Step 7. Centrifuge the cell suspension from step 6 at 1200 rpm for 5 min, discard the supernatant, add 3-5 times the volume of erythrocyte lysis buffer to the cell pellet, resuspend, and let stand at room temperature or 4°C for 10 min.

[0058] Step 8. After the red cleavage is complete, centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

[0059] Step 9. Wash the cell pellet with magnetically activated cell sorting buffer (PBS, supplemented with 0.5% bovine serum albumin and 2 mM EDTA) and resuspend (per 10 mM). 7 Each cell requires 90 μL of buffer.

[0060] Step 10. Add magnetic beads and incubate at 4°C for 15 minutes. Centrifuge to collect cells, wash the cell pellet with magnetic cell sorting buffer and resuspend; use a magnetic sorting column and OctoMACS. TM The separator performs magnetic separation, loading the cell suspension onto the column and washing it with magnetically activated cell sorting buffer. The cell suspension that did not bind to the magnetic beads is collected, i.e., the negative sorting buffer. The magnetic microbeads used in this step are Anti-Ter-119 MicroBeads, CD45 MicroBeads, and CD31 MicroBeads, with each type used in a 103 liter. 7 10 μL per cell.

[0061] Step 11. Collect the cell pellet from the negative sorting solution in Step 10, resuspend it in the Dead Cell Removal Kit, and incubate for 15 minutes at room temperature in the dark to complete magnetic labeling. Centrifuge to collect cells, wash the cell pellet with magnetically activated cell sorting buffer, and resuspend; then use the magnetic sorting column and OctoMACS again. TM The separator performs magnetic separation and collects the negative separation solution.

[0062] Step 12. Collect the cell pellet from the negative sorting solution in Step 11, which is mouse jawbone bone marrow mesenchymal stem cells; resuspend in complete culture medium, count the cells, select an appropriate culture dish according to the number, and continue culturing in a 37℃, 5% CO2 incubator.

[0063] The colony map of mouse jawbone bone marrow mesenchymal stem cells obtained in this embodiment is attached. Figure 2 As shown.

[0064] Comparative Example 1 Compared to Example 1, Comparative Example 1 did not screen for CD31, CD45, and TER119 markers. The specific steps are as follows: Steps 1 to 9: Same as steps 1 to 9 in Example 1; Step 10. Add the Dead Cell Removal Kit (DCDK) magnetic beads to complete the magnetic labeling. Centrifuge to collect cells, wash the cell pellet with magnetically activated cell sorting buffer and resuspend; use a magnetic sorting column and OctoMACS. TM The separator performs magnetic separation and collects the negative separation solution.

[0065] Step 11. Collect the cell pellet from the negative sorting solution, i.e., mouse jawbone bone marrow mesenchymal stem cells; resuspend in complete culture medium, count and seed in appropriate culture dishes, and continue to culture in a 37°C, 5% CO2 incubator.

[0066] Experimental Example 1 Mesenchymal stem cells from mouse jawbone bone marrow were induced to differentiate into osteoblasts, adipocytes, and chondrocytes, respectively, to obtain osteocytes, adipocytes, and chondrocytes. Specific experiments are as follows: 1. The experiment used third-generation mouse jawbone bone marrow mesenchymal stem cells.

[0067] 2. Osteogenic induction: Cells were treated with osteogenic differentiation medium (complete medium, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate), and the medium was changed every two days for 7 days.

[0068] 3. Adipogenic induction: On days 1 and 2, cells were treated with adipocyte differentiation medium (complete medium, 500 μM IBMX, 1 μM dexamethasone, 5 μg / ml insulin and 1 μM rosiglitazone); for the next 4 days, the medium was changed to adipocyte differentiation basal medium (complete medium, 5 μg / ml insulin and 1 μM rosiglitazone) every two days.

[0069] 4. Chondrogenic induction: Mouse jawbone bone marrow mesenchymal stem cells were inoculated at a rate of 3 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / tube in 1.5 mL microcentrifuge tubes to form three-dimensional chondrocytes. The chondrocyte differentiation medium was changed every two days. Results are attached. Figure 3 As shown in the attached figure. The results indicate that the mouse jawbone bone marrow mesenchymal stem cells obtained using the method of this invention possess the ability to differentiate into osteogenic, adipogenic, and chondrogenic lines, consistent with the biological characteristics of stem cells. Figure 3 .

