Stem cells, preparation methods and applications thereof, and labeling compositions and applications thereof

Abstract

This application discloses a stem cell, a preparation method and application thereof, and a labeling composition and application thereof. The stem cell expresses markers including CX43 and FGFR2. This application identifies and isolates a new stem cell with strong osteogenic and chondrogenic differentiation capabilities, and its osteogenic capacity is even stronger than that of traditional bone marrow mesenchymal stem cells (BMSCs). This stem cell can be used to repair bone defects in animals or humans. Using this stem cell, bone defects can be effectively and rapidly treated, shortening bone repair time and significantly improving prognosis.

Description

Technical Field

[0001] The present application relates to the field of stem cells, and in particular to a stem cell, a preparation method and application thereof, and a labeling composition and application thereof. Background Art

[0002] Severe bone structure defects caused by trauma, congenital deformities or extensive tumor surgery usually require surgical reconstruction. Bone defects have always been a major concern in trauma orthopedics. At the same time, bone defects can also lead to nonunion, making the patient's prognosis even worse. This not only undermines the patient's confidence in overcoming the disease, but also makes the repair of bone defects more and more popular. The current preferred method for treating bone defects is autologous bone or allogeneic bone transplantation. Although autologous bone transplantation has low immunogenicity, the material is limited, bone harvesting is invasive, and complications occur after bone harvesting. Allogeneic bone transplantation is limited in its wide clinical application due to the frequent immune response. Currently, stem cell-based regenerative medicine treatment is expected to become the next important treatment direction. Summary of the Invention

[0003] In view of this, the present application provides a stem cell and a preparation method and application thereof, as well as a labeling composition and application thereof. The present application aims to provide a stem cell with strong bone repair ability.

[0004] The embodiment of the present application is implemented as follows:

[0005] In a first aspect, the present application provides a stem cell, wherein the markers expressed by the stem cell include CX43 and FGFR2.

[0006] Optionally, in some embodiments of the present application, the stem cells are CX43 and FGFR2 positive.

[0007] Optionally, in some embodiments of the present application, the stem cells contain at least one of the following gene markers: FKBP11, PTN, TNN, TNC, OMD, ACTA2 and MARCKS.

[0008] Optionally, in some embodiments of the present application, the stem cells are mesenchymal stem cells; and / or,

[0009] The stem cells are derived from human stem cells or animal stem cells.

[0010] In a second aspect, the present application proposes a method for preparing stem cells, comprising the following steps:

[0011] Providing a cell population sample and an antibody composition comprising an anti-CX43 antibody and an anti-FGFR2 antibody;

[0012] staining the cell population sample using the antibody composition;

[0013] The cells that are positive for CX43 and FGFR2 in the cell population sample are separated to obtain stem cells.

[0014] In a third aspect, the present application further proposes a marker composition for screening or identifying stem cells, wherein the marker composition comprises CX43 and FGFR2.

[0015] Optionally, in some embodiments of the present application, the expression product of at least one gene among FKBP11, PTN, TNN, TNC, OMD, ACTA2 and MARCKS is also included.

[0016] In a fourth aspect, the present application further proposes a use of the labeling composition as described above in preparing a kit for isolating or identifying stem cells.

[0017] In a fifth aspect, the present application further provides a use of stem cells in preparing a product for treating bone defects, wherein the stem cells include the stem cells described above, or the stem cells include stem cells prepared by the preparation method described above, or the stem cells include stem cells expressing the marker composition described above;

[0018] The bone defect treatment products include medicines for treating bone defects, materials for treating bone defects or kits for treating bone defects.

[0019] Optionally, in some embodiments of the present application, the drug for treating bone defects comprises the stem cells and a pharmaceutically acceptable carrier; and / or,

[0020] The material for treating bone defects includes any one of tissue engineering bone materials, bone defect scaffold materials, and bone defect composite repair materials; and / or,

[0021] The application object of the bone defect treatment product is humans or animals.

