Use of itm2a-positive skeletal / tendon stem cells in treatment of skeletal / tendon injuries

Abstract

The present invention provides a pharmaceutical composition, comprising: i) isolated skeletal or tendon stem cells, wherein the skeletal or tendon stem cells are Itm2a-positive; and ii) a pharmaceutically acceptable carrier. The present invention also provides a method for isolating Itm2a-positive skeletal or tendon stem cells and a method for treating a skeletal or tendon injuries or defects using Itm2a-positive skeletal or tendon stem cells.

Description

Use of Itm2a-positive skeletal / tendon stem cells in the treatment of bone / tendon injuries Technical Field The present invention relates to cell biology and stem cell therapy. In particular, the present invention relates to a method for enriching skeletal stem cells by using Itm2a protein, and a method for treating bone fractures using Itm2a-positive skeletal stem cells. Background Art Skeletal injuries are a frequent occurrence, particularly among adolescents, athletes, and the elderly, placing a strain on social security. Current clinical approaches to autologous bone transplantation for skeletal injuries have several limitations, including variability in graft quality (particularly in patients with osteoporosis) and limited donor bone availability. Harvesting autologous grafts often increases operative time, and complications of varying severity occur at the graft donor site in 10% to 35% of patients. Cell therapy strategies can also be adopted, in which the search and separation of seed cells for cell therapy is particularly important. The fracture repair process includes two classic bone formation methods: intramembranous ossification and endochondral ossification. When the bone fixation at the wound site is very strong, the body will quickly carry out early repair through intramembranous ossification, and then initiate endochondral ossification for subsequent repair (Einhorn & Gerstenfeld, Fracture healing: mechanisms and interventions. Nat Rev Rheumatol. 2015 Jan; 11(1): 45-54). In addition to the degree of fixation, the source of the stem cells involved in the repair also determines the repair method. Endosteal stem cells will directly form osteoblasts in the bone marrow cavity through intramembranous ossification and cross-link the broken bone. Periosteal stem cells will form intramembranous ossification on the cortical bone surface without moving stress, and endochondral ossification in the fracture gap (Claes et al., Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol, 2012, 8, 133–143). The stem cells involved in fracture repair mainly come from mesenchymal stem cells (MSCs) in the periosteum, bone marrow and endosteum (Raggatt et al., Cellular and molecular mechanisms of bone remodeling. J Biol Chem. 2010; 285(33): 25103-8). SDF1 is an important factor that can recruit MSCs to the fracture injury site. MSCs have CXCR4 receptors on their surface that can bind to SDF1. Knocking out CXCR4 in MSCs will affect their cell migration, thereby delaying fracture repair (Kawakami et al., Calf circumference as a surrogate marker of muscle mass for diagnosing sarcopenia in Japanese men and women. Geriatr Gerontol Int. 2015 Aug; 15(8): 969-76; Kawanami et al., Effects of a decrease in mechanical stress on femoral regional bone mineral density and osteoblast microstructure: Comparison in a model of freely mobile and cast immobilized rats. Japanese Journal of Physical Fitness and Sports Medicine, 2009; 58: 305-316).MSCs begin to proliferate immediately after migrating to the fracture injury area. PDGF secreted by platelets in the fracture injury area can further promote angiogenesis and MSC proliferation (Caplan & Correa, PDGF in bone formation and regeneration: new insights into a novel mechanism involving MSCs. J Orthop Res. 2011 Dec; 29(12): 1795-803; Caverzasio et al., The p38α MAPK positively regulates osteoblast function and postnatal bone acquisition. Cell Mol Life Sci. 2012 Sep; 69(18): 3115-25). In the early stages of fracture repair, platelet-derived lectins (TSPs) from platelets and MSCs can inhibit angiogenesis (Taylor et al., Developing ADHD. J Child Psychol Psychiatry. 2009 Jan;50(1-2):126-32). When cartilage is formed, the cartilage callus is essentially filled with dense collagen and matrix, in an avascular state. As chondrocytes hypertrophy, they gradually begin to secrete VEGF, PEG-1α, and PDGF to promote the formation and invasion of blood vessels. The blood vessels then feed back to promote cartilage calcification and bone formation. With the migration and expansion of MSCs, a new vascular network begins to form in the fracture area. This new vascular network brings oxygen, nutrients, and precursor cells to the damaged tissue, accelerating the repair process. Skeletal stem cells (SSCs) are tissue-specific stem cells that can self-renew and are located at the top of the bone differentiation hierarchy, generating mature skeletal cell types required for bone growth, maintenance and repair. Bone development, homeostasis and repair require the self-renewal and differentiation of skeletal stem cells to function. SSCs are able to self-renew in vitro to form clones and have the ability to differentiate into osteoblasts, intramedullary fat, cartilage and hematopoietic support matrix, playing an important role in bone development, homeostasis and injury repair. The dysfunction of SSCs is caused by stress conditions such as aging and inflammation, and is becoming a cause of bone pathology (such as the pathogenesis of fracture nonunion). Recent lineage tracing experiments have shown that SSCs are present in the bone marrow, periosteum and resting areas of the growth plate. The growth plate plays a major role in longitudinal bone extension, while the periosteum is crucial for bone thickening and repair. The identity of periosteal stem cells has only begun to be slowly unraveled in recent years. During development, periosteal stem cells originate from mesenchymal cells and have multipotency. Prrx1-Cre widely marks mesenchymal cells in the limbs and other parts of the body. The expression of a group of marker molecules related to bone marrow mesenchymal stem cells (BMSCs), including Pdgfrα, Grem1, Cxcl12 and Nestin, associated with Prrx1-Cre-labeled periosteal cells, is co-localized in the periosteum (Bianco et al., Osteoprogenitors and the hematopoietic microenvironment. Best Pract Res Clin Haematol. 2011 Mar; 24(1): 37-47; and Worthley et al., Skeletal stem cells in space and time. Cell. 2015 Jan 15; 160(1-2): 17-9). Prrx1-Cre-labeled periosteal cells transplanted into the fracture site can generate chondrocytes and osteoblasts, demonstrating their multipotency.Osx-CreER, Gli1-CreER, Lepr-Cre, Sox9-CreER, and Hoxa11-CreER also mark periosteal stem cells, which form osteoblasts and chondrocytes to participate in repair after fracture (Bianco et al., Osteoprogenitors and the hematopoietic microenvironment. Best Pract Res Clin Haematol. 2011 Mar; 24(1): 37-47; Duchamp de Lageneste et al., Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin. Nat Commun. 2018 Feb 22; 9(1): 773; Mo et al., Single-cell transcriptomics of LepR-positive skeletal cells reveals heterogeneous stress-dependent stem and progenitor pools. EMBO J. 2022 Feb 15; 41(4): e108415; Pineault ... al., Hox11-expressing regional skeletal stem cells are progenitors for osteoblasts, chondrocytes, and adipocytes throughout life. Nat Commun. 2019 Jul 18; 10(1): 3168; Sacchetti et al., Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell. 2007 Oct 19; 131(2): 324-36). However, since these Cre markers mark a wide range of mesoderm-derived cell populations, it has not yet been determined which population within the periosteum has stronger skeletal stem cell properties.Surprisingly, cathepsin K (Ctsk), which was previously used as a marker for bone-resorbing osteoclasts, was found to mark periosteal stem cells (Debnath et al., Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature 562(2018): 133-139; Han et al., Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation. J Clin Invest. 2019 May 1; 129(5): 1895-1909; Yang et al., Ptpn11 deletion in a novel progenitor causes metachondromatosis by inducing hedgehog signalling. Nature. 2013 July). 25; 499(7459):491-5), Ctsk+ cells were isolated from the femoral periosteum of young mice and found to exhibit self-renewal properties, with monoclonal formation (CFU-F) ability and multi-lineage differentiation potential (bone, cartilage and fat cells, but no stromal cells). The absence of the osteogenic differentiation transcription factor Osx in Ctsk-Cre-marked animals leads to poor fracture repair and severe damage to the bone cortex. However, it should be noted that, similar to Prrx1, Gli1, etc., Ctsk-Cre marks a wide range of periosteal cells, only some of which are skeletal stem cells. In 2019, Park et al. identified a group of long-term Mx1+αSMA+ periosteal stem cells in vivo by constructing Mx1-Cre&αSMA-GFP mice. Combined with dynamic imaging technology, it was tracked in vivo that Mx1+αSMA+periosteal stem cells were involved in fracture and bone injury repair, and the migration of these periosteal stem cells was regulated by CCR5. It also suggested that the combination of different markers may separate skeletal stem cell subpopulations with different characteristics (Ortinau et al., Identification of Functionally Distinct Mx1+αSMA+Periosteal Skeletal Stem Cells. Cell Stem Cell. 2019 Dec 5; 25(6): 784-796.e5). Tendons are dense connective tissues rich in collagen that transmit the tensile forces between muscle and bone[1]. Tendons lack cells and blood vessels, resulting in insufficient endogenous repair capacity. Approximately 30% of musculoskeletal diseases involve tendon injuries[2]. Tendon injuries are a difficult clinical challenge because the destruction of tendon structure impairs tendon function and may lead to poor healing, such as heterotopic ossification[3,4]. Although surgical treatment can restore the integrity of tendon tissue, injured tendons are often unable to return to their pre-injury state due to scar formation and fibrosis, making them more susceptible to rupture. Various treatments have been proposed for tendon injuries, such as the use of platelet-rich plasma, hyaluronic acid, and corticosteroid injections. However, these methods mainly reduce inflammation and pain, and are not very effective in repairing the tendon itself[2,4,5]. Stem cell-based treatments hold great promise, as exogenous cell transplantation or endogenous cell activation in injured or diseased tendons has demonstrated promising results in promoting repair [6]. Currently, the cell sources studied include tendon stem cells (TSC), bone marrow stem cells (BMSC), adipose-derived stem cells (ADSC), and embryonic stem cells (ESC). Tendon stem cells have the characteristics of rapid proliferation and a greater tendency to form tenocytes, making them more suitable for stem cell therapy of tendon injuries [7-9]. Tendon stem cells play a very important role in tendon development and homeostasis as well as injury repair. Tendon stem cells, also known as tendon stem / progenitor cells (TSPC), show different tendencies in differentiation potential due to their heterogeneity [7]. In 2007, tendon stem cells were defined as a group of cells with monoclonal formation ability, self-renewal potential and differentiation multipotency.