[0070] Experimental Example 2 In this experiment, single-cell sequencing was performed on mesenchymal stem cells from mouse jawbone bone marrow. The specific procedures are as follows: 1. Based on the 10X Genomics 3' transcriptome sequencing method, gel beads containing barcode information are combined with a mixture of cells and enzymes, and then encapsulated by oil surfactant droplets in a microfluidic system to form a microdroplet structure that encapsulates the gel beads in an oil phase emulsion (GEM).

[0071] 2. The gel beads in the GEM dissolve, releasing the barcode sequence, which is then reverse transcribed to produce barcoded cDNA for sequencing. After the liquid oil layer is disrupted, PCR amplification is performed using the cDNA as a template. The amplified product is purified and quality-checked. Once the quality-checked product passes, a library is constructed.

[0072] 3. High-throughput sequencing of the constructed library was performed using the paired-end sequencing mode of the Illumina sequencing platform.

[0073] The results are attached. Figure 4 and appendix Figure 5 As shown, the results indicate that mouse jawbone bone marrow mesenchymal stem cell-specific markers are highly expressed in the extracted mouse jawbone bone marrow cells.

[0074] Experimental Example 3 This experiment investigated the cell count of mouse jawbone bone marrow mesenchymal stem cells isolated using different methods.

[0075] 1. Extraction Method 1: PBS Washing Method The method for preparing mouse mandibular bone samples is the same as in Example 1 of this invention. The shredded mouse mandibular bone is rinsed with PBS, the rinsing solution is collected, and the cell pellet is obtained by centrifugation.

[0076] 2. Extraction Method 2: Collagenase combined with dispersant digestion method The preparation method for mouse mandible samples was the same as in Example 1 of this invention. Collagenase and dispersant enzyme were dissolved in αMEM medium to prepare a digestion solution with a collagenase concentration of 3 mg / ml and a dispersant enzyme concentration of 4 mg / ml. The shredded mouse mandible was placed in the digestion solution and digested at 37°C for 1 hour. Subsequently, the digestion solution was filtered through a 70 μm filter, and the remaining tissue was washed with complete culture medium and then filtered into the above filtrate to terminate digestion. The cell pellet was obtained by centrifugation.

[0077] 3. Extraction Method 3: The method of Embodiment 1 of the present invention.

[0078] The results are attached. Figure 6 and appendix Figure 7 As shown in the figure. The results indicate that the mouse jawbone bone marrow mesenchymal stem cells extracted using the method of this invention have significantly more viable cells and greater cell colony-forming ability than those extracted using the other two methods.

[0079] Test Example 4 In this experimental case, flow cytometry was used to perform fluorescence analysis on the mouse jawbone bone marrow mesenchymal stem cells extracted in Example 1. The specific procedure is as follows: Transgenic mice of Gli1CreER; tdTomato were induced to express red fluorescent protein in Gli1-positive cells after tamoxifen injection. Mandibular bones of the transgenic mice were collected, and bone marrow mesenchymal stem cells were extracted using the method described in Example 1. The extracted cells were resuspended in αMEM medium and washed with 5% BSA. Flow cytometry was performed using FACSCalibur. Each dataset was analyzed using FlowJo software.

[0080] The results are attached. Figure 8 and attached Figure 9 As shown, the Gli1-positive cells accounted for up to 90% of the mouse jawbone bone marrow mesenchymal stem cells extracted by the method of this invention, and were labeled with red fluorescence. Gli1 is a stem cell marker, and the above results indicate that the method of this invention can efficiently enrich and obtain high-purity jawbone bone marrow mesenchymal stem cells.

[0081] Experimental Example 5 In this experiment, the expression of osteogenic and chondrogenic genes in third-generation long bone BMSCs induced by osteogenic fusion and third-generation mouse OMSCs obtained in Example 1 of this invention was detected by qPCR technology.

[0082] In this experimental example, BMSCs were obtained by extracting primary cells. The specific method is as follows: long bones of mice were collected, cut, and centrifuged to collect bone marrow. Red blood cells were lysed according to steps 7 and 8 of Example 1, and the cell pellet was collected, resuspended in complete culture medium, and cultured to the third generation.

[0083] The specific procedures for gene detection in this experiment are as follows: Two cell lines were cultured for 7 days using the induction culture method described in "2. Osteogenic Induction" of Experiment 1. Total RNA was extracted and reverse transcribed into cDNA using a quantitative PCR-specific reverse transcription reagent for subsequent qRT-PCR experiments. The reaction system was prepared according to the SYBR Green qRT-PCR reagent instructions. 96-well plates were loaded with the reagents and subjected to pre-denaturation, denaturation, annealing, and melting curve analysis. Results were corrected using the expression level of an internal control gene. ΔΔCT Methods for calculating gene expression levels.