[0022] Beneficial effects:

[0023] In the technical solution provided in this application, a new stem cell is identified and isolated. The stem cell has strong osteogenic and chondrogenic differentiation ability, and its osteogenic ability is stronger than that of traditional bone marrow mesenchymal stem cells (BMSCs). It can be used to repair bone defects in animals or humans. Using this stem cell, bone defects can be effectively and quickly treated, shortening bone repair time and significantly improving prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 This is a morphological rendering of a stem cell proposed in an embodiment of the present application;

[0026] Figure 2 This is a violin plot of stem cell gene marker expression proposed in an embodiment of the present application;

[0027] Figure 3 It is a 10× single-cell transcriptome map of the regenerating tissue of mouse P3 fingertips and deer antler bud base;

[0028] Figure 4 This is a diagram showing the qualitative test results of osteogenic and chondrogenic differentiation abilities in Experimental Example 1;

[0029] Figure 5 This is a graph showing the quantitative PCR test results for osteogenic and chondrogenic differentiation efficiency in Experimental Example 1;

[0030] Figure 6 1 is a graph showing the results of monoclonal ability testing of stem cells obtained in Example 1 and Comparative Examples 2-4;

[0031] Figure 7 This is the result of Ponceau staining of new bone under the renal capsule in Experimental Example 2;

[0032] Figure 8 This is a comparison chart of the statistical results of the new bone area in Experimental Example 2;

[0033] Figure 9 1 is a micro-CT image of the lateral condyle of the defect group, the comparative example treatment group, and the example stem cell treatment group in Experimental Example 3;

[0034] Figure 10 Statistical graph of the volume fraction of newly formed bone at the defect site in the defect group, the comparative example treatment group, and the example stem cell treatment group in Experimental Example 3;

[0035] Figure 11 This is a statistical diagram of the number of newly formed bone trabeculae at the defect site in the defect group, the comparative example treatment group, and the example stem cell treatment group in Experimental Example 3;

[0036] Figure 12 3 is the HE staining of the lateral condyle of the defect group, the comparative example treatment group and the example stem cell treatment group in Experimental Example 3;

[0037] Figure 13 This is a Safranin Fast Green staining image of the lateral condyle of the defect group, the comparative treatment group, and the example stem cell treatment group in Experimental Example 3. DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. In addition, it should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0040] In addition, in the description of the specification of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0041] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by methods known in the art.

[0042] In this application, "and / or" describes the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0043] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0044] In the present application, the bone defect includes bone defects caused by trauma, surgery or disease. The bone defect caused by the disease includes bone-related diseases caused by diabetes, rheumatoid arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, multiple sclerosis, periodontitis, inflammatory bowel disease, mucositis, colitis or sepsis.

[0045] The technical solution of this application is as follows:

[0046] In the first aspect, the embodiments of the present application propose a stem cell, which is a pluripotent stem cell with the following characteristics: the stem cell has a strong osteogenic and chondrogenic differentiation ability, and its osteogenic ability is stronger than that of traditional bone marrow mesenchymal stem cells (BMSCs), and can be used for repairing bone defects in animals or humans. Using this stem cell, bone defects can be effectively and quickly treated, shortening the bone repair time and significantly improving the prognosis.

[0047] The stem cells proposed in this application include expression markers including CX43 and FGFR2. The expression markers may refer to stem cells expressing specific marker genes, or expressing specific marker proteins, or expressing marker genes and proteins. The stem cells can be identified and isolated by the above-mentioned marker combination. The stem cells isolated based on the above-mentioned marker combination have strong osteogenic and chondrogenic differentiation abilities, and their osteogenic abilities are stronger than those of conventional BMSCs. It is understood that for the judgment of whether stem cells express each marker, in terms of gene expression, for example, it can be performed by existing known methods such as gene chip arrays, polymerase chain reaction (reverse transcriptase PCR, real-time PCR, existing PCR). In addition, in terms of protein expression, it can be performed by existing known methods such as FACS analysis (flow cytometry) and enzyme-linked immunosorbent assay (ELISA) using antibodies that specifically bind to each marker protein. In any case, cells that do not express each marker gene or protein are used as negative controls to determine whether or not the expression is present, and the level of expression is determined. Specifically, in a cell sample, cells that are positive for both CX43 and FGFR2 are stem cells proposed in this application.

[0048] Specifically:

[0049] CX43 refers to Recombinant Connexin 43, which is located on the outer surface of the cell membrane of the present application's stem cells. This protein plays an important role in embryonic development. Mutations in this gene can lead to skeletal malformations.

[0050] FGFR2 refers to fibroblast growth factor receptor 2. The FGFR2 protein is located on the outer surface of the cell membrane of the stem cells of the present application and is encoded by the FGFR2 gene located on chromosome 10. It plays an important role in tissue repair, bone and angiogenesis.