[0010] Since then, tendon stem cells have received much attention because current treatments for tendon diseases often fail to produce satisfactory results. Extensive in vitro studies of tendon stem cells have been conducted, and a series of stem cell markers involved in tendon injury repair have been discovered, such as CD146 (human and mouse), CD105 (mouse), CD90 (human), CD44 (human), Sca1 (mouse), and PDGFRα (mouse) [11-15]. To identify tendon stem cells in vivo, Cre-ERT2 combined with tamoxifen-induced lineage tracing methods have been widely used, but the fundamental problem has not been solved. In αSMA-CreERT2 transgenic mice, αSMA labels multiple cell types around the tendon, but it does not label stem cells because the second harmonic generation (SHG) signal generated by the collagen matrix secreted by tenocytes after injury cannot be detected [16,17]. On the other hand, Scleraxis (Scx)-labeled tenocytes can participate in tendon repair, but only in infancy. In adulthood, Scx-positive tenocytes do not participate in tendon repair but instead transdifferentiate into osteoblasts and chondrocytes

[0018] . In 2019, a study found that Tppp3+PDGFRα+ tendon stem cells migrate to the damaged area, differentiate into tenocytes, and lose their stem cell characteristics. Tppp3+PDGFRα+ tendon stem cells are stored in the tendon sheath and proliferate and maintain their proportion after injury. It was also found that the PDGF signaling pathway is crucial for their participation in tendon injury repair

[0017] . However, Tppp3 is a microtubule polymerization protein rather than a membrane protein, and it is impossible to isolate live Tppp3-positive cells, so their findings cannot be applied to tendon stem cell therapy. Previous membrane protein markers such as Sca1 and CD44 are not specific enough. The current research bottleneck is the lack of specific membrane protein markers and related model animals for tendon stem cells [5]. Therefore, in-depth analysis of the specific membrane protein markers and characteristics of tendon stem cells, their interactive microenvironment and participation in the repair process can promote future clinical applications. Integral membrane protein 2A (Itm2a) is a type II integral membrane protein. The coding sequence of the mouse Itm2a gene is 792 bp long, and the encoded protein is approximately 30 kD in size. The sequence homology between mouse and human ITM2A proteins is as high as 95%. Itm2a is primarily expressed in ribs, vertebrae, long bones, skeletal muscle, T cells, hair follicles, skin, and tongue [19,20]. Previous studies have found that Itm2a is regulated by PAX3, a transcription factor important for muscle, nerve, and facial development in vertebrates

[0021] . In situ hybridization experiments in newborn mouse embryos and bones have shown that Itm2a expression precedes known marker genes for endochondral ossification, such as alkaline phosphatase, collagenase 3, and osteocalcin. Therefore, Itm2a is thought to mark chondrocytes in both resting and proliferative zones

[0022] . In addition, Itm2a is also involved in the differentiation of mesenchymal stem cells into chondrocytes

[0020] . However, there are currently no reports on the study of Itm2a in stem cells. Therefore, there is a need to provide skeletal stem cells with high osteogenic ability and tendon stem cells with high tenogenic ability, as well as to identify new molecular markers to isolate skeletal stem cells with high osteogenic ability from bone tissue such as periosteum and tendon stem cells with high tenogenic ability from tendon or tendon sheath tissue. Summary of the Invention In a first aspect, the present invention provides a pharmaceutical composition comprising: i) isolated skeletal stem cells, wherein the skeletal stem cells are Itm2a positive; and ii) a pharmaceutically acceptable carrier. In some embodiments, the skeletal stem cells are human skeletal stem cells, and the skeletal stem cells are CD45-CD31-TER119-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, the skeletal stem cell population has the ability to differentiate into osteoblasts. In some embodiments, the skeletal stem cell population is self-renewing and clonogenic. In some embodiments, the skeletal stem cells are enriched from bone tissue samples. In some embodiments, the skeletal stem cell population is expanded in vitro. In some embodiments, the pharmaceutical composition is formulated for local administration, such as local administration at a fracture site. In a second aspect, the present invention provides a method for preparing an isolated skeletal stem cell population, wherein the skeletal stem cells are Itm2a positive, the method comprising: a) providing a cell pool from bone tissue; b) selecting Itm2a-positive cells from the cell pool derived from bone tissue; and c) optionally expanding the cells selected in step b) in culture. In some embodiments, step b) comprises selecting with a binding molecule against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody. In some embodiments, step b) comprises selecting by flow cytometry. In some embodiments, the skeletal stem cells are human skeletal stem cells, and the method further comprises selecting CD45-CD31-TER119-CD235-CD146-PDPN+CD73+CD164+ cells from the cell pool from bone tissue. In a third aspect, the present invention provides a method of treating a bone injury or defect in a subject in need thereof, the method comprising a) providing a cell pool from bone tissue; b) selecting Itm2a-positive cells from the cell pool derived from bone tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof. In some embodiments, the bone tissue is autologous bone tissue of the subject in need. In some embodiments, the bone tissue is allogeneic bone tissue. In some embodiments, step b) comprises selecting using a binding molecule against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody. In some embodiments, step b) comprises selecting by flow cytometry. In some embodiments, the expanded cells are administered locally at the site of bone injury or defect. In some embodiments, the skeletal stem cells are human skeletal stem cells, and the method further comprises selecting CD45-CD31-TER119-CD235-CD146-PDPN+CD73+CD164+ cells from the cell pool from bone tissue. The present invention also provides a method of treating bone damage or defect in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present invention. In some embodiments, the pharmaceutical composition is administered locally at the bone fracture site. In some embodiments, the skeletal stem cells are autologous or allogeneic to the subject in need thereof. The present invention also provides a pharmaceutical composition of the present invention for use in treating bone damage or defects in a subject in need thereof. The present invention also provides the use of the pharmaceutical composition of the present invention or the isolated skeletal stem cell population prepared by the method of the present invention in the preparation of a medicament for treating bone damage or defect in a subject in need. In some embodiments, the bone injury or defect is a bone fracture. In a fourth aspect, the present invention provides a pharmaceutical composition comprising: i) isolated tendon stem cells, wherein the tendon stem cells are Itm2a positive; and ii) a pharmaceutically acceptable carrier. In some embodiments, the tendon stem cells are human tendon stem cells, and the tendon stem cells are CD45-CD31-CD235a- cells. In some embodiments, the tendon stem cell population has the ability to differentiate into tenocytes. In some embodiments, the tendon stem cell population is self-renewing and clonogenic. In some embodiments, the tendon stem cells are enriched from tendon or tendon sheath tissue samples. In some embodiments, the tendon stem cell population is expanded ex vivo. In some embodiments, the pharmaceutical composition is formulated for local administration, for example, local administration at the site of tendon injury or defect. In a fifth aspect, the present invention provides a method for preparing an isolated tendon stem cell population, wherein the tendon stem cells are Itm2a positive, the method comprising: a) providing a pool of cells from tendon or tendon sheath tissue; b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue; and c) optionally expanding the cells selected in step b) in culture. In some embodiments, step b) comprises selecting with a binding molecule against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody. In some embodiments, step b) comprises selecting by flow cytometry. In some embodiments, the tendon stem cells are human tendon stem cells, and the method further comprises selecting CD45-CD31-CD235a- cells from the pool of cells from tendon or tendon sheath tissue. In a sixth aspect, the present invention provides a method of treating a tendon injury or defect in a subject in need thereof, the method comprising a) providing a pool of cells from tendon or tendon sheath tissue; b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof. In some embodiments, the tendon or tendon sheath tissue is autologous tendon or tendon sheath tissue of the subject in need. In some embodiments, the tendon or tendon sheath tissue is allogeneic tendon or tendon sheath tissue. In some embodiments, step b) includes selecting with a binding molecule against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody. In some embodiments, step b) includes selecting by flow cytometry. In some embodiments, the expanded cells are administered locally at the site of tendon injury or defect. In some embodiments, the tendon stem cells are human tendon stem cells, and the method further comprises selecting CD45-CD31-CD235a- cells from the pool of cells from tendon or tendon sheath tissue. The present invention also provides a method of treating tendon injury or defect in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present invention. In some embodiments, the pharmaceutical composition is administered locally at the site of tendon injury or defect. In some embodiments, the tendon stem cells are autologous or allogeneic to the subject in need thereof. The present invention also provides a pharmaceutical composition of the present invention for use in treating a tendon injury or defect in a subject in need thereof. The present invention also provides use of the pharmaceutical composition of the present invention or the isolated tendon stem cell population prepared by the method of the present invention in the preparation of a medicament for treating tendon injury or defect in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows single-cell sequencing of Prrx1-Cre-positive periosteum in 4-week-old mice. A: Prrx1-Cre+ periosteal cell single-cell sequencing process. After wrapping the two ends of the femur with low-melting point agarose and digesting with collagenase, Prrx1-Ai9-positive periosteal cells were obtained by flow cytometry sorting for single-cell sequencing; B: t-SNE dimensionality reduction shows 13 cell subpopulations in single-cell sequencing: stem / progenitor cells (cell subpopulations 0 to 3, 7 and 10), osteoblasts (cell subpopulations 4 and 12), endothelial cells (cell subpopulations 5, 6 and 8), muscle cells (cell subpopulations 9 and 11); C to F: Feature maps show the difference between the two groups. Genes specifically expressed in the same cell cluster, with two representative genes selected for each cell cluster: periosteal stem / progenitor cells (C) (Col3a1 and Itgbl1), osteoblasts (D) (Sp7 and Bglap), muscle cells (E) (Pax7 and Myob5), and endothelial cells (F) (Emcn and Pecam1); and G: Pseudo-chronic analysis of Prrx1-Ai9-positive periosteal cells, showing that the cells can be divided into three states; (H) Distribution of cells in the three states on the pseudo-chronic timeline. FIG2 shows the enrichment of skeletal stem cells from Prrx1-positive periosteal cells using Itm2a molecules. A: Feature plot showing the expression distribution of skeletal stem cell markers CD105 and CD200 in Prrx1-Cre positive cells; B: t-SNE dimensionality reduction showing the distribution of periosteal stem / progenitor cell populations (cell state 2, cell subpopulations 0, 1, 2, 3, 7 and 10); C: Feature plot showing the expression distribution of Itm2a molecules in periosteal skeletal stem cells; D: Flow cytometric analysis of the expression of Itm2a and skeletal stem cell marker molecules CD105 and CD200 in the periosteum; and E: Statistics of the proportion of skeletal stem cells in Itm2a negative / positive cells. FIG3 shows the in vitro functional characterization of Itm2a-positive cells. A: Confocal microscopy imaging shows the distribution of ITM2A protein in 293 cells after overexpression of mItm2a and hITM2A plasmids; B: Crystal violet staining was used to compare the differences in the in vitro clonogenic ability of Lin-Itm2a- and Lin-Itm2a+ periosteal cells; C: Statistics of the in vitro clonogenic ability of Lin-Itm2a- and Lin-Itm2a+ periosteal cells, data represent mean ± SD, n = 3, ***P < 0.001; and D: Comparison of the in vitro chondrogenic (left, Alcian blue staining), adipogenic (middle, Oil Red O staining), and osteogenic (right, Alizarin Red staining) abilities of Lin-Itm2a- and Lin-Itm2a+ periosteal cell-derived monoclonal cells. FIG4 shows the in vivo functional characterization of Itm2a-positive periosteal skeletal stem cells. A: Schematic diagram of the process of functional verification experiments in which Actin-GFP-positive Itm2a-SSC and Itm2a+SSC were transplanted into bone defects and under the renal capsule in vitro; B: μ-CT results showed the repair of bone defects by Actin-GFP-positive Itm2a-SSC and Itm2a+SSC; C: Statistics of new bone formation at the bone defect site 7 days after Actin-GFP-positive Itm2a-SSC and Itm2a+SSC transplantation, data are mean ± SD, n = 3, ***P < 0.001; D: Confocal microscopy imaging showed that Actin-GFP-positive Itm2a-SSC and Itm2a+SSC periosteal cells were transplanted into bone defects. A: Co-localization of GFP and bone marker (OPN) in the newly formed bone after injury; E: μ-CT results showing the ability of Actin-GFP-positive Itm2a-SSC and Itm2a+SSC to form bone organoids under the renal capsule; F: Statistics of bone organoids under the renal capsule 2 months after Actin-GFP-positive Itm2a-SSC and Itm2a+SSC transplantation, data represent mean ± SD, n = 3, ***P < 0.001; and G: Confocal microscopy imaging shows the co-localization of GFP and bone marker (OPN) in bone organoids after Actin-GFP-positive Itm2a-SSC and Itm2a+SSC periosteal cells were transplanted into the renal capsule. Figure 5 shows the ability of Itm2a-positive periosteal skeletal stem cells to self-renew and differentiate into the entire skeletal stem cell lineage. A: Flow cytometric analysis showing the distribution of Itm2a and skeletal stem cell markers in bone organoids derived from Itm2a-positive SSCs; and B: Flow cytometric analysis showing the distribution of Itm2a and skeletal stem cell markers in bone organoids derived from Itm2a-negative SSCs. Figure 6 shows the distribution of ITM2A cells in human periosteum samples A: Photograph of human periosteum samples; B: Confocal microscopy imaging showing the distribution of ITM2A-positive cells in human periosteum samples; and C: Flow cytometry analysis of the proportion of skeletal stem cells and ITM2A-positive cells in human periosteal cells. FIG7 shows functional characterization of ITM2A cells in human periosteum samples. A: Comparison of the in vitro chondrogenic (left, Alcian blue staining), adipogenic (middle, Oil Red O staining) and osteogenic (right, Alizarin Red staining) abilities of ITM2A-negative and ITM2A-positive human periosteal cells; B: Comparison of the in vitro clonogenic abilities of ITM2A-negative and ITM2A-positive human periosteal cells by crystal violet staining; C: Quantitative analysis of the in vitro clonogenic abilities of ITM2A-negative and ITM2A-positive human