[0084] The results are attached. Figure 10 As shown, the results indicate that OMSCs obtained by the method of this invention have significantly higher expression of osteogenic and chondrogenic genes than BMSCs, suggesting that they have stronger potential for osteogenic and chondrogenic differentiation.

[0085] Experimental Example 6 This experiment analyzed single-cell RNA sequencing (scRNA-seq) data from long bone-derived cells and jawbone-derived cells of this invention. Specifically, the scRNA-seq dataset of mouse jawbone bone marrow mesenchymal stem cells obtained in Experiment 2 was merged with the scRNA-seq dataset of 12-week-old mouse long bone Col2+ cells (ZHONG L, YAO L, TOWER RJ, et al. Single cell transcriptomics identifies a unique adipose lineage cellpopulation that regulates bone marrow environment [J]. Elife, 2020, 9. DOI:10.7554 / eLife.54695.). After quality control and standardization, the data were integrated for analysis. Regarding phenotypic marker identification, differential gene expression analysis was performed on mouse OMSCs subpopulations and long bone-derived BMSCs subpopulations.

[0086] The results are attached. Figure 11 ~Appendix Figure 13 As shown: (1) tSNE clustering diagram of cells annotated according to sample source (jawbone and long bone) (attached) Figure 11 This indicates that the distribution areas of the cell populations derived from the jawbone and long bones have almost no overlap, and there are significant differences between the two.

[0087] (2) GO enrichment analysis bubble chart of differentially expressed genes (DEGs) upregulated by jawbone MSCs compared with long bone MSCs (attached) Figure 12 The results showed that the genes upregulated in OMSCs were significantly enriched in osteogenic differentiation-related biological processes, and osteogenic-related genes (such as Ibsp, Spp1, Dlx5) and chondrogenic-related genes (such as Sox9) were all significantly overexpressed in OMSCs.

[0088] (3) The results showed that Barx1, Ifitm5 and Fbln2 genes were specifically highly expressed in jawbone bone marrow mesenchymal stem cells, but almost not expressed in long bone-derived BMSCs (see attached). Figure 13 and appendix Figure 14 (As shown).

[0089] The results indicate that OMSCs may maintain their stemness by activating multiple signaling pathways and possess stronger osteogenic and chondrogenic differentiation potential.

[0090] Experimental Example 7 To verify the enrichment effect of the screening method described in Example 1 on the target cell population, this experiment analyzed and compared the cells obtained in Example 1 and Comparative Example 1. Specifically, the cell samples obtained by screening in Example 1 and the cell samples of Comparative Example 1 that were not screened for CD31, CD45, and TER119 markers were detected by flow cytometry and scRNA-seq, respectively, to evaluate the impact of the screening steps on the purity and cell population composition of Gli1+ mesenchymal stem cells.

[0091] The results are as follows: (1) Flow cytometry results showed that after screening with CD31, CD45 and TER119 markers, the proportion of Gli1+ mesenchymal stem cells in the sample could reach about 90%, indicating that the target mesenchymal stem cells were significantly enriched.

[0092] (2) The results of single-cell transcriptome sequencing combined with tSNE dimensionality reduction visualization analysis are attached. Figure 15 As shown. tSNE plot of the unselected group (attached) Figure 15 The right figure shows that Gli1+ cells, represented by purple dots, are scattered among a large number of non-target cells, accounting for approximately 5%; the remaining cells are mostly CD31+, CD45+, and TER119+ cell populations. This result indicates that, without screening, the proportion of target Gli1+ mesenchymal stem cells in the original cell population is low.

[0093] The results show that after screening with CD31, CD45 and TER119 markers, the proportion of Gli1+ mesenchymal stem cells increased from about 5% to about 90% without screening, indicating that the screening step can effectively remove non-target cells and significantly improve the purity of the target cell population.

[0094] This demonstrates that the screening method described in Example 1 can effectively enrich Gli1+ mesenchymal stem cells, which plays an important role in obtaining a high-purity target cell population and can provide a foundation for subsequent cell identification, functional studies and related applications.