[0051] During separation, cells that are both CX43-positive and FGFR2-positive can be selected to obtain the stem cells described in this application. Furthermore, CX43 and FGFR2 are located on the outer surface of the cell membrane, making antibody staining more accurate, effective, and rapid.

[0052] Furthermore, the stem cells also contain at least one of the following gene markers: FKBP11 (FKBPProlyl Isomerase 11), PTN (Pleiotrophin), TNN (Tenascin N), TNC (Tenascin C), OMD (Osteomodulin), ACTA2 (Actin Alpha 2, Smooth Muscle) and MARCKS (Myristoylated alanine-rich C-kinase substrate). Further identification of whether the stem cells isolated based on the above markers have at least one of the above gene markers is helpful for further identification of the stem cells; at the same time, based on the above characteristics, the stem cells can secrete the expression products of at least one gene among FKBP11, PTN, TNN, TNC, OMD, ACTA2 and MARCKS, and thus have the functions of these expression products. It should be noted that the aforementioned gene markers include corresponding gene markers from humans or animals, or gene markers with nucleotide sequence homology of at least 98%. Taking MARCKS as an example, its nucleotide sequence includes, but is not limited to, the nucleotide sequence of the human MARCKS gene, a nucleotide sequence with at least 98% homology to the nucleotide sequence of the human MARCKS gene, a nucleotide sequence of an animal MARCKS gene, or a nucleotide sequence with at least 98% homology to the nucleotide sequence of the animal MARCKS gene. Animals include, but are not limited to, deer and mice. Depending on the source of the stem cells, the gene markers contained therein correspond to the corresponding gene markers of the source organism, or gene markers with nucleotide sequence homology of at least 98% to the corresponding gene markers of the source organism. Knowing the organism from which the stem cells originate, those skilled in the art can obtain their sequence information (and sequences with at least 98% homology) and characteristics through conventional methods (e.g., searching on NCBI).

[0053] In a specific embodiment:

[0054] The nucleotide sequence of MARCKS includes the sequence with accession number XM_043889887.1 on NCBI and sequences with a homology of not less than 98% to the sequence (for example, the nucleotide sequence shown in SEQ ID NO.1);

[0055] The nucleotide sequence of FKBP11 includes the sequence with accession number XM_006054550.3 on NCBI and sequences with a homology of not less than 98% to the sequence (for example, the nucleotide sequence shown in SEQ ID NO. 2);

[0056] The nucleotide sequence of PTN includes the sequence with accession number XM_043873485.1 on NCBI and sequences with a homology of not less than 98% to the sequence (for example, the nucleotide sequence shown in SEQ ID NO. 3);

[0057] The nucleotide sequence of TNN includes the sequence with accession number XM_043924613.1 on NCBI and sequences with a homology of not less than 98% to the sequence (for example, the nucleotide sequence shown in SEQ ID NO. 4);

[0058] The nucleotide sequence of OMD includes the sequence with accession number XM_043453757.1 on NCBI and sequences with a homology of not less than 98% to the sequence (e.g., the nucleotide sequence shown in SEQ ID NO. 5);

[0059] The nucleotide sequence of ACTA2 includes the sequence with accession number XM_043476084.1 on NCBI and sequences with a homology of not less than 98% to the sequence (for example, the nucleotide sequence shown in SEQ ID NO. 6);

[0060] The nucleotide sequence of TNC includes the sequence with accession number XM_005251975.5 on NCBI and sequences with a homology of not less than 98% to the sequence (for example, the nucleotide sequence shown in SEQ ID NO. 7).

[0061] The protein expressed by MARCKS is a myristoylated alanine-rich C kinase substrate, which is believed to be related to cell motility, phagocytosis, membrane trafficking and mitogenesis, and plays an important role in limb regeneration. MARCKS is located on the intracellular membrane and is highly expressed in this stem cell. In practical applications, it can also be used as a marker to identify this stem cell. The protein expressed by FKBP11 is FKBP prolyl isomerase 11, which is related to bone development; the protein expressed by PTN is heparin-binding growth factor, and the protein encoded by this gene plays an important role in cell growth and survival, cell migration, angiogenesis and tumorigenesis; the protein expressed by TNN is tenascin-W, which is involved in multiple biological processes, including the generation of neurons, negative regulation of the canonical Wnt signaling pathway of osteoblast differentiation, and negative regulation of osteoblast differentiation, and is related to cartilage formation; the protein expressed by TNC is tenascin-C, which constitutes the extracellular matrix of tissues and participates in multiple biological processes such as bone and nerve development; the protein expressed by OMD is bone regulatory protein, which is mainly involved in intercellular matrix formation and is related to bone mineralization; the protein expressed by ACTA2 is α-smooth muscle actin, a highly conserved protein that participates in cell movement, structural composition and intercellular signal transduction.