periosteal cells, data are expressed as mean ± SD, n = 5, ***P < 0.001; D: μ-CT results showing the ability of ITM2A-negative and ITM2A-positive human periosteal cells to form bone organoids transplanted into the subrenal capsule; and E: Quantitative analysis of subrenal bone organoids 2 months after transplantation of ITM2A-negative and ITM2A-positive human periosteal cells, data are expressed as mean ± SD, n = 4, ***P < 0.001. FIG8 shows lineage tracing of periosteal skeletal stem cells in Itm2a-CreER mice. A: Construction strategy of Itm2a-CreER mice; B: Confocal microscopy imaging shows the distribution of Itm2a-positive cells in the periosteum of mice 2 days after induction at 4 weeks of age; C: Confocal microscopy imaging shows the co-localization of Itm2a-positive cells and the osteoblast marker OPN in the periosteum; D: Confocal microscopy imaging shows the co-localization of Itm2a-positive cells and the stem cell marker CD200 in the periosteum; and E: Flow cytometric analysis of the proportion of skeletal stem cells in Itm2a-negative and -positive periosteal cells. FIG9 shows that Itm2a-positive periosteal stem cells participate in the repair of fracture injuries. A: Flowchart of tamoxifen injection, fracture injury modeling and sampling in Itm2a-CreER; Ai6 mice; B: Confocal microscopy imaging showing the distribution of Itm2a-positive cell populations (ZsGreen signal positive) in the periosteum 3 days after fracture injury; C: Confocal microscopy imaging showing the colocalization of Itm2a-positive cells and the chondrocyte marker COL2A1 protein in the soft callus 7 days after fracture injury; D: Confocal microscopy imaging showing the colocalization of Itm2a-positive cells and the osteoblast marker OPN protein in the hard callus 14 days after fracture injury; E: Confocal microscopy imaging showing the colocalization of Itm2a-positive cells and the bone marrow stromal cell marker LEPR protein in the medullary cavity of the callus after fracture injury; and F: Quantitative analysis of the proportion of Itm2a-positive cells in the cartilage, osteoblasts and bone marrow stromal cells formed during the fracture injury repair process, data are expressed as mean ± SD, n = 3. FIG10 shows that knockout of Bmp2 in Itm2a lineage cells results in impaired fracture repair. A: Bar graph showing the signaling pathways enriched in upregulated genes in Itm2a-positive skeletal stem cells; B: Heat map showing the expression of BMP signaling pathway-related genes in Itm2a-negative and -positive periosteal skeletal stem cells; C: Schematic diagram of the tamoxifen-induced, fracture, and sample collection process in control mice and Bmp2 conditional knockout mice; D: X-ray results showing callus formation in control mice and Bmp2 conditional knockout mice 7 days, 14 days, and 21 days after fracture; E: Statistics of callus parameters in control mice and Bmp2 conditional knockout mice 7 days, 14 days, and 21 days after fracture, data are expressed as mean ± SD, n = 4; F: μ-CT and safranin fast green staining results in control mice and Bmp2 conditional knockout mice 14 days after fracture; and G: Quantitative analysis of new bone mass in callus in control mice and Bmp2 conditional knockout mice 14 days after fracture, data are expressed as mean ± SD, n = 4. Figure 11 shows that depletion of Itm2a lineage cells leads to impaired fracture repair. A: Schematic diagram of the tamoxifen-induced, fracture, and sample collection process in control mice and Itm2a-CreER-DTA mice; B: Confocal microscopy imaging showing the distribution of Itm2a-positive cells in the periosteum after tamoxifen induction; C: μ-CT and safranin fast green staining results in control mice and Itm2a-CreER-DTA mice on day 28 after fracture; and D: Quantitative analysis of fracture repair in control mice and Itm2a-CreER-DTA mice on day 28 after fracture, *P = 0.025, Fisher's exact test. Figure 12 shows that TNC protein enhances the bone repair function of Itm2a+ skeletal stem cells. A: Schematic diagram of the TNC protein and Itm2a+ skeletal stem cell transplantation process; B: μ-CT results showing the osteogenesis after TNC protein and Itm2a+ cell transplantation on the 7th day after injury; C: Statistics of bone volume on the 7th day after TNC protein and Itm2a+ cell transplantation, data are expressed as mean ± SD, n = 6, ***P < 0.001, **P < 0.01, *P < 0.05, ns - no significant difference. Figure 13 shows single-cell RNA sequencing analysis of tenosynovial cells. A: U-map clustering of single-cell sequencing of uninjured tendon in adult mice; B: Gene expression profiles of different cell clusters; C: Single-cell sequencing clustering of uninjured and injured tendons in adult mice; D: Ratio of cell clusters in single-cell sequencing of uninjured and injured tendons in adult mice; E: Pseudo-time series analysis of tendon sheaths and tenocytes; F: Single-cell RNA velocity analysis of uninjured and injured tendons in adult mice on day 7 after injury; G: Gene expression profiles of tenosynovial cells; H: Confocal laser microscopy imaging of tendons in PDGFRa-CreER, ScxGFP, and R26-Ai9 mice; I: Distribution of the Itm2a gene before and after injury; J: Immunofluorescence staining of Itm2a in tendons. Figure 14 shows the involvement of Itm2a+ tendon sheath stem / progenitor cells in tendon repair. A: Schematic diagram of the construction of Itm2a-CreER mice; B: mCherry expression and localization of the vascular marker CD31 in tendons of Itm2a-CreER mice; C: Confocal laser scanning microscopy images of Itm2a-CreER;R26-LSL-Ai6 mice at different time points after tamoxifen induction; D: Schematic diagram of the construction of Itm2a-DreER;ScxGFP;R26-RSR-tdTomato mice; E: Confocal laser scanning microscopy images of Itm2a-DreER;ScxGFP;R26-RSR-tdTomato mice at different time points after tendon injury; F: SHG signals in Itm2a-DreER;ScxGFP;R26-RSR-tdTomato mice before and after tendon injury. Figure 15 shows the self-renewal capacity and multipotency of Itm2a+ tendon sheath stem / progenitor cells. A: Sorting of Itm2a+ cells from adult Itm2a-DreER; ScxGFP; R26-RSR-tdTomato mouse tendons; B: Clonal proliferation of Itm2a+ cells from mouse tendons on day 4 after sorting; C: Crystal violet staining of Itm2a+ cells from mouse tendons after proliferation; D: Staining of Itm2a+ cells from mouse tendons for osteogenic differentiation (Alizarin Red S), chondrogenic differentiation (SOFG), and adipogenic differentiation (Oil Red O); E: Confocal laser microscopy imaging of Itm2a+ cells from mouse tendons transplanted into the renal capsule; F: Confocal laser microscopy imaging of tendon organoids from Itm2a+ cells from mouse tendons; G: Confocal laser microscopy imaging of Itm2a+ cells from mouse tendons transplanted into injured tendons. Figure 16 shows that depletion of Itm2a+ tendon sheath stem / progenitor cells inhibits tendon repair. A: Construction strategy of Itm2a-CreER;R26-DTA mice; B: Induction strategy of Itm2a-CreER;R26-DTA mice; C: Depletion of Itm2a+ cells from tendons of Itm2a-CreER;R26-DTA mice; D-E: H&E staining (D) and SHG fluorescence (E) images of tendons of Itm2a-CreER;R26-DTA mice and control mice 28 days after tendon injury. Figure 17: A Image of uninjured human Achilles tendon B U-map clustering of single-cell sequencing of uninjured human Achilles tendon C Single-cell sequencing clustering of tenocytes and ITM2A of uninjured human Achilles tendon D Pseudo-time analysis of tendon sheath and tenocytes E Immunofluorescence staining of ITM2A of human tendon F Sorting of Lin-ITM2A+ cells of human tendon G Crystal violet staining of Lin-ITM2A+ cells and Lin-ITM2A- cells of human tendon after proliferation H Photographs of osteogenic differentiation (Alizarin Red S), chondrogenic differentiation (Alcian blue), and adipogenic differentiation (Oil red O) staining of Lin-ITM2A+ cells and Lin-ITM2A- cells of human tendon I Fluorescence image of tendon organoids of Lin-ITM2A+ cells and Lin-ITM2A- cells of human tendon. Detailed Description of the Invention definition In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (referred to as "Sambrook"). At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below. As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C." As used herein, the term "Itm2a" refers to integral membrane protein 2A, a type II integral membrane protein. The coding sequence of the mouse Itm2a gene is 792 base pairs (bp) long, encoding a protein of approximately 30 kilodaltons (kDa). The amino acid sequence of mouse Itm2a shares 95% homology with that of human ITM2A. Itm2a is primarily expressed in ribs, vertebrae, long bones, skeletal muscle, T cells, hair follicles, skin, and tongue. However, prior art has not reported on the relationship between Itm2a and stem cells. Stem cells are cells that retain the ability to renew their own kind by cell mitosis, and their daughter cells can differentiate into a variety of specialized cell types. Two major types of mammalian cells are: embryonic stem cells (ESC) found in blastocysts, and adult stem cells found in adult tissues. In developing embryos, ESCs can differentiate into all specialized embryonic tissues. In adult organisms, adult stem cells and progenitor cells serve as the body's repair system, replenishing specialized cells and also maintaining the normal functioning of regenerated organs, such as blood, skin, and bone tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers. As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of proliferating and producing more progenitor cells that are capable of producing a large number of mother cells that can in turn produce differentiated or differentiable daughter cells known as precursor cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types while also retaining one or more cells with the developmental potential of the parent. The term "stem cell" also refers to a subpopulation of progenitor cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype under certain circumstances, and in certain cases also retain the ability to proliferate without substantially differentiating. The term "skeletal stem cells" encompasses stem cells that are capable of differentiating into cells of the skeletal lineage and ultimately forming skeletal tissue. The term "lineage" is used herein to describe cells with a common ancestor or cells with a common developmental fate, for example, chondrocyte precursor cells, osteoblast precursor cells, chondrocytes, and osteoblasts. Skeletal stem cells have the ability to self-renew, have clonal multipotency, and still have the ability to differentiate into different lineages after transplantation. As used herein, the expressions "isolated skeletal stem cell population, wherein the skeletal stem cells are Itm2a positive," and "isolated Itm2a positive skeletal stem cell population," "isolated Itm2a positive cell population" encompass heterogeneous or homogeneous populations of skeletal stem cells. A population comprising at least two different cell types is referred to herein as a "heterogeneous population." A population comprising only one cell type (e.g., Itm2a positive skeletal stem cells) is referred to herein as a "homogeneous population" of cells. Tendon stem cells, also known as tendon stem / progenitor cells (TSPCs), play a crucial role in tendon development and homeostasis, as well as injury repair. Tendon stem cells encompass those capable of differentiating into tenocytes and ultimately forming tendon tissue. Tendon stem cells possess self-renewal and clonal multipotency. As used herein, the expressions "isolated tendon stem cell population, wherein the tendon stem cells are Itm2a positive," and "isolated Itm2a-positive tendon stem cell population," "isolated Itm2a-positive cell population" encompass heterogeneous or homogeneous populations of tendon stem cells. A population comprising at least two different cell types is referred to herein as a "heterogeneous population." A population comprising only one cell type (e.g., Itm2a-positive tendon stem cells) is referred to herein as a "homogeneous population" of cells. As used herein, the term "isolated cell" refers to a cell that has been removed from the organism, organ or tissue in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro. "Self-renewal" refers to the ability of a cell to divide and produce at least one daughter cell with the same characteristics of the parent cell. The second daughter cell can enter a specific differentiation pathway. Daughter cells that enter the differentiation pathway usually have lost their self-renewal ability and, when divided, produce two daughter cells that exhibit a more differentiated (i.e., restricted) phenotype. As used herein, the term "isolation" refers to the process of removing a cell or cell population from a subject or biological sample in which it is initially found. As used herein, the term "isolated population" refers to a cell population removed and separated from a biological sample, or a mixed or heterogeneous population of cells found in such a sample. As used herein, the term "enrichment" refers to an increase in the amount, concentration, density or proportion of a cell type relative to the initial biological sample, culture or preparation, for example, an increase of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% or at least 75%, preferably an increase of at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. For example, if the proportion of a cell type in the initial sample is 3%, and the proportion of the cell type in the enriched sample is 30%, then the "enrichment" increases the proportion of the cell type by 1000%. As used herein, "marker" describes the characteristics and / or phenotype of a cell. Markers can be used to select cells containing the characteristics of interest. Markers vary with specific cells. A marker is a characteristic that is unique to a cell type, whether morphological, functional, or biochemical (enzymatic), or a molecule expressed by a cell type. Preferably, such markers are proteins, and more preferably, have epitopes of antibodies or other binding molecules available in the art. However, the marker can be composed of any molecule found in the cell, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or proterties include but are not limited to proterties, size, appearance (e.g., smooth, translucent), and nuclear-cytoplasmic ratios. Examples of functional characteristics or proterties include but are not limited to the ability to adhere to a specific substrate, the ability to absorb or exclude specific dyes, the ability to migrate under specific conditions, and the ability to differentiate along a specific lineage. Markers can be detected by any method available to those skilled in the art. Therefore, as used herein, "cell surface marker" refers to any molecule expressed on the cell surface. Cell surface expression generally requires that the molecule has a transmembrane domain. Some molecules that are not usually found on the cell surface can be engineered to be expressed on the cell surface by recombinant technology. Many naturally occurring cell surface markers are named "CD" or "cluster of differentiation" molecules. Cell surface markers often provide antigenic determinants that antibodies can bind to. As used herein, the term "antibody" refers to a complete immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having an Fc (fragment crystallizable) region or an FcRn-binding fragment of the Fc region. Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. "Antigen-binding fragments" include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, single-domain antibodies, chimeric antibodies, bispecific antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide. Such antibodies or antigen-binding fragments are commercially available from suppliers such as R&D Systems, BD Biosciences, e-Biosciences, Merck / millipore, Invitrogen, and ABCAM, or can be produced by methods known to those skilled in the art against these cell surface markers or other intracellular markers. The term "subject" refers to an animal, for example, a human from whom cells for use in the methods described herein can be obtained (i.e., a donor subject) and / or a human to whom treatment (including prophylactic treatment) with the cells described herein is provided, i.e., a recipient subject. For treatment of disease conditions or morbidities that are specific to a particular animal, such as a human subject, the term subject refers to that particular animal. "Non-human animals" and "non-human mammals," used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. However, advantageously, the subject is a mammal such as a human, or other mammals such as domestic animals, for example, dogs, cats, horses, etc., or food-producing mammals, for example, cattle, sheep, pigs, etc. As used herein, the term "extracellular matrix" (ECM) refers to a complex network of various macromolecules surrounding cells in multicellular organisms. The extracellular matrix is mainly composed of five types of substances, namely collagen, non-collagenous proteins, elastin, proteoglycans and aminoglycans. Proteins in the extracellular matrix are collectively referred to as "extracellular matrix proteins" or "ECM proteins". Examples of extracellular matrix proteins include, but are not limited to, tenascin and periostin. Tenascin is a large, multimeric extracellular matrix (ECM) protein. Four tenascin proteins are present in the connective tissue of vertebrates: tenascin-C (TNC), tenascin-R (TNR), tenascin-X (TNX), and tenascin-W (TNW). Mice lacking TNC and TNR exhibit abnormalities in their nervous systems. Furthermore, mice lacking TNC exhibit defects in multiple regenerative processes. Mice lacking TNX exhibit hyperelastic skin, which is very similar to that seen in Ehlers Danlos patients with mutations in the TNX gene. Because TNC is highly overexpressed in tumor stroma, antibodies targeting TNC have been used for tumor diagnosis and treatment, but there are currently no reports of tenascin promoting bone repair. Periostin is a multifunctional soluble intercellular matrix protein that is widely expressed in stress-bearing fibrous connective tissues such as bones, periodontal ligaments, skin, and heart valves. It is closely related to bone metabolism, cardiovascular disease, bronchial asthma, and injury repair. As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and generally do not produce toxic or allergic or similar untoward reactions when administered to humans. Itm2a-positive skeletal stem cells The present invention is based on the discovery of Itm2a-positive skeletal stem cells. The inventors discovered that Itm2a-positive skeletal stem cells possess characteristics of adult stem cells, namely self-renewal and clonogenicity. The inventors also discovered that Itm2a-positive skeletal stem cells can reconstitute the entire skeletal stem cell lineage and possess the ability to form bone, cartilage, and fat, specifically possessing a stronger osteogenesis capacity than Itm2a-negative cells. It is well known that, because of their characteristics, stem cells produce all cells and tissues of the body. Therefore, stem cells can be used to repair or accelerate the repair of damaged and / or defective bones. If a sufficient amount of SSCs can be obtained, damaged and / or defective bones can be repaired by building new tissues in the bones. In defective and / or damaged bones, SSCs may be few or non-existent. Because adult SSCs self-renew, implanted adult SSCs will colonize at the location of bone damage or defects. Through cloning, self-renewal and differentiation, implanted SSCs will produce new bone tissue. Therefore, isolated SSC populations or compositions comprising isolated SSC populations can be used to treat bone damage or defects in a subject, such as fractures. Thus, the present invention provides isolated skeletal stem cells, wherein the skeletal stem cells are Itm2a positive. The present invention also provides isolated skeletal stem cell populations, wherein the population is enriched for Itm2a positive skeletal stem cells, for example, the population primarily comprises SSCs (e.g., at least 60%, 70%, 80%, 90% or more). In some embodiments, the population comprises very few Itm2a negative cells, or no Itm2a negative cells. In some embodiments, the skeletal stem cells are Lin- (ie, CD45 / CD31 / TER119 negative) cells (CD45-CD31-TER119- cells). In some embodiments, the skeletal stem cells are mouse skeletal stem cells. In some embodiments, the skeletal stem cells are CD200 positive (CD200+) cells. In some embodiments, the skeletal stem cells are Ctsk positive (Ctsk+) cells. In some embodiments, the skeletal stem cells are CD105 negative (CD105-) cells. In some embodiments, the skeletal stem cells are 6C3 negative (6C3-) cells. In some embodiments, the skeletal stem cells are CD90.2 negative (CD90.2-) cells. In some embodiments, the mouse skeletal stem cells are CD200+CD105- cells. In some embodiments, the mouse skeletal stem cells are CD200+Ctsk+CD105- cells. In some embodiments, the mouse skeletal stem cells are 6C3-CD90.2-CD200+CD105- cells. In some embodiments, the mouse skeletal stem cells are Lin-6C3-CD90.2-CD200+CD105- cells. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the skeletal stem cells are CD45 negative (CD45-) cells. In some embodiments, the skeletal stem cells are CD235 negative (CD235-) cells. In some embodiments, the skeletal stem cells are CD146 negative (CD146-) cells. In some embodiments, the skeletal stem cells are PDPN positive (PDPN+) cells. In some embodiments, the skeletal stem cells are CD73 positive (CD73+) cells. In some embodiments, the skeletal stem cells are CD164 positive (CD164+) cells. In some embodiments, the human skeletal stem cells are CD45-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, the human skeletal stem cells are Lin-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, the skeletal stem cells have the ability to differentiate into osteoblasts. In some embodiments, the skeletal stem cell population is self-renewing and clonogenic. In some embodiments, the skeletal stem cells are enriched from a bone tissue sample. In some embodiments, the skeletal stem cell population is expanded ex vivo. In some embodiments, the skeletal stem cells are modified to increase the level of BMP2 protein. The expression level of a specific protein in a cell can be increased by techniques known in the art, including but not limited to introducing a BMP2 overexpression vector (e.g., a viral vector, such as rAAV) in a cell, targeting a BMP2 regulatory sequence (e.g., a promoter) of a transcriptional activation system (e.g., a transcriptional activation domain (TAD) fused to a sequence-specific DNA binding domain such as a zinc finger domain, TALE, or dCas, or an epigenetic regulatory portion, such as a polypeptide that reduces the level of DNA methylation, such as a TET protein or its functional fragment). Isolation, expansion, and modification of Itm2a-positive skeletal stem cells The inventors discovered that Itm2a is expressed on the cell membrane of skeletal stem cells, providing a new molecular marker for identifying and isolating skeletal stem cells. Therefore, the present invention provides a method for preparing an isolated skeletal stem cell population, wherein the skeletal stem cells are Itm2a positive, the method comprising: a) providing a pool of cells from bone tissue; and b) selecting Itm2a-positive cells from the bone tissue-derived cell pool. In some embodiments, the method further comprises selecting Lin- (ie, CD45 / CD31 / TER119 negative) cells (CD45-CD31-TER119- cells). In some embodiments, the skeletal stem cells are mouse cells. In some embodiments, the method further comprises selecting CD200 positive (CD200+) cells. In some embodiments, the method further comprises selecting Ctsk positive (Ctsk+) cells. In some embodiments, the method further comprises selecting CD105 negative (CD105-) cells. In some embodiments, the method further comprises selecting 6C3 negative (6C3-) cells. In some embodiments, the method further comprises selecting CD90.2 negative (CD90.2-) cells. In some embodiments, the method further comprises selecting CD200+CD105- cells. In some embodiments, the method further comprises selecting CD200+Ctsk+CD105- cells. In some embodiments, the method further comprises selecting 6C3-CD90.2-CD200+CD105- cells. In some embodiments, the method further comprises selecting Lin-6C3-CD90.2-CD200+CD105- cells. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the method further comprises selecting CD45 negative (CD45-) cells. In some embodiments, the method further comprises selecting CD235 negative (CD235-) cells. In some embodiments, the method further comprises selecting CD146 negative (CD146-) cells. In some embodiments, the method further comprises selecting PDPN positive (PDPN+) cells. In some embodiments, the method further comprises selecting CD73 positive (CD73+) cells. In some embodiments, the method further comprises selecting CD164 positive (CD164+) cells. In some embodiments, the method further comprises selecting CD45-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, the method further comprises selecting Lin-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, step a) comprises obtaining bone tissue from a subject. In some embodiments, the subject is a human, including adolescents, adults, and the elderly. In some embodiments, the bone tissue is periosteum. In some embodiments, the method further comprises enzymatically digesting the bone tissue, e.g., eliminating non-cellular structures in the tissue, to obtain a pool of cells from the bone tissue. In some embodiments, the method further comprises removing red blood cells, e.g., lysing the red blood cells. In some embodiments, the method further comprises filtering to remove cell aggregates. In some embodiments, step b) comprises selecting with a binding molecule directed against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule against CD45, CD31 and / or TER119, such as an anti-CD45, anti-CD31 and / or anti-TER119 antibody or an antigen-binding fragment thereof. In some embodiments, the skeletal stem cells are mouse cells.In some embodiments, the method further comprises selecting with a binding molecule to 6C3, CD90.2, CD200 and / or CD105, such as an anti-6C3, anti-CD90.2, anti-CD200 and / or anti-CD105 antibody or an antigen-binding fragment thereof. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the method further comprises selecting with a binding molecule to CD235, CD146, PDPN, CD73 and / or CD164, such as an anti-CD235, anti-CD146, anti-PDPN, anti-CD73 and / or anti-CD164 antibody or antigen-binding fragment thereof. In some embodiments, the binding molecule is conjugated to solid particles, such as magnetic beads. In some embodiments, the binding molecule is labeled, such as with a fluorescent dye. In some embodiments, step b) comprises selection by flow cytometry.In some embodiments, step b) comprises selection by magnetic selection. In a specific embodiment, the method comprises: obtaining periosteum (e.g., 1 cm×1 cm) from a subject; digesting the periosteum with collagenase to obtain single cells; collecting the single cells by centrifugation and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; incubating the cells with an anti-Itm2a antibody; and isolating cells bound to the antibody. In some embodiments, the method further comprises c) expanding the cells selected in step b) in a culture medium. In some embodiments, the cells are cultured on a biocompatible scaffold, preferably a 3D scaffold. In some embodiments, after expansion, the number of Itm2a-positive cells is at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10,000-fold, 20,000-fold, 50,000-fold, or more. The number of cells in culture can be determined by any method known in the art, for example, by using a Coulter counter. Such methods are well known to those skilled in the art. In some embodiments, the cells selected in step b) (ie, Itm2a-positive cells) are stored at low temperature (eg, -80°C). In some embodiments, the expanded cells are stored at low temperatures (eg, -80° C.) for storage purposes. When needed, the frozen cells are thawed and then, for example, used for implantation into a subject in need thereof. In some embodiments, before cryopreservation, the cell suspension is mixed with a cryoprotectant. Methods for cryopreserving tissues and cells at low temperatures with cryoprotectants are well known in the art. Frozen samples can be carried out in the presence of one or more different cryoprotectants to minimize cell damage during freeze-thaw. For example, dimethyl sulfoxide (DMSO), trehalose or sucrose can be used. The invention also provides a method for expanding or proliferating Itm2a-positive skeletal stem cells, comprising incubating isolated Itm2a-positive skeletal stem cells in a culture medium. In some embodiments, the method further comprises culturing the Itm2a-positive skeletal stem cells on a biocompatible scaffold, preferably a 3D scaffold. In some embodiments, after expansion or proliferation, the number of Itm2a-positive cells is at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10,000-fold, 20,000-fold, 50,000-fold, or more. The present invention also provides a kit for isolating skeletal stem cells, comprising an Itm2a binding molecule. In some embodiments, the binding molecule is an anti-Itm2a antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the kit further comprises a binding molecule against CD45, CD31 and / or TER119, such as an anti-CD45, anti-CD31 and / or anti-TER119 antibody or an antigen-binding fragment thereof. In some embodiments, the skeletal stem cells are mouse cells.In some embodiments, the method further comprises selecting with a binding molecule to 6C3, CD90.2, CD200 and / or CD105, such as an anti-6C3, anti-CD90.2, anti-CD200 and / or anti-CD105 antibody or an antigen-binding fragment thereof. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the kit further comprises a binding molecule to CD235, CD146, PDPN, CD73 and / or CD164, such as an anti-CD235, anti-CD146, anti-PDPN, anti-CD73 and / or anti-CD164 antibody or an antigen-binding fragment thereof. In some embodiments, the binding molecule is conjugated to solid particles, such as magnetic beads. In some embodiments, the binding molecule is labeled, such as with a fluorescent dye. The present invention also provides a method for modifying isolated skeletal stem cells, comprising increasing the level of skeletal stem cell BMP2 protein. In some embodiments, the method includes introducing a BMP2 overexpression vector (e.g., a viral vector, such as rAAV) into the skeletal cells. In some embodiments, the method includes increasing the expression or activity of endogenous BMP2, such as introducing a transcriptional activation system targeting a BMP2 regulatory sequence (e.g., a promoter) (e.g., a transcriptional activation domain (TAD) fused to a sequence-specific DNA binding domain such as a zinc finger domain, TALE, or dCas, or an epigenetic regulatory portion, such as a polypeptide that reduces DNA methylation levels, such as a TET protein or a functional fragment thereof). Pharmaceutical composition The present invention provides a pharmaceutical composition comprising: i) isolated skeletal stem cells, wherein the skeletal stem cells are Itm2a positive; and ii) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a population enriched for Itm2a-positive skeletal stem cells, i.e., the population comprises primarily Itm2a-positive SSCs (e.g., at least 60%, 70%, 80%, 90% or more). In some embodiments, the population comprises few Itm2a-negative cells, or no Itm2a-negative cells. In some embodiments, the skeletal stem cells are Lin- (ie, CD45 / CD31 / TER119 negative) cells (CD45-CD31-TER119- cells). In some embodiments, the skeletal stem cells are mouse skeletal stem cells. In some embodiments, the skeletal stem cells are CD200 positive (CD200+) cells. In some embodiments, the skeletal stem cells are Ctsk positive (Ctsk+) cells. In some embodiments, the skeletal stem cells are CD105 negative (CD105-) cells. In some embodiments, the skeletal stem cells are 6C3 negative (6C3-) cells. In some embodiments, the skeletal stem cells are CD90.2 negative (CD90.2-) cells. In some embodiments, the mouse skeletal stem cells are CD200+CD105- cells. In some embodiments, the mouse skeletal stem cells are CD200+Ctsk+CD105- cells. In some embodiments, the mouse skeletal stem cells are 6C3-CD90.2-CD200+CD105- cells. In some embodiments, the mouse skeletal stem cells are Lin-6C3-CD90.2-CD200+CD105- cells. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the skeletal stem cells are CD45 negative (CD45-) cells. In some embodiments, the skeletal stem cells are CD235 negative (CD235-) cells. In some embodiments, the skeletal stem cells are CD146 negative (CD146-) cells. In some embodiments, the skeletal stem cells are PDPN positive (PDPN+) cells. In some embodiments, the skeletal stem cells are CD73 positive (CD73+) cells. In some embodiments, the skeletal stem cells are CD164 positive (CD164+) cells. In some embodiments, the human skeletal stem cells are CD45-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, the skeletal stem cells have the ability to differentiate into osteoblasts. In some embodiments, the skeletal stem cell population is self-renewing and clonogenic. In some embodiments, the skeletal stem cells are enriched from a bone tissue sample. In some embodiments, the skeletal stem cell population is expanded ex vivo. In some embodiments, the pharmaceutical composition is formulated for local administration, eg, local administration at the site of a bone fracture. In some embodiments, the pharmaceutical composition comprises a 3D culture of the skeletal stem cell population. The 3D culture can be obtained by culturing the skeletal stem cell population on a biocompatible 3D tissue scaffold. In some embodiments, the pharmaceutical composition is formulated as a gel. The composition may comprise additional bioactive agents, including but not limited to pharmaceutically active compounds, hormones, growth factors, enzymes, DNA, RNA, siRNA, hyaluronic acid, antibodies, antibiotics, anti-inflammatory molecules, extracellular matrix proteins (such as tenosynovins, e.g., TNC, and periostin), or combinations thereof. In some embodiments, the bioactive agent comprises BMP2 or an isolated polynucleotide (e.g., mRNA) or expression vector encoding BMP2. In some embodiments, the bioactive agent comprises "pro-angiogenic factors" including, but not limited to, epidermal growth factor (EGF), E-cadherin, VEGF, angiogenic proteins, angiopoietin-1, fibroblast growth factor (FGF, including aFGF and bFGF), hepatocyte growth factor (HGF), angiogenin, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), and inflammatory cytokines and chemokines (which are inducers of angiogenesis and increased blood vessel formation), for example, interleukin-3 (IL-3), interleukin-8 (IL-8), CCL2 (MCP-1), interleukin-8 (IL-8), and CCL5 (RANTES). Treating bone injuries or defects The present invention provides a method for treating a bone injury or defect, such as a bone fracture, in a subject in need thereof, the method comprising a) providing a cell pool from bone tissue; b) selecting Itm2a-positive cells from the cell pool derived from bone tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof. In some embodiments, the bone tissue is bone tissue from a subject in need thereof. In some embodiments, the bone tissue is allogenic bone tissue. In some embodiments, the subject in need thereof is a human, including adolescents, adults, and the elderly. In some embodiments, the bone tissue is periosteum. In some embodiments, the method further comprises enzymatically digesting the bone tissue, e.g., eliminating non-cellular structures in the tissue, to obtain a pool of cells from the bone tissue. In some embodiments, the method further comprises removing red blood cells, e.g., lysing the red blood cells. In some embodiments, the method further comprises filtering to remove cell aggregates. In some embodiments, the method further comprises selecting Lin- (ie, CD45 / CD31 / TER119 negative) cells (CD45-CD31-TER119- cells). In some embodiments, the skeletal stem cells are mouse cells. In some embodiments, the method further comprises selecting CD200 positive (CD200+) cells. In some embodiments, the method further comprises selecting Ctsk positive (Ctsk+) cells. In some embodiments, the method further comprises selecting CD105 negative (CD105-) cells. In some embodiments, the method further comprises selecting 6C3 negative (6C3-) cells. In some embodiments, the method further comprises selecting CD90.2 negative (CD90.2-) cells. In some embodiments, the method further comprises selecting CD200+CD105- cells. In some embodiments, the method further comprises selecting CD200+Ctsk+CD105- cells. In some embodiments, the method further comprises selecting 6C3-CD90.2-CD200+CD105- cells. In some embodiments, the method further comprises selecting Lin-6C3-CD90.2-CD200+CD105- cells. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the method further comprises selecting CD45 negative (CD45-) cells. In some embodiments, the method further comprises selecting CD235 negative (CD235-) cells. In some embodiments, the method further comprises selecting CD146 negative (CD146-) cells. In some embodiments, the method further comprises selecting PDPN positive (PDPN+) cells. In some embodiments, the method further comprises selecting CD73 positive (CD73+) cells. In some embodiments, the method further comprises selecting CD164 positive (CD164+) cells. In some embodiments, the method further comprises selecting CD45-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, the method further comprises selecting Lin-CD235-CD146-PDPN+CD73+CD164+ cells. In some embodiments, step b) comprises selecting with a binding molecule directed against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule against CD45, CD31 and / or TER119, such as an anti-CD45, anti-CD31 and / or anti-TER119 antibody or an antigen-binding fragment thereof. In some embodiments, the skeletal stem cells are mouse cells.In some embodiments, the method further comprises selecting with a binding molecule to 6C3, CD90.2, CD200 and / or CD105, such as an anti-6C3, anti-CD90.2, anti-CD200 and / or anti-CD105 antibody or an antigen-binding fragment thereof. In some embodiments, the skeletal stem cells are human skeletal stem cells. In some embodiments, the method further comprises selecting with a binding molecule to CD235, CD146, PDPN, CD73 and / or CD164, such as an anti-CD235, anti-CD146, anti-PDPN, anti-CD73 and / or anti-CD164 antibody or antigen-binding fragment thereof. In some embodiments, the binding molecule is conjugated to solid particles, such as magnetic beads. In some embodiments, the binding molecule is labeled, such as with a fluorescent dye. In some embodiments, step b) comprises selection by flow cytometry.In some embodiments, step b) comprises selection by magnetic selection. In a specific embodiment, the method comprises: obtaining periosteum (e.g., 1 cm×1 cm) from a subject; digesting the periosteum with collagenase to obtain single cells; collecting the single cells by centrifugation and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; incubating the cells with an anti-Itm2a antibody; and isolating cells bound to the antibody. In some embodiments, after amplification, the number of Itm2a-positive cells is at least 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10,000 times, 20,000 times, 50,000 times or more. The number of cells in the culture can be determined by any method known in the art, for example, by utilizing a Coulter counter. These methods are well known to those skilled in the art. In some embodiments, the cells selected in step b) (ie, Itm2a-positive cells) are stored at low temperature (eg, -80°C). In some embodiments, step c) comprises culturing the cells on a biocompatible scaffold, preferably a 3D scaffold. In some embodiments, the expanded cells are stored at low temperatures (eg, -80°C) for storage purposes. When needed, the frozen cells are thawed and then implanted into a subject in need. In some embodiments, before cryopreservation, the cell suspension is mixed with a cryoprotectant. Methods for cryopreserving tissues and cells at low temperatures with cryoprotectants are well known in the art. Frozen samples can be carried out in the presence of one or more different cryoprotectants to minimize cell damage during freeze-thaw. For example, dimethyl sulfoxide (DMSO), trehalose or sucrose can be used. In some embodiments, the expanded cells are administered locally at the site of a fracture or bone defect. In some embodiments, the cells are administered in the form of a gel. The cells can be administered in combination with additional bioactive agents, including but not limited to pharmaceutically active compounds, hormones, growth factors, enzymes, DNA, RNA, siRNA, hyaluronic acid, antibodies, antibiotics, anti-inflammatory molecules, extracellular matrix proteins (such as tenosynovins, e.g., TNC, and periostin), or combinations thereof. In some embodiments, the bioactive agent comprises "pro-angiogenic factors" including, but not limited to, epidermal growth factor (EGF), E-cadherin, VEGF, angiogenic proteins, angiopoietin-1, fibroblast growth factor (FGF, including aFGF and bFGF), hepatocyte growth factor (HGF), angiogenin, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), and inflammatory cytokines and chemokines (which are inducers of angiogenesis and increased blood vessel formation), for example, interleukin-3 (IL-3), interleukin-8 (IL-8), CCL2 (MCP-1), interleukin-8 (IL-8), and CCL5 (RANTES). In some embodiments, the method further comprises modifying the isolated skeletal stem cells to increase the level of BMP2 protein. In some embodiments, the method comprises introducing a BMP2 overexpression vector (e.g., a viral vector, such as rAAV) into the skeletal cells. In some embodiments, the method comprises increasing the expression or activity of endogenous BMP2, such as introducing a transcriptional activation system targeting a BMP2 regulatory sequence (e.g., a promoter) (e.g., a transcriptional activation domain (TAD) fused to a sequence-specific DNA binding domain such as a zinc finger domain, TALE, or dCas, or an epigenetic regulatory portion, such as a polypeptide that reduces DNA methylation levels, such as a TET protein or a functional fragment thereof). The present invention also provides a method of treating bone damage or defects, such as bone fractures, in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present invention. In some embodiments, the pharmaceutical composition is administered locally at the fracture site. In some embodiments, the skeletal stem cells are autologous or allogeneic to the subject in need thereof. The present invention also provides an enriched isolated Itm2a-positive skeletal stem cell population or a pharmaceutical composition comprising the population for treating bone injuries or defects, such as fractures, in a subject in need thereof. The present invention also provides the use of an enriched isolated Itm2a-positive skeletal stem cell population or a pharmaceutical composition containing the population in the preparation of a medicament for treating bone injuries or defects, such as fractures, in a subject in need. The present invention also provides a method for screening drugs for treating bone injuries or defects, such as bone fractures, comprising: i) contacting the compound with Itm2a-positive skeletal stem cells, for example, culturing the skeletal stem cells in a culture medium containing the compound; ii) detecting the growth and / or differentiation of the skeletal stem cells; iii) comparing the growth and / or differentiation of skeletal stem cells to that of a control; and iv) selecting compounds that promote the growth and / or differentiation of said skeletal stem cells. The control skeletal stem cells are Itm2a-positive skeletal stem cells that have not been contacted with the compound. The compound can be a small molecule compound or a protein. In some embodiments, the differentiation is osteogenic differentiation. In some embodiments, the detection in step ii) includes in vitro detection and / or in vivo detection. For example, the cells can be cultured in vitro and / or induced to differentiate, and their proliferation capacity and / or osteogenic differentiation capacity can be detected; the skeletal stem cells can also be transplanted into animals to detect their proliferation capacity and / or osteogenic differentiation capacity. The present invention also provides a method for screening drugs for treating bone injuries or defects, such as bone fractures, comprising: i) contacting a compound with a population of skeletal stem cells, such as by culturing the population in a culture medium comprising the compound; ii) detecting the proportion, proliferation capacity and / or osteogenic differentiation capacity of Itm2a-positive cells in the skeletal stem cell population; iii) comparing the proportion of Itm2a-positive cells in a control skeletal stem cell population; and iv) Selecting compounds that increase the proportion of Itm2a-positive cells in a skeletal stem cell population. The control skeletal stem cell population is a skeletal stem cell that has not been exposed to the compound. The compound can be a small molecule compound or a protein. In some embodiments, the detection in step ii) includes in vitro detection and / or in vivo detection. For example, the cells can be cultured in vitro and / or induced to differentiate, and their proliferation capacity and / or osteogenic differentiation capacity can be detected; the skeletal stem cells can also be transplanted into an animal and their proliferation capacity and / or osteogenic differentiation capacity can be detected. The present invention also provides a method for screening drugs for treating bone injuries or defects, such as bone fractures, comprising: i) administering the compound to a subject; ii) isolating a skeletal stem cell population from the subject, and detecting the proportion, proliferation capacity, and / or osteogenic differentiation capacity of Itm2a-positive skeletal stem cells; iii) comparing the proportion, proliferation capacity and / or osteogenic differentiation capacity of Itm2a-positive skeletal stem cells in a skeletal stem cell population isolated from a control subject; and iv) selecting compounds that increase the proportion, proliferation capacity and / or osteogenic differentiation capacity of Itm2a-positive cells in a skeletal stem cell population isolated from a subject. The control animal is a skeletal stem cell that has not been administered the compound. The compound can be a small molecule compound or a protein. In some embodiments, the testing in step ii) includes in vitro testing and / or in vivo testing. For example, the cells can be cultured in vitro and / or induced to differentiate, and their proliferation capacity and / or osteogenic differentiation capacity can be tested; or the skeletal stem cells can be transplanted into an animal and their proliferation capacity and / or osteogenic differentiation capacity can be tested. Itm2a-positive tendon stem cells Combining scRNA-seq and lineage tracing techniques to investigate the tendon healing process, the inventors discovered a subpopulation of stem / progenitor cells within the tendon sheath, marked by the membrane protein Itm2a. Through in vitro culture, differentiation, and transplantation experiments, they demonstrated that Itm2a+ cells possess multipotency and self-renewal abilities. Human tendons also contain ITM2A+ tendon stem / progenitor cells, which exhibit properties similar to those of mouse Itm2a+ tendon stem