[0095] In summary, the bone marrow mesenchymal stem cells (OMSCs) obtained from mouse jawbone using the method of this invention represent a novel cell subpopulation that differs fundamentally from conventional long bone-derived bone marrow mesenchymal stem cells (BMSCs) in terms of population composition, molecular phenotype, and functional potential. Specifically: 1) OMSCs exhibit a unique, almost non-overlapping, independent distribution in single-cell transcriptome mapping, confirming their distinct cell population; 2) This population displays stronger expression characteristics of osteogenic and chondrogenic differentiation-related genes, indicating superior osteogenic differentiation potential; 3) This invention is the first to identify the specific high expression of genes such as Barx1, Ifitm5, and Fbln2 in OMSCs, providing specific molecular markers that distinguish them from known BMSCs. Furthermore, the method of this invention effectively enriches the target cell subpopulation, a necessary step in obtaining high-purity mesenchymal stem cells. Without screening, the cell purity requirements for subsequent research or applications cannot be met. Therefore, the method described in this invention is essentially an effective means of specifically obtaining and enriching the jawbone bone marrow mesenchymal stem cell subpopulation with unique advantages.

[0096] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalent alterations can be made within the spirit and scope defined by the claims of the present invention, but all such changes will fall within the protection scope of the present invention.

Claims

1. A subset of jawbone bone marrow mesenchymal stem cells, characterized in that, The jawbone bone marrow mesenchymal stem cell subpopulation was obtained by sequentially digesting jawbone tissue with protease and collagenase P, followed by immunomagnetic bead sorting to select a cell population that was negative for CD31, CD45, and TER119, and finally removing dead cells. The cell surface of the jawbone bone marrow mesenchymal stem cell subpopulation also highly expressed jawbone-specific markers Barx1, Ifitm5, and Fbln2.

2. The method for isolating a subset of jawbone bone marrow mesenchymal stem cells according to claim 1, characterized in that, Includes the following steps: S1. Sequential enzyme digestion: After cleaning and cutting the mandible into small pieces, digest it first with a digestive solution containing protease at 37°C for 10-40 minutes; then digest it with a digestive solution containing collagenase P at 37°C for 30-140 minutes. S2. Cell collection: After digestion is complete, centrifuge and discard the supernatant; resuspend in erythrocyte lysis buffer; after erythrocyte lysis is complete, centrifuge again and discard the supernatant, and collect the cell pellet; S3. Stem cell sorting: The collected cell pellet was resuspended in magnetically activated cell sorting buffer, and cell populations that were negative for CD31, CD45, and TER119 were sorted by immunomagnetic beads. Dead cells were removed to efficiently enrich the jawbone bone marrow mesenchymal stem cell subpopulation.

3. The method for isolating a subset of jawbone bone marrow mesenchymal stem cells according to claim 2, characterized in that, The digestive solution containing protease was prepared by dissolving the protease in αMEM medium, wherein the concentration of the protease was 2-8 mg / ml; The digestion solution containing collagenase P was prepared by dissolving collagenase P in αMEM medium, wherein the concentration of collagenase P was 1~4 mg / ml.

4. The method for isolating a subset of jawbone bone marrow mesenchymal stem cells according to claim 2, characterized in that, In step S1, the digestion is carried out on a shaker.

5. The method for isolating a subset of jawbone bone marrow mesenchymal stem cells according to claim 2, characterized in that, In step S2, after adding red blood cell lysis buffer and resuspending, let stand for 5-20 minutes, and then centrifuge.

6. The method for isolating a subset of jawbone bone marrow mesenchymal stem cells according to claim 2, characterized in that, In step S3, the immunomagnetic bead sorting specifically includes: The cell pellet collected in step S2 was resuspended in magnetically activated cell sorting buffer, and incubated with a primary antibody conjugated with magnetic microbeads. The primary antibody was CD31 antibody, CD45 antibody and TER119 antibody. After primary antibody incubation, magnetic separation was performed, and the negative separation solution was collected. Cells were collected by centrifugation of the negative sorting solution, and the cell pellet was washed with magnetically activated cell sorting buffer. After removing the supernatant, the cell pellet was resuspended, and dead cell removal beads were added for magnetic labeling and incubation. Magnetic separation was performed again, the negative sorting solution was collected, centrifuged, and the cell pellet was the jawbone bone marrow mesenchymal stem cell subpopulation.

7. The method for isolating a subset of jawbone bone marrow mesenchymal stem cells according to claim 6, characterized in that, After adding the primary antibody coupled with magnetic microbeads, incubate at 4°C for 0.5–2 hours; After adding dead cells to remove the microbeads, incubate at 4°C in the dark for 5-30 minutes.

8. The application of the jawbone bone marrow mesenchymal stem cell subpopulation according to claim 1, characterized in that, The application of the jawbone bone marrow mesenchymal stem cell subpopulation in the preparation of drugs or medical materials for treating bone defects.

9. The application of the jawbone bone marrow mesenchymal stem cell subset according to claim 8, characterized in that, The bone defect is a jawbone defect.

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