[0062] In some embodiments of the present application, the stem cells are mesenchymal stem cells. In the present application, the mesenchymal stem cells refer to cells that have the ability to differentiate into cells belonging to the mesenchyme, such as osteocytes, cardiomyocytes, chondrocytes, tendon cells, adipocytes, and are capable of proliferating while maintaining the differentiation ability, for example, mesenchymal stem cells derived from bone marrow, fat, blood, periosteum, dermis, umbilical cord, placenta, amnion, chorion, decidua, muscle, endometrium, dermis, dental follicle, periodontal tissue, dental pulp, tooth germ, etc., preferably mesenchymal stem cells derived from umbilical cord, from fat, or from bone marrow. It is understood that "derived from" means that the above-mentioned cells are obtained, grown, or obtained by in vitro operation of the tissue as the supply source.

[0063] In some embodiments of the present application, the stem cells are derived from human stem cells or animal stem cells. Specifically, the stem cells proposed in the present application can be directly isolated from human stem cells or animal stem cells, or obtained through artificial induction. As an example, the method of obtaining the present stem cells may include but is not limited to: in some embodiments, the present stem cells are obtained from the mesenchymal tissue at the top of the antler bud base or growth point. In the specific operation, only limited invasive surgery is required to obtain the tissue and then isolate the present stem cells. The operation is simple, and the mesenchymal stem cells obtained from the tissue can be cultured and passaged without differentiation. Therefore, after simple optimization, they can be efficiently recovered under GMP conditions, the source is stable, and the medical burden is significantly reduced; in other embodiments, the present stem cells are obtained from the regenerated tissue of the P3 fingertip of mice, which is a stable and easy source; in yet other embodiments, the present stem cells can also be induced from human stem cells through chemical induction methods or gene editing methods. For example, human stem cells are transformed using CRISPER-Cas9 technology to have similar chondrogenic and osteogenic abilities as the present stem cells. Compared with animal sources, human stem cells are more suitable for the treatment of human bone defects and have higher safety.

[0064] It should be noted that in this article, when it comes to the implementation of CRISPER-Cas9 technology transformation, tissue acquisition, cell staining, etc., if the specific experimental methods and experimental raw materials are not specified, they are all carried out in accordance with conventional methods in the field and are not described in detail here.

[0065] In a second aspect, the present application further provides a method for preparing stem cells, the method comprising the following steps:

[0066] Step S10, providing a cell population sample and an antibody composition, wherein the antibody composition comprises an anti-CX43 antibody and an anti-FGFR2 antibody;

[0067] Step S20, staining the cell population sample using the antibody composition;

[0068] Step S30, separating the CX43-positive and FGFR2-positive cells from the cell population sample to obtain stem cells.

[0069] In a third aspect, the present application further proposes the use of the aforementioned stem cells in the preparation of a product for treating bone defects. The characteristics and isolation methods of the stem cells have been described above and will not be repeated here.

[0070] The bone defect treatment products include drugs for treating bone defects, materials for treating bone defects, or kits for treating bone defects.

[0071] The drug for treating bone defects refers to a drug used to treat and repair bone defects, and has the effect of treating and repairing bone defects. Specifically, the drug includes a drug that promotes bone regeneration, and the drug for treating bone defects includes the stem cells. For example, a composite biogel for repairing bone defects, or a biological preparation that promotes osteogenic differentiation of bone marrow mesenchymal stem cells, etc. Specifically, the composite biogel is obtained by planting the stem cells on a bioactive gel after in vitro amplification and culture, and the biological preparation contains the stem cells. The preparation method of the drug can refer to the preparation of common bone defect drugs in the field, and will not be described in detail here.