cells. It is well known that, due to their properties, stem cells produce all cells and tissues of the body. Therefore, stem cells can be used to repair or accelerate the repair of damaged and / or defective tendons. If a sufficient amount of TSCs can be obtained, damaged and / or defective tendons can be repaired by building new tissue in the tendon. In defective and / or damaged tendons, TSCs may be few or absent. Because adult TSCs self-renew, implanted adult TSCs will colonize at the site of tendon damage or defect. Through cloning, self-renewal and differentiation, implanted TSCs will produce new tendon tissue. Therefore, isolated TSC populations or compositions comprising isolated TSC populations can be used to treat tendon damage or defects in a subject. Thus, the present invention provides isolated tendon stem cells, wherein the tendon stem cells are Itm2a positive. The present invention also provides isolated tendon stem cell populations, wherein the population is enriched for Itm2a positive tendon stem cells, for example, the population primarily comprises TSCs (e.g., at least 60%, 70%, 80%, 90% or more). In some embodiments, the population comprises very few Itm2a negative cells, or does not comprise Itm2a negative cells. In some embodiments, the tendon stem cells are Lin- (ie, CD45 / CD31 / CD235a negative) cells (CD45-CD31-CD235a- cells). In some embodiments, the tendon stem cells are mouse tendon stem cells. In some embodiments, the tendon stem cells are CD146 positive cells (CD146+ cells). In some embodiments, the tendon stem cells are CD105 positive cells (CD105+ cells). In some embodiments, the tendon stem cells are Sca1 positive cells (Sca1+ cells). In some embodiments, the tendon stem cells are PDGFRα positive cells (PDGFRα+ cells). In some embodiments, the tendon stem cells are CD146+CD105+Sca1+PDGFRα+ cells. In some embodiments, the tendon stem cells are human tendon stem cells. In some embodiments, the tendon stem cells are CD146 positive cells (CD146+ cells). In some embodiments, the tendon stem cells are CD90 positive cells (CD90+ cells). In some embodiments, the tendon stem cells are CD44 positive cells (CD44+ cells). In some embodiments, the tendon stem cells are CD146+CD90+CD44+ cells. In some embodiments, the tendon stem cells have the ability to differentiate into tenocytes. In some embodiments, the tendon stem cell population is self-renewing and clonogenic. In some embodiments, the tendon stem cells are enriched from a tendon or tendon sheath tissue sample. In some embodiments, the tendon stem cell population is expanded ex vivo. Isolation, expansion, and modification of Itm2a-positive tendon stem cells The inventors discovered that Itm2a is expressed on the cell membrane of tendon stem cells, providing a new molecular marker for identifying and isolating tendon stem cells. Therefore, the present invention provides a method for preparing an isolated tendon stem cell population, wherein the tendon stem cells are Itm2a positive, the method comprising: a) providing a pool of cells from tendon or tendon sheath tissue; and b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue. In some embodiments, the method further comprises selecting Lin- (ie, CD45 / CD31 / CD235a negative) cells (CD45-CD31-CD235a- cells). In some embodiments, the tendon stem cells are mouse tendon stem cells. In some embodiments, the method further comprises selecting CD146 positive cells (CD146+ cells). In some embodiments, the method further comprises selecting CD105 positive cells (CD105+ cells). In some embodiments, the method further comprises selecting Sca1 positive cells (Sca1+ cells). In some embodiments, the method further comprises selecting PDGFRα positive cells (PDGFRα+ cells). In some embodiments, the method further comprises selecting CD146+CD105+Sca1+PDGFRα+ cells. In some embodiments, the tendon stem cells are human tendon stem cells. In some embodiments, the method further comprises selecting CD146 positive cells (CD146+ cells). In some embodiments, the method further comprises selecting CD90 positive cells (CD90+ cells). In some embodiments, the method further comprises selecting CD44 positive cells (CD44+ cells). In some embodiments, the method further comprises selecting CD146+CD90+CD44+ cells. In some embodiments, step a) comprises obtaining tendon or tendon sheath tissue from a subject. In some embodiments, the subject is a human, including adolescents, adults, and the elderly. In some embodiments, the tendon or tendon sheath tissue is a tendon or tendon sheath. In some embodiments, the method further comprises enzymatically digesting the tendon or tendon sheath tissue, e.g., eliminating non-cellular structures in the tissue, to obtain a pool of cells from the tendon or tendon sheath tissue. In some embodiments, the method further comprises removing red blood cells, e.g., lysing the red blood cells. In some embodiments, the method further comprises filtering to remove cell aggregates. In some embodiments, step b) comprises selecting with a binding molecule directed against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD45, CD31 and / or CD235a, such as an anti-CD45, anti-CD31 and / or anti-CD235a antibody or antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD146, CD105, Sca1 and / or PDGFRα, such as an anti-CD146, anti-CD105, anti-Sca1 and / or anti-PDGFRα antibody or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD146, CD90 and / or CD44, such as an anti-CD146, CD90 and / or CD44 antibody or antigen-binding fragment thereof. In some embodiments, the binding molecule is conjugated to solid particles, such as magnetic beads. In some embodiments, the binding molecule is labeled, such as with a fluorescent dye. In some embodiments, step b) comprises selection by flow cytometry.In some embodiments, step b) comprises selection by magnetic selection. In a specific embodiment, the method comprises: obtaining tendon or tendon sheath tissue from a subject; digesting the tendon or tendon sheath tissue with collagenase (e.g., collagenase II) and dispase (e.g., dispase II) to obtain single cells; collecting the single cells by centrifugation and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; incubating the cells with an anti-Itm2a antibody; and isolating cells bound to the antibody. In some embodiments, the method further comprises c) expanding the cells selected in step b) in culture. In some embodiments, the cells are cultured on a biocompatible scaffold, preferably a 3D scaffold. In some embodiments, after expansion, the number of Itm2a-positive cells is at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10,000-fold, 20,000-fold, 50,000-fold, or more. The number of cells in culture can be determined by any method known in the art, for example, by using a Coulter counter. Such methods are well known to those skilled in the art. In some embodiments, the cells selected in step b) (ie, Itm2a-positive cells) are stored at low temperature (eg, -80°C). In some embodiments, the expanded cells are stored at low temperatures (eg, -80° C.) for storage purposes. When needed, the frozen cells are thawed and then, for example, used for implantation into a subject in need thereof. In some embodiments, before cryopreservation, the cell suspension is mixed with a cryoprotectant. Methods for cryopreserving tissues and cells at low temperatures with cryoprotectants are well known in the art. Frozen samples can be carried out in the presence of one or more different cryoprotectants to minimize cell damage during freeze-thaw. For example, dimethyl sulfoxide (DMSO), trehalose or sucrose can be used. The present invention also provides a method for expanding or proliferating Itm2a-positive tendon stem cells, comprising incubating the isolated Itm2a-positive tendon stem cells in a culture medium. In some embodiments, the method further comprises culturing the Itm2a-positive tendon stem cells on a biocompatible scaffold, preferably a 3D scaffold. In some embodiments, after expansion or proliferation, the number of Itm2a-positive cells is at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10,000-fold, 20,000-fold, 50,000-fold, or more. The present invention also provides a kit for isolating tendon stem cells, comprising an Itm2a binding molecule. In some embodiments, the binding molecule is an anti-Itm2a antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the kit further comprises a binding molecule against CD45, CD31 and / or CD235a, such as an anti-CD45, anti-CD31 and / or anti-CD235a antibody or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD146, CD105, Sca1 and / or PDGFRα, such as an anti-CD146, anti-CD105, anti-Sca1 and / or anti-PDGFRα antibody or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD146, CD90 and / or CD44, such as an anti-CD146, CD90 and / or CD44 antibody or antigen-binding fragment thereof. In some embodiments, the binding molecule is conjugated to solid particles, such as magnetic beads. In some embodiments, the binding molecule is labeled, such as with a fluorescent dye. Pharmaceutical composition The present invention provides a pharmaceutical composition comprising: i) isolated tendon stem cells, wherein the tendon stem cells are Itm2a positive; and ii) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a population enriched for Itm2a-positive tendon stem cells, i.e., the population comprises primarily Itm2a-positive TSCs (e.g., at least 60%, 70%, 80%, 90% or more). In some embodiments, the population comprises few Itm2a-negative cells, or no Itm2a-negative cells. In some embodiments, the tendon stem cells are Lin- (ie, CD45 / CD31 / CD235a negative) cells (CD45-CD31-CD235a- cells). In some embodiments, the tendon stem cells are mouse tendon stem cells. In some embodiments, the tendon stem cells are CD146 positive cells (CD146+ cells). In some embodiments, the tendon stem cells are CD105 positive cells (CD105+ cells). In some embodiments, the tendon stem cells are Sca1 positive cells (Sca1+ cells). In some embodiments, the tendon stem cells are PDGFRα positive cells (PDGFRα+ cells). In some embodiments, the tendon stem cells are CD146+CD105+Sca1+PDGFRα+ cells. In some embodiments, the tendon stem cells are human tendon stem cells. In some embodiments, the tendon stem cells are CD146 positive cells (CD146+ cells). In some embodiments, the tendon stem cells are CD90 positive cells (CD90+ cells). In some embodiments, the tendon stem cells are CD44 positive cells (CD44+ cells). In some embodiments, the tendon stem cells are CD146+CD90+CD44+ cells. In some embodiments, the tendon stem cells have the ability to differentiate into tenocytes. In some embodiments, the tendon stem cell population is self-renewing and clonogenic. In some embodiments, the tendon stem cells are enriched from a tendon or tendon sheath tissue sample. In some embodiments, the tendon stem cell population is expanded ex vivo. In some embodiments, the pharmaceutical composition is formulated for local administration, for example, at the site of a tendon injury or defect. In some embodiments, the pharmaceutical composition comprises a 3D culture of the tendon stem cell population. The 3D culture can be obtained by culturing the tendon stem cell population on a biocompatible 3D tissue scaffold. In some embodiments, the pharmaceutical composition is formulated as a gel. The composition may comprise additional bioactive agents including, but not limited to, pharmaceutically active compounds, hormones, growth factors, enzymes, DNA, RNA, siRNA, hyaluronic acid, antibodies, antibiotics, anti-inflammatory molecules, extracellular matrix proteins (such as tenascins, e.g., TNC, and periostin), or combinations thereof. In some embodiments, the bioactive agent includes, but is not limited to, bFGF, GDF5, GDF6 (BMP13), GDF7 (BMP12), IGF1, PDGF, TGF-b1, TGF-b2, and VEGF. Treating tendon injuries or defects The present invention provides a method of treating a tendon injury or defect in a subject in need thereof, the method comprising a) providing a pool of cells from tendon or tendon sheath tissue; b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof. In addition, tendon injuries can cause complications such as tendon adhesion and heterotopic ossification, which can lead to pain and impaired tendon recovery. Improving tendon repair can prevent and treat complications of tendon injuries. Therefore, the present invention also provides a method for preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification) in a subject in need thereof, the method comprising a) providing a pool of cells from tendon or tendon sheath tissue; b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof. In some embodiments, the tendon or tendon sheath tissue is autologous tendon or tendon sheath tissue of the subject in need. In some embodiments, the tendon or tendon sheath tissue is allogeneic tendon or tendon sheath tissue. In some embodiments, the subject in need is a human, including adolescents, adults, and the elderly. In some embodiments, the tendon or tendon sheath tissue is a tendon or tendon sheath. In some embodiments, the method further comprises enzymatically digesting the tendon or tendon sheath tissue, e.g., eliminating non-cellular structures in the tissue, to obtain a pool of cells from the tendon or tendon sheath tissue. In some embodiments, the method further comprises removing red blood cells, e.g., lysing the red blood cells. In some embodiments, the method further comprises filtering to remove cell aggregates. In some embodiments, the method further comprises selecting Lin- (ie, CD45 / CD31 / CD235a negative) cells (CD45-CD31-CD235a- cells). In some embodiments, the tendon stem cells are mouse tendon stem cells. In some embodiments, the method further comprises selecting CD146 positive cells (CD146+ cells). In some embodiments, the method further comprises selecting CD105 positive cells (CD105+ cells). In some embodiments, the method further comprises selecting Sca1 positive cells (Sca1+ cells). In some embodiments, the method further comprises selecting PDGFRα positive cells (PDGFRα+ cells). In some embodiments, the method further comprises selecting CD146+CD105+Sca1+PDGFRα+ cells. In some embodiments, the tendon stem cells are human tendon stem cells. In some embodiments, the method further comprises selecting CD146 positive cells (CD146+ cells). In some embodiments, the method further comprises selecting CD90 positive cells (CD90+ cells). In some embodiments, the method further comprises selecting CD44 positive cells (CD44+ cells). In some embodiments, the method further comprises selecting CD146+CD90+CD44+ cells. In some embodiments, step b) comprises selecting with a binding molecule directed against Itm2a. In some embodiments, the binding molecule is an anti-Itm2a antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule directed against CD45, CD31 and / or CD235a, such as an anti-CD45, anti-CD31 and / or anti-CD235a antibody or antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD146, CD105, Sca1 and / or PDGFRα, such as an anti-CD146, anti-CD105, anti-Sca1 and / or anti-PDGFRα antibody or an antigen-binding fragment thereof. In some embodiments, the method further comprises selecting with a binding molecule to CD146, CD90 and / or CD44, such as an anti-CD146, CD90 and / or CD44 antibody or antigen-binding fragment thereof. In some embodiments, the binding molecule is conjugated to solid particles, such as magnetic beads. In some embodiments, the binding molecule is labeled, such as with a fluorescent dye. In some embodiments, step b) comprises selection by flow cytometry.In some embodiments, step b) comprises selection by magnetic selection. In a specific embodiment, the method comprises: obtaining tendon or tendon sheath tissue from a subject; digesting the tendon or tendon sheath tissue with collagenase (e.g., collagenase II) and dispase (e.g., dispase II) to obtain single cells; collecting the single cells by centrifugation