[0072] The drug for treating bone defects may further include a pharmaceutically acceptable carrier, where the "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle, and the "pharmaceutically acceptable carrier" is suitable for contact with human and / or other animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0073] The material for treating bone defects refers to a material used to treat and repair bone defects. For example, the material can be a tissue engineering bone material for bone defect regeneration and repair, a bone defect scaffold material, a bone defect composite repair material, a functional composite scaffold material, etc. The material includes stem cells and implant materials. The implant materials can be selected from, but not limited to, at least one of natural polymers, synthetic polymers, biomedical metal materials, and inorganic materials. As an example, a bone defect scaffold material can include the stem cells and medical bone materials. Specifically, 1×10 4 -1×10 5 The bone defect scaffold material can be obtained by adding the stem cells.

[0074] The kit is a kit that can prepare a drug or material for treating bone defects. In some embodiments of the present application, the kit includes the stem cells and a carrier, and the carrier can be a medical sponge, a medical dressing, a gel composition that can be made into a gel, etc. For example, in a specific embodiment, the kit includes a stem cell suspension (cell concentration is 10 4 -10 6 The kit is used as follows: before use, the stem cell suspension is added to the gelatin sponge to prepare a scaffold filling material, the scaffold filling material is filled into the bone defect, and the muscle and skin at the bone defect are sutured.

[0075] The stem cells proposed in this application can be applied to humans or animals. Accordingly, the application objects of the bone defect treatment product can be humans or animals, and it has broad development prospects.

[0076] Fifthly, the present application also proposes a marking composition, which includes but is not limited to CX43 and FGFR2. The marking composition can screen and identify stem cells. The stem cells screened or identified based on the marking composition have strong osteogenic and chondrogenic differentiation capabilities, and their osteogenic ability is stronger than that of traditional BMSCs. The use of these stem cells can effectively and quickly treat bone defects, shorten bone repair time, and significantly improve the prognosis.

[0077] It is understood that the CX43 and FGFR2 may be corresponding gene features or expressed proteins. For example, in some embodiments, the nucleotide sequence of FGFR2 includes the sequence with accession number XM_043926212.1 on NCBI and a sequence with at least 98% homology to the sequence (e.g., the nucleotide sequence shown in SEQ ID NO. 8); the nucleotide sequence of CX43 includes the sequence with accession number XM_OU343105.1 on NCBI and a sequence with at least 98% homology to the sequence (e.g., the nucleotide sequence shown in SEQ ID NO. 9).

[0078] In addition, the marker composition also includes the expression product of at least one of the genes FKBP11, PTN, TNN, TNC, OMD, ACTA2, and MARCKS. Further identification of stem cells isolated based on these markers to determine whether they possess at least one of these gene markers can help further improve identification accuracy.

[0079] In a sixth aspect, the present application further proposes a use of the above-mentioned labeling composition in the preparation of a kit for isolating or identifying stem cells.

[0080] Specifically, the kit includes: anti-Connexin 43 (10 μL), anti-FGFR2 (10 μL), fluorescently labeled secondary antibody FITC (10 μL) and fluorescently labeled secondary antibody Cy3 (10 μL).

[0081] To use this kit, deer antler mesenchymal stem cells were isolated from 5-day-old antler tissue and incubated with the primary antibodies anti-Connexin 43 and anti-FGFR2 (1:200) for 1 hour. The cells were then washed three times with PBS. The two fluorescently labeled secondary antibodies were diluted 1:200 and mixed and incubated for 1 hour. The proportion of double-fluorescence-positive cells (cells that emit fluorescent signals) was determined using flow cytometry, and the double-positive cells were separated using flow cytometry to obtain the primary screening cells.

[0082] The technical solutions and technical effects of the present application are described in detail below through specific embodiments. The following embodiments are only some embodiments of the present application and do not specifically limit the present application.

[0083] Example Preparation of Stem Cells

[0084] (1) Preparation of antler mesenchymal stem cells: 5-day-old antler antlers were collected. Dust and blood stains on the antler surface were removed with sterile gauze. After skin preparation, the antlers were disinfected with 75% alcohol. Then, 3 cm thick regenerated tissue on the antler bud base was cut with a disposable sterile tissue slice blade, minced with a blade, and digested with collagenase I (100 μg / ml) / collagenase II (100 μg / ml) / collagenase IV (30 μg / ml) solution (Gibco, 17100017, 17101015, and 17104019) at 37°C for 60 minutes (or until the tissue was completely digested) with intermittent shaking. The single cell suspension was filtered through 70 μm and 40 μm cell strainers (Corning Falcon, 352350 and 352340) in sequence and centrifuged at 500×g for 5 minutes. The cell pellet was resuspended in PBS and treated with RBC lysis buffer (Solarbio, R1010) to remove red blood cells. The cells were then washed twice with PBS, resuspended in 0.04% ultrapure BSA (Invitrogen), and counted using a Countess automated cell counter (Bio-Rad).

[0085] The cells were washed with a flow cytometry buffer at 0-4°C and centrifuged at 1000 g for 5 minutes, and the process was repeated twice to obtain antler mesenchymal stem cells.

[0086] (2) Isolation of stem cells:

[0087] a. Perform immunofluorescence staining on the antler mesenchymal stem cells using the primary antibody anti-Connexin 43 (Biorbyt, orb995524); then detect the proportion of positive cells (cells emitting fluorescent signals) using a flow cytometer, and separate the CX43-positive cells using the flow cytometer to obtain primary screening cells.

[0088] b. Immunofluorescence staining of the antler mesenchymal stem cells was performed using an anti-FGFR2 (LSBio, LS-C214232-200) solution; the proportion of positive cells (cells emitting fluorescent signals) was detected using a flow cytometer, and the FGFR2-positive cells were separated using a flow cytometer to obtain CX43 + FGFR2 + Stem cells.

[0089] (1) CX43 + FGFR2 + The morphological effect of stem cells is shown in the figure Figure 1 As shown, it can be seen from the figure that the cells have a typical long spindle shape.

[0090] (2) Gene marker detection

[0091] CX43 obtained in Example was analyzed by 10×genomic technology. + FGFR2 + Single-cell transcriptome sequencing of stem cells was performed, with a sequencing data volume of 100g. The sequencing data were aligned to the sika deer reference genome using Cellranger software to quantify the expression of key genes.

[0092] After testing, the stem cells contained the following gene markers: MARCKS having the nucleotide sequence shown as SEQ ID NO.1, FKBP11 having the nucleotide sequence shown as SEQ ID NO.2, PTN having the nucleotide sequence shown as SEQ ID NO.3, TNN having the nucleotide sequence shown as SEQ ID NO.4, OMD having the nucleotide sequence shown as SEQ ID NO.5, ACTA2 having the nucleotide sequence shown as SEQ ID NO.6, and TNC having the nucleotide sequence shown as SEQ ID NO.7.

[0093] In addition, the expression of the above group markers is as follows Figure 2 As shown in the figure, it can be seen that the above gene markers are in CX43 + FGFR2 + They are highly expressed in stem cells.

[0094] (3) Collect the regenerative tissue of mouse P3 fingertips, collect 10× single cell transcriptome maps of the regenerative tissue of mouse P3 fingertips and the regenerative tissue on the antler bud base in step (1) of Example 1, integrate the cell maps of the two by the CCA method in Seurat software, then perform dimensionality reduction clustering of the data and display it using the UMAP method. The results are as follows Figure 3 As shown in the figure, CX43 + FGFR2 + The cell population exists in both the mouse P3 fingertip regeneration tissue and the deer antler regeneration tissue.

[0095] Comparative Example 1

[0096] Traditional BMSCs were used as comparative example 1.

[0097] Comparative Example 2

[0098] This comparative example is basically the same as Example 1, except that:

[0099] In step (2), CX43 positive and FGFR2 negative cells were separated by flow cytometry, and the separated stem cells (CX43 + FGFR2 - Stem cells) are used as products for subsequent experiments.

[0100] Comparative Example 3

[0101] This comparative example is basically the same as Example 1, except that:

[0102] In step (2), CX43 negative and FGFR2 positive cells were separated by flow cytometry, and the separated stem cells (CX43 - FGFR2 -+ Stem cells) are used as products for subsequent experiments.

[0103] Comparative Example 4

[0104] This comparative example is basically the same as Example 1, except that:

[0105] In step (2), cells that are negative for both CX43 and FGFR2 are screened out, and the isolated stem cells (CX43 - FGFR2 - Stem cells) are used as products for subsequent experiments.

[0106] Experimental Example 1

[0107] (1) The osteogenic and chondrogenic differentiation abilities of the stem cells isolated in Example 1 and the stem cells isolated in Comparative Example 1 were measured. The detection method was as follows:

[0108] Osteogenic differentiation assay: Stem cells were seeded in 6-well plates and differentiated for 28 days using osteogenic differentiation induction medium (Cyagen Biosciences, GUXMX-90021) (medium was changed every 2 days). RNA was extracted and quantified by qPCR for RUNX2 and SP7 genes. Cells were also stained with Alizarin Red.