and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; incubating the cells with an anti-Itm2a antibody; and isolating cells bound to the antibody. In some embodiments, after amplification, the number of Itm2a-positive cells is at least 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times, 2000 times, 5000 times, 10,000 times, 20,000 times, 50,000 times or more. The number of cells in the culture can be determined by any method known in the art, for example, by utilizing a Coulter counter. These methods are well known to those skilled in the art. In some embodiments, the cells selected in step b) (ie, Itm2a-positive cells) are stored at low temperature (eg, -80°C). In some embodiments, step c) comprises culturing the cells on a biocompatible scaffold, preferably a 3D scaffold. In some embodiments, the expanded cells are stored at low temperatures (eg, -80°C) for storage purposes. When needed, the frozen cells are thawed and then implanted into a subject in need. In some embodiments, before cryopreservation, the cell suspension is mixed with a cryoprotectant. Methods for cryopreserving tissues and cells at low temperatures with cryoprotectants are well known in the art. Frozen samples can be carried out in the presence of one or more different cryoprotectants to minimize cell damage during freeze-thaw. For example, dimethyl sulfoxide (DMSO), trehalose or sucrose can be used. In some embodiments, the expanded cells are administered locally at the site of tendon injury or defect. In some embodiments, the cells are administered in the form of a gel. The cells can be administered in combination with additional bioactive agents, including but not limited to pharmaceutically active compounds, hormones, growth factors, enzymes, DNA, RNA, siRNA, hyaluronic acid, antibodies, antibiotics, anti-inflammatory molecules, extracellular matrix proteins (such as tenosynovins, e.g., TNC, and periostin), or combinations thereof. In some embodiments, the bioactive agent comprises "pro-angiogenic factors" including, but not limited to, epidermal growth factor (EGF), E-cadherin, VEGF, angiogenic proteins, angiopoietin-1, fibroblast growth factor (FGF, including aFGF and bFGF), hepatocyte growth factor (HGF), angiogenin, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), and inflammatory cytokines and chemokines (which are inducers of angiogenesis and increased blood vessel formation), for example, interleukin-3 (IL-3), interleukin-8 (IL-8), CCL2 (MCP-1), interleukin-8 (IL-8), and CCL5 (RANTES). The present invention also provides a method of treating tendon injury or defect in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition of the present invention. The present invention also provides a method for preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification) in a subject in need thereof, comprising administering the pharmaceutical composition of the present invention to the subject in need thereof. In some embodiments, the pharmaceutical composition is administered locally at the site of tendon injury or defect. In some embodiments, the tendon stem cells are autologous or allogeneic to the subject in need thereof. The present invention also provides an enriched population of isolated Itm2a-positive tendon stem cells or a pharmaceutical composition comprising the population for use in treating tendon injury or defect in a subject in need thereof. The present invention also provides an enriched population of isolated Itm2a-positive tendon stem cells or a pharmaceutical composition comprising the population for preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification) in a subject in need thereof. The present invention also provides the use of an enriched isolated Itm2a-positive tendon stem cell population or a pharmaceutical composition comprising the population in the preparation of a medicament for treating tendon injury or defect in a subject in need thereof. The present invention also provides the use of an enriched isolated Itm2a-positive tendon stem cell population or a pharmaceutical composition comprising the population in the preparation of a medicament for preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification) in a subject in need thereof. The present invention also provides a method for screening drugs for treating tendon injury or defect, or preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification), the method comprising i) contacting the compound with Itm2a-positive tendon stem cells, for example, culturing the tendon stem cells in a culture medium containing the compound; ii) detecting the growth and / or differentiation of the tendon stem cells; iii) comparing the growth and / or differentiation of tendon stem cells to that of a control; and iv) selecting a compound that promotes the growth and / or differentiation of said tendon stem cells. The control tendon stem cells are Itm2a-positive tendon stem cells that have not been contacted with the compound. The compound can be a small molecule compound or a protein. In some embodiments, the compound promotes the differentiation of the tendon stem cells into tendon cells. In some embodiments, the detection in step ii) includes in vitro detection and / or in vivo detection. For example, the cells can be cultured in vitro and / or induced to differentiate, and their proliferation ability and / or ability to differentiate into tendon cells can be detected; the tendon stem cells can also be transplanted into animals to detect their proliferation ability and / or ability to differentiate into tendon cells. The present invention also provides a method for screening drugs for treating tendon injury or defect, or preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification), the method comprising i) contacting a compound with a population of tendon stem cells, such as culturing the population in a culture medium comprising the compound; ii) detecting the proportion of Itm2a-positive cells, proliferation capacity and / or ability to differentiate into tenocytes in the tendon stem cell population; iii) comparing the proportion of Itm2a-positive cells in a control tendon stem cell population; and iv) selecting a compound that increases the proportion of Itm2a-positive cells in the tendon stem cell population. The control tendon stem cell population is tendon stem cells that have not been exposed to the compound. The compound can be a small molecule compound or a protein. In some embodiments, the testing in step ii) includes in vitro testing and / or in vivo testing. For example, the cells can be cultured in vitro and / or induced to differentiate, and their proliferation capacity and / or ability to differentiate into tenocytes can be tested; or the tendon stem cells can be transplanted into an animal and their proliferation capacity and / or ability to differentiate into tenocytes can be tested. The present invention also provides a method for screening drugs for treating tendon injury or defect, or preventing or treating complications of tendon injury or defect (such as tendon adhesion and tendon heterotopic ossification), the method comprising i) administering the compound to a subject; ii) isolating a tendon stem cell population from the subject, and detecting the proportion of Itm2a-positive tendon stem cells, their proliferation capacity, and / or their ability to differentiate into tenocytes; iii) comparing the proportion of Itm2a-positive tendon stem cells, their proliferation capacity and / or their ability to differentiate into tenocytes in a tendon stem cell population isolated from a control subject; and iv) selecting a compound that increases the proportion of Itm2a-positive cells, the proliferation capacity and / or the ability to differentiate into tenocytes in a tendon stem cell population isolated from a subject. The control animal is a tendon stem cell that has not been administered the compound. The compound can be a small molecule or a protein. In some embodiments, the testing in step ii) includes in vitro testing and / or in vivo testing. For example, the cells can be cultured in vitro and / or induced to differentiate, and their proliferation capacity and / or ability to differentiate into tenocytes can be tested. Alternatively, the tendon stem cells can be transplanted into an animal and their proliferation capacity and / or ability to differentiate into tenocytes can be tested. Implementation Plan Embodiment 1, a pharmaceutical composition comprising: i) isolated tendon stem cells, wherein the tendon stem cells are Itm2a positive; and ii) a pharmaceutically acceptable carrier. Embodiment 2. The pharmaceutical composition of embodiment 1, wherein the tendon stem cells are human cells, and wherein the tendon stem cells are CD45-CD31-CD235a- cells. Embodiment 3, the pharmaceutical composition of embodiment 1 or 2, wherein the tendon stem cells have the ability to differentiate into tenocytes. Embodiment 4. The pharmaceutical composition of any one of embodiments 1-3, wherein the tendon stem cells are self-renewing and clonogenic. Embodiment 5: The pharmaceutical composition of any one of Embodiments 1-4, wherein the tendon stem cells are enriched from tendon or tendon sheath tissue. Embodiment 6. The pharmaceutical composition of embodiment 5, wherein the tendon stem cells are expanded ex vivo. Embodiment 7. The pharmaceutical composition of any one of embodiments 1-6, wherein the pharmaceutical composition is formulated for local administration, for example, local administration at the site of tendon injury. Embodiment 8. A method for preparing an isolated tendon stem cell population, wherein the tendon stem cells are Itm2a positive, the method comprising: a) providing a pool of cells from tendon or tendon sheath tissue; b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue; and c) optionally expanding the cells selected in step b) in culture. Embodiment 9. The method of embodiment 8, wherein step b) comprises selecting with a binding molecule against Itm2a. Embodiment 10. The method of embodiment 9, wherein the binding molecule is an anti-Itm2a antibody or an antigen-binding fragment thereof. Embodiment 11. The method of any one of embodiments 8-10, wherein step b) comprises selecting by flow cytometry. Embodiment 12. The method of any one of embodiments 8-11, wherein the tendon stem cells are human tendon stem cells, and wherein the method further comprises selecting from the pool of cells from tendon or tendon sheath tissue CD45-CD31-CD235a- cells. Embodiment 13. A method of treating a tendon injury or defect in a subject in need thereof, the method comprising a) providing a pool of cells from tendon or tendon sheath tissue; b) selecting Itm2a-positive cells from the cell pool derived from tendon or tendon sheath tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof. Embodiment 14. The method of embodiment 13, wherein the tendon or tendon sheath tissue is autologous tendon or tendon sheath tissue of the subject in need thereof. Embodiment 15. The method of embodiment 13, wherein the tendon or tendon sheath tissue is allogeneic tendon or tendon sheath tissue. Embodiment 16. The method of any one of embodiments 13-15, wherein step b) comprises selecting with a binding molecule against Itm2a. Embodiment 17. The method of embodiment 16, wherein the binding molecule is an anti-Itm2a antibody. Embodiment 18. The method of any one of embodiments 13-17, wherein step b) comprises selecting by flow cytometry. Embodiment 19. The method of any one of embodiments 13-18, wherein the tendon stem cells are human tendon stem cells, and wherein the method further comprises selecting from the pool of cells from tendon or tendon sheath tissue CD45-CD31-CD235a- cells. Embodiment 20. The method of any one of embodiments 13-19, wherein the expanded cells are administered locally at the site of the tendon injury or defect. Embodiment 21. A method of treating a tendon injury or defect in a subject in need thereof, comprising administering to the subject in need thereof the pharmaceutical composition of any one of embodiments 1-7. Embodiment 22. The method of embodiment 21, wherein the pharmaceutical composition is administered locally at the site of the tendon injury or defect. Embodiment 23, the method of embodiment 21 or 22, wherein the tendon stem cells are autologous or allogeneic to the subject in need thereof. Embodiment 24. The pharmaceutical composition of any one of embodiments 1-7, for use in treating tendon injury or defect in a subject in need thereof. Embodiment 25. Use of the pharmaceutical composition of any one of embodiments 1-7 or the isolated tendon stem cell population prepared by the method of any one of embodiments 8-12 in the preparation of a medicament for treating tendon injury or defect in a subject in need thereof. Beneficial effects of the present invention The present invention identifies Itm2a as a molecular marker for sorting and isolating bone or tendon stem cells. Itm2a-positive bone or tendon stem cells have excellent expansion (self-renewal) and differentiation capabilities. The molecular markers provided by the present invention allow for the transplantation of bone or tendon stem cells obtained from the patient's own body, significantly reducing the risk of rejection during cell / tissue transplantation. Therefore, the present invention provides suitable seed cells for repairing defects in the skeletal system, such as bone tissue and tendons. Example The following examples are provided for illustration only and are not intended to limit the present application in any way. Example 1. Materials and methods 1.1 Experimental Animals The mice used in the examples of this application were all of C57BL6 background. Itm2a-CreERT-IRES-mCherry (Itm2a-CreER) mice were constructed by Jicui Pharmaceutical Co., Ltd. R26-Ai6 mice, Bmp2 fl / fl Mice and R26-DTA mice were provided by Dr. Zeng Yi from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. Actin-GFP mice and Ki67-CreER mice were provided by Dr. Zhou Bin from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. R26-Ai9 mice were provided by Dr. Qiu Zilong from the Institute of Neurobiology, Chinese Academy of Sciences. R26-RSR-tdTomato mice were provided by Dr. Zhou Bo from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Scx-GFP transgenic mice were provided by Professor Ouyang Hongwei from Zhejiang University. PDGFRα-CreER mice were provided by Dr. Hu Ping from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Nude mice were purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. Prrx1-Cre mice (purchased from Jackson Lab) were crossed with Rosa26-Ai9 mice to breed Prrx1-Cre; Rosa26-Ai9 mice. Mouse cortical bone cells were digested and isolated at 4 weeks of age, and Ai9-positive cells were sorted for single-cell sequencing. Itm2a-CreER mice were crossed with R26-Ai6 mice to breed Itm2a-CreER;R26-Ai6 mice, which were induced with tamoxifen at different ages. The skeletal system of the mice was collected and flow cytometry analysis and frozen sections were performed to observe the labeling of Itm2a in the skeletal system. Four-week-old Itm2a-CreER; Rosa26-Ai6 mice were induced with tamoxifen. One week after drug withdrawal, the mice were subjected to fracture injury, and the involvement of Itm2a in endochondral ossification at different time periods after injury was collected. Four-week-old Itm2a-CreER; Rosa26-Ai6 mice were induced with tamoxifen. One week after drug withdrawal, Ai6-positive cells in the mouse cortical bone were sorted, wrapped with matrix gel and filled into the subrenal capsule of the mouse to observe the ability of Ai6-positive cells to form heteroosteomalasty under the renal capsule. Tamoxifen induction was performed by intraperitoneal injection of 2 mg of tamoxifen dissolved in corn oil, three times with one day intervals for a total of one week. 1.2 Antibodies The antibodies used in the examples of this application are shown in Tables 1 and 2. Table 1. Primary antibodies used in the examples Table 2. Secondary