[0109] Chondrogenic differentiation ability test: 2.5×10 5 Each stem cell was placed in a 15ml centrifuge tube and centrifuged at 1000 rpm. The cells were then cultured in chondrogenic differentiation induction medium (Cyagen Biosciences, GUXMX-90041). After 24 hours, chondrocytes formed and continued to differentiate for 3 weeks (medium was changed every 2 days). RNA was extracted and quantitative PCR was performed to detect SOX9 and COL2A1 genes. The chondrocytes were then embedded, frozen, and sectioned. The sections were then stained with Alcian blue.

[0110] The results are as follows Figure 4 and Figure 5 As shown, Figure 4 In the figure, from top to bottom are the alizarin red staining images of cells in the osteogenic differentiation ability test and the alcian blue staining images of chondrocytes in the chondrogenic differentiation ability test; from left to right are the stem cells of comparative example 1 and the embodiment.

[0111] from Figure 4 It can be seen that compared with BMSCs, CX43 + FGFR2 + Stem cells have stronger osteogenic and chondrogenic differentiation capabilities. Figure 5 As can be seen in Figure 2, the differentiation efficiency was quantified by quantitative real-time PCR (qPCR) analysis of osteogenic (RUNX2 and SP7) and chondrogenic (SOX9 and COL2A1) marker genes, and the quantitative results were consistent with those of Figure 4 The results were consistent with those shown in Figure 3, namely, compared with BMSCs, CX43 + FGFR2 + The stem cells have stronger osteogenic and chondrogenic differentiation abilities. The stem cells isolated from the illustrated example show better osteogenic and chondrogenic differentiation abilities than the traditional BMSCs stem cells of comparative example 1.

[0112] (2) Detecting the monoclonal capacity of the stem cells isolated in Example 1 and the stem cells isolated in Comparative Examples 2 to 4. The detection method is as follows:

[0113] Stem cells were seeded in 6-well plates (4.5×10 32 ml of DMEM medium (supplemented with 10% FBS and 1% penicillin / streptomycin solution) was added to each well and incubated at 37°C in a 5% CO2 cell culture incubator. The medium was changed every 2 days. On the 10th day, the cells were fixed and stained with crystal violet stain. Quantification of adherent colonies with more than 50 cells was performed.

[0114] The results are as follows Figure 6 As shown, with CX43 - FGFR2 - Stem cells, CX43 + FGFR2 - Stem cells, CX43 - FGFR2 + Compared with stem cells, CX43 + FGFR2 + Stem cells showed higher colony formation efficiency and more and larger colonies, indicating that CX43 + FGFR2 + Stem cells have stronger clonal expansion capabilities and are more effective in repairing bone damage.

[0115] Experimental Example 2

[0116] The stem cells isolated in Example 1 and the stem cells in Comparative Example 1 were used for subrenal transplantation bone formation experiments.

[0117] The experimental steps are as follows:

[0118] Provide 0.05 mL of Matrigel (BD Matrigel, 356234), add 5×10 6 The stem cells are resuspended and mixed to obtain a composite material.

[0119] Ten immunodeficient mice (male, 8-10 weeks) were used and divided into two groups: BMSCs group and CX43 + FGFR2 + Immunodeficient mice were anesthetized and sterilized, and a 1 cm incision was made on the right side of the back with a scalpel to remove the right kidney. A small incision was made below the kidney, and 5 μL of the composite material (containing 5×10 5 The renal capsule was sealed with a cautery device and the skin incision was sutured. After 8 weeks, the animals were euthanized, the kidneys were removed, fixed, dehydrated, frozen and stained with Ponceau red. Figure 7 and Figure 8 As shown, Figure 7 This is the result of Ponceau staining of new bone under the renal capsule. Figure 8 This is a comparison chart of the statistical results of the new bone area.

[0120] from Figure 7 and Figure 8 It can be seen that not only can the stem cells isolated in Example 1 successfully form new bone under the renal capsule of mice, but also, compared with Comparative Example 1, the experimental group corresponding to the stem cells isolated in Example 1 has a larger area of ​​new bone formed, indicating that compared with traditional BMSCs, the stem cells isolated in Example 1 have stronger bone formation ability.

[0121] Experimental Example 3

[0122] (1) A kit for treating bone defects is provided, wherein the kit comprises 2 mL of a stem cell suspension containing stem cells prepared in Example (cell concentration is 2×10 4 Before use, 500 μL of stem cell suspension was added to a piece of gelatin sponge to make a scaffold containing 1×10 7 stem cells.

[0123] (2) A lateral femoral condyle bone defect model was constructed using 15 New Zealand male rabbits (2 months old). The specific method was to use an electric drill to create a defect with a diameter of 8 mm and a depth of 6 mm on the right lateral femoral condyle of the rabbit. The 15 New Zealand male rabbits were randomly divided into three groups (5 in each group): a defect group, a control group (BMSCs), and an example stem cell treatment group (CX43 + FGFR2 + ). Among them: the defect group was only modeled without any treatment; the control group was filled with 1×10 7 The gelatin sponge was filled with BMSCs cells; the stem cell treatment group was the scaffold prepared in the defect filling step (1). After 8 weeks, the sample was collected and then subjected to micro-CT examination. The results are shown in Figure 2. Figure 9-13 As shown, where:

[0124] Figure 9 The micro-CT images of the lateral condyle of the defect group, the comparative example treatment group and the example stem cell treatment group are shown in FIG. Figure 10 Statistical graph of new bone volume fraction at the defect site in the defect group, comparative example treatment group and example stem cell treatment group. Figure 11 Statistical graph of the number of newly formed bone trabeculae at the defect site in the defect group, the comparative example treatment group, and the example stem cell treatment group; Figure 12 HE staining of the lateral condyle of the defect group, the comparative example treatment group and the example stem cell treatment group. Figure 13 Safranin fast green staining of the lateral condyle of the defect group, the comparative example treatment group and the example stem cell treatment group.

[0125] Result Analysis: Example CX43 +FGFR2 + The stem cell treatment group successfully formed new bone in the defect of the right lateral femoral condyle of the rabbit; Figure 9-10 It can be seen that in the same time, the example CX43 + FGFR2 + The volume fraction of new bone in the defect of the stem cell treatment group was significantly higher than that of the BMSCs treatment group. + FGFR2 + The number of new bone trabeculae in the defect of the stem cell treatment group was also significantly higher than that of the BMSCs treatment group. Figure 12 and 13 It can be seen that the new bone is well integrated with the adjacent natural tissue and has normal morphology. This shows that the stem cells provided by this application have stronger osteogenic capacity than traditional BMSCs, can more effectively treat bone defects, shorten bone repair time, and significantly improve prognosis.

[0126] The above describes in detail the stem cells, preparation methods, and applications thereof, as well as the labeling compositions and applications thereof, provided in the examples of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The descriptions of the above examples are intended only to help understand the methods and core concepts of the present application. Furthermore, those skilled in the art will appreciate that specific implementation methods and application scopes may vary based on the concepts of the present application. In summary, the contents of this specification should not be construed as limiting the present application.

Claims

1. A stem cell for preparing a product for treating bone defects, characterized in that: The markers expressed by the stem cells include CX43 and FGFR2; the stem cells further contain at least one of the following gene markers: FKBP11, PTN, TNN, TNC, OMD, ACTA2, and MARCKS; The stem cells are CX43 and FGFR2 positive, and the stem cells are antler mesenchymal stem cells.

2. A method for preparing stem cells for use in preparing products for treating bone defects, characterized in that: The following steps are involved: Providing a cell population sample and an antibody composition comprising an anti-CX43 antibody and an anti-FGFR2 antibody; staining the cell population sample using the antibody composition; Isolating CX43-positive and FGFR2-positive cells from the cell population sample to obtain stem cells for preparing a product for treating bone defects; Wherein, the cell population sample is antler mesenchymal stem cells.

3. An application of stem cells in the preparation of a product for treating bone defects, characterized in that: The stem cells are the stem cells for preparing a product for treating bone defects according to claim 1, or the stem cells are the stem cells for preparing a product for treating bone defects obtained by the preparation method according to claim 2; The bone defect treatment products include medicines for treating bone defects, materials for treating bone defects or kits for treating bone defects.

4. The use according to claim 3, characterized in that The drug for treating bone defects comprises the stem cells and a pharmaceutically acceptable carrier; and / or, The material for treating bone defects includes any one of tissue engineering bone materials, bone defect scaffold materials, and bone defect composite repair materials; and / or, The application object of the bone defect treatment product is humans or animals.

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