antibodies used in the examples 1.3. Periosteal Cell Digestion and Analysis C57BL6 mice aged 4-6 weeks were euthanized with CO . The articular cartilage between the pelvis and the femoral head was cut and the mouse hind limbs were isolated. The mouse foot was removed, the femur and tibia were retained, and the muscles were removed. The isolated bones were placed in PBS in a six-well plate, which was placed on ice. In a clean bench, autoclaved low-melting-point agarose (Corning, 356231) was placed in a 6 cm culture dish and wrapped around the two ends of the long bone when the agarose was semi-solid, exposing the diaphysis containing the periosteum. The agarose-encapsulated bones were placed in a 15 ml centrifuge tube and 5 ml of digestion solution (α-MEM (Corning, 10-022-CVR) containing 1 mg / ml collagenase (Sigma, C0130), 2 mg / ml dispase II (Sigma, D4693), and 2% penicillin / streptomycin) was added. Digestion was repeated twice in a shaker at 280 rpm for 1 h at 37°C. The supernatant was mixed on ice with 10 ml of pre-chilled C3H10 medium (α-MEM (Corning, 10378-016) containing 10% fetal bovine serum (FBS) (Ausbian, VS500T) and 1% penicillin / streptomycin (Gibco, 10378-016)) to terminate the digestion. The mixture was centrifuged at 1,200 g for 4 min at 4°C, and the supernatant was discarded. Add 5 ml of red blood cell lysis buffer (Beyotime, C3702) and mix well. Lyse at room temperature for 5-10 minutes. After the solution no longer turns red, add 10 ml of C3H10 medium to neutralize the reaction. Centrifuge again at 1,200 g for 5 minutes and discard the supernatant (the white precipitate indicates complete lysis of the red blood cells). Resuspend the precipitate in C3H10 medium and count the cells. Dilute the suspension to 1×10 7 cells / ml, 3 ml of cell solution was added to each well of a 6-well plate and cultured at 37°C. The cultured cell suspension was centrifuged at 4°C, 1,300g for 3 minutes and the supernatant was discarded. The pellet was resuspended in 2 ml of flow cytometry buffer (PBS containing 2% FBS), and the suspension was filtered through a 40 μm cell strainer, then centrifuged at 4°C, 1,300g for 3 minutes and the supernatant was discarded. The pellet was resuspended in 2 ml of red blood cell lysis buffer, the suspension was placed on ice for 25 minutes, and then mixed with 5 ml of PBS to terminate the lysis. The mixture was centrifuged at 4°C, 1,300g for 3 minutes and the supernatant was discarded. The pellet was resuspended in 1 ml of flow cytometry buffer, and the cells were counted, and the suspension was diluted to 0.1 to 1×10 7 cells / ml and aliquoted into 100 μl (each containing 0.1 to 1 × 10 6 cells). Add antibodies according to the cell number (record the cell number, antibody concentration and volume, 10 6Each cell was stained in a 100 μl volume (using an antibody concentration of 0.1 μg / mL) on ice for 30 minutes in the dark. Centrifuge at 1,300 g for 5 minutes at 4°C, discard the supernatant, and wash the cells with 1 ml of flow cytometry buffer. Centrifuge at 1,300 g for 5 minutes at 4°C, discard the supernatant, and resuspend the cells in 100 μl of flow cytometry buffer. If the primary antibody is biotinylated, add a streptavidin-conjugated secondary antibody and stain on ice for 20 minutes in the dark. Centrifuge at 700 g for 2 minutes at 4°C, discard the supernatant, and wash the cells with 1 ml of flow cytometry buffer. Centrifuge at 700 g for 2 minutes at 4°C, discard the supernatant, and resuspend the cells in 100 μl of flow cytometry buffer. Detect or sort the cells using a flow cytometer (Arial Sorp Cell Sorter, BD Biosciences). 1.4. Fixed Fracture Surgery in Mice Mice were anesthetized by intraperitoneal injection of chloral hydrate (400 mg / kg). The anesthetized mice were shaved and the skin was prepared on the lateral side of the right femur and disinfected with alcohol wipes. The skin at the femur was incised, and the soft tissue was bluntly dissected using forceps. The patella was moved to expose the femoral condyle, and an intramedullary nail was inserted from the femoral condyle into the femoral medullary cavity for fixation. The muscles in the middle of the femur were bluntly dissected using forceps to expose the femur. A dental drill (0.9 mm diameter) was used to transect or drill a hole in the middle of the femur (only one side was drilled). For transected mice, the intramedullary nail was retained to fix the femur. The muscles and femur were sutured separately. The mice were placed in a warm cage and, after awakening, transferred to an IVC cage for culture. Postoperatively, the mice were observed weekly for fracture repair using X-ray (Faxtrion, USA, model MX-20, exposure 6 seconds). 1.5. Skeletal stem cell transplantation under the renal capsule The cell suspension was centrifuged and the supernatant was discarded. The cells were resuspended in less than 10 μl of Matrigel (Corning, 356231) on ice (Matrigel melts at about 0°C and solidifies at -20°C or room temperature 23-25°C). The mice were weighed and anesthetized by injection of 2% chloral hydrate (20x + 50 μl for mice weighing x g). The anesthetized mice were shaved at the surgical site and transferred to a foam pad. The mice were fixed in lateral recumbency and the skin was disinfected with 70% ethanol and iodine. Using scissors and tweezers, a 1 cm incision was made in the skin parallel to the spine and the skin was separated from the abdominal wall by blunt dissection. The kidneys can be observed through the body wall adjacent to the spleen. The body wall was lifted with tweezers and an incision was made that was smaller than the kidney (5-8 mm). The kidney was gently protruded through the incision through the abdominal wall by massaging the surrounding abdominal wall. The kidneys were kept moist with warm sterile PBS. A shallow 1-2 mm incision was made on the surface of the renal capsule on the side of the kidney using the bevel of a 31-gauge sterile needle. Use a microsyringe to draw up the Matrigel-cell mixture and inject it into the renal capsule through the cannula over the indwelling needle. Once the mixture solidifies, remove the needle. Return the kidney to the peritoneal cavity by lifting the body wall. Add 1 ml of PBS to the peritoneal cavity to prevent postoperative dehydration. Suture the muscle layer with 4-0 absorbable sutures and the skin with 4-0 silk sutures. Clean and disinfect. Mice were kept at 37°C until awake, transferred to new cages, and inspected for wounds daily. After at least 4 weeks of maintenance, mice were sacrificed using CO2 and analyzed for grafts. 1.6 Immunofluorescence of frozen bone sections Select mouse frozen sections with equivalent section positions (sections with a thickness of 12-16 μm obtained by Leica CM3050S cryostat), place them in a sterile staining box (up to three sections per box, with the same orientation), wash them three times with PBS, each for 5 minutes, and then discard the PBS. Perform antigen retrieval according to the following protocol: Method 1: Place the sections in boiling antigen retrieval solution (0.01M sodium citrate buffer, pH 6.0) in a pressure cooker with the lid closed. Maintain high pressure for 10 minutes, turn off the power and keep warm for 10 minutes, then vent and allow to cool naturally. Option 2: Place the slices in antigen retrieval solution (0.01 M sodium citrate buffer, pH 6.0) preheated at 95°C and incubate for 20 minutes, then cool naturally (suitable for antibodies that are easy to make on bone slices). Solution 3: Proteinase K repair, 10 μg / ml proteinase K solution (50 mM Tris-HCl, 5 mM EDTA, pH 8.0), digestion at 37°C for 15 min (suitable for bone slices of older rats that are prone to detachment). After antigen retrieval, wash the sections three times with PBS for 10 minutes each. Remove excess liquid from the slides and block the sections with approximately 50 μl of blocking solution (10% horse serum (Gibco, 16050122) in PBS) for 45 minutes at room temperature. Dilute the primary antibody in antibody diluent (5% horse serum in PBS) (select the optimal dilution for each antibody through a series of dilutions; for example, anti-SOX9 should be diluted at 1:2000) and incubate with the sections (approximately 30-50 μl per section) overnight at 4°C. Wash the sections three times with PBS for 10 minutes each. Dilute the secondary antibody in antibody diluent (for example, goat anti-rabbit Cy3 at a 1:1000 dilution) and incubate with the sections at room temperature for 1 hour in the dark. Wash the sections twice with PBS for 10 minutes each, protected from light. Stain with DAPI (1 μg / ml diluted 1:1000 in PBS) for 10 minutes in the dark. Wash the sections twice with PBS for 10 minutes each, protected from light. Each section was mounted with approximately 50 μl of Mowiol mounting medium, allowed to dry, and then sealed with nail polish. The sections were imaged using a laser confocal / fluorescence microscope (FV3000 confocal microscope and BX51 fluorescence microscope, Olympus). 1.7. Bone Histological Staining (Safranin Fast Green Staining) Reagents: 0.05% fast green solution: 0.05 g fast green (Shanghai Jinsui, JS11367) dissolved in 100 ml ddH2O; 1% acetic acid differentiation solution: 1 ml glacial acetic acid dissolved in 99 ml ddH2O; and 0.5% Safranin O staining solution: 0.5 g Safranin O (Sigma, S2255) was dissolved in 100 ml ddH2O. Sections were dewaxed and hydrated, then stained in 0.05% Fast Green for 1.5 minutes. Next, they were treated with acetic acid for 10 seconds and then stained in 0.5% Safranin O for 1 minute. The sections were then differentiated in 95% ethanol for several seconds, rinsed in running water, cleared with xylene, and mounted with neutral gum. 1.8 Tendon Histological Analysis Paraffin sections: Achilles tendons were fixed in 4% precooled PFA at 4°C for 48 hours and decalcified in 10% EDTA (pH 7.5). After washing with PBS, the samples were dehydrated in 70%, 95%, and 100% ethanol, immersed in xylene, embedded in paraffin, and cut into 7 μm thick sections (Leica CM3050S). Sections were stained with hematoxylin and eosin (H&E) according to standard staining procedures

[0023] . Achilles tendons were fixed in 4% PFA at 4°C for 48 h. After complete decalcification in 10% EDTA, they were dehydrated in 30% sucrose overnight, embedded in OCT, and sectioned at 18 μm using a Leica CM3050 S cryostat. 1.9 Tendon Immunofluorescence The frozen sections were air-dried and rehydrated with PBS, and antigen retrieval was performed according to the following protocol: 10 μg / ml proteinase K solution (50 mM Tris-HCl, 5 mM EDTA, pH 8.0), digested at 37° C. for 15 min. The cells were blocked and permeabilized with PBS containing 10% horse serum (Gibco, 16050122) and 0.2% Triton X-100 for 1 hour at room temperature, and then washed three times with PBS for 10 minutes each time. The cells were then incubated with the following primary antibodies: goat anti-CD31 (R&D, AF3628, 1:200), rabbit anti-COL1 (Rockland, 41642, 1:200), and rabbit anti-Itm2a (abclonal, a10385, 1:200) (diluted in PBS containing 5% horse serum) at 4°C overnight. The cells were washed three times with PBS for 10 min each. Fluorescently labeled secondary antibodies (donkey anti-goat or donkey anti-rabbit, Thermofisher, catalog A-21447 or A32795, 1:1000, diluted in PBS containing 5% horse serum) were incubated for 1 hour at room temperature in the dark. Cell nuclei were stained with DAPI (Sigma, D9542). Slides were mounted using fluorescent mounting medium (Dako, S3023). Images were acquired using an Olympus FV3000 confocal microscope. 1.10 Tenocyte Isolation and Culture Primary tenocytes were isolated from the Achilles tendons of 8-week-old adult mice or humans. Specifically, the tendons were cut into small segments and digested with collagenase II (1 mg / ml, Sigma) and dispase II (2 mg / ml, Roche) at 37°C for 2 hours. The digested products were collected, and the red blood cells in the obtained tendon-derived cells were removed using RBC lysis buffer (Beyotime, C3702), followed by centrifugation at 1200 rpm for 5 minutes. The isolated cells are cultured in α-MEM (Corning) containing 10% FBS, for example, in a 24-well plate or culture dish. 1.11. Fluorescence assisted cell sorting (FACS): Achilles tendon cells were isolated from the Achilles tendons of Itm2a-DreER; ScxGFP; R26-RSR-tdTomato mice, incubated on ice for 30 minutes, washed with PBS, and then sorted using a Sony MA900 flow cytometer. For human tenocytes, Lin-antibody (BD, 1:200) and anti-Itm2a antibody (abclonal, a10385, 1:50) were incubated for 1 hour, then washed with PBS, and donkey anti-rabbit secondary antibody (1:1000) was added for incubation for 40 minutes. Then, the cells were centrifuged and washed with PBS, and sorted using a flow cytometer Sony MA900. 1.12. Colony formation and in vitro pluripotent differentiation assays: For colony formation experiments, Itm2a+ cells were isolated from the Achilles tendons of Itm2a-DreER;ScxGFP;R26-RSR-tdTomato mice and cultured for 7 days. The cells were then fixed with 4% PFA and stained with crystal violet

[0024] , and colonies were counted. For osteogenic differentiation, briefly, approximately 2x10 4 Cells were plated in 96-well plates and cultured in α-MEM medium containing 10% FBS supplemented with 1% penicillin / streptomycin (Thermo Fisher Scientific). After 12 hours, the medium was changed to osteogenic differentiation medium (α-MEM medium containing 10% FBS, supplemented with 50 μg / ml ascorbic acid (Sigma-Aldrich) and 1 mg / ml glycerophosphate (Sigma-Aldrich)). The medium was changed every two days. After 21 days of induction, osteogenic differentiation was confirmed by Alizarin Red S staining

[0024] . For adipogenic differentiation, the differentiation medium consisted of solution A and solution B. Solution A contained α-MEM with 10% FBS, 50 mM dexamethasone (Sigma-Aldrich), 100 nM rosiglitazone (Sigma-Aldrich), 500 nM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich), 10 mg / ml insulin (Sigma-Aldrich), and 1% penicillin / streptomycin (Sigma-Aldrich). Solution B contained α-MEM with 10% FBS, 10 mg / ml insulin (Sigma-Aldrich), and 1% penicillin / streptomycin (Sigma-Aldrich). Solution A and solution B were induced alternately, with each solution replaced daily. After 7 days, adipogenic differentiation ability was determined by staining with 0.5% Oil Red O (Sigma-Aldrich). For chondrogenic differentiation, cells were collected and resuspended in α-MEM containing 10% FBS and 1% penicillin / streptomycin. 5 A droplet of cells (15 μl) was carefully placed in the middle of each well of a 24-well plate. After the cells adhered for 2-4 hours at 37°C, 500 ml of chondrogenic medium (α-MEM medium containing 10% FBS, supplemented with 1% insulin-transferrin-selenium solution (ITS, Sigma-Aldrich), 10 ng / ml TGF-β3 (Peprotech), 100 nM dexamethasone (added Sigma-Aldrich), 40 μg / ml proline (Sigma-Aldrich), 50 μg / ml 1-ascorbic acid 2-phosphate (Sigma-Aldrich) and 1 mM sodium pyruvate (Thermo Fisher Scientific)) was added. The medium was changed every 2 days. On day 21, the differentiated cells were stained with Safranin O Fast Green (SOFG). Briefly, the reagents were prepared as follows: 0.05% Fast Green solution: 0.05 g Fast Green (Shanghai Jinsui, JS11367) dissolved in 100 ml ddH2O; 1% acetic acid differentiation solution: 1 ml glacial acetic acid dissolved in 99 ml ddH2O; and 0.5% Safranin O staining solution: 0.5 g Safranin O (Sigma, S2255) was dissolved in 100 ml ddH2O. Sections were dewaxed and hydrated, then stained in 0.05% Fast Green for 1.5 minutes. Next, they were treated with acetic acid for 10 seconds and then stained in 0.5% Safranin O for 1 minute. The sections were then differentiated in 95% ethanol for several seconds, rinsed in running water, cleared with xylene, and mounted with neutral gum. 1.13 Tendon Organoid Experiment The sorted Itm2a+ cells were cultured in α-MEM medium containing 10% FBS until they were confluent in a 6cm dish. They were then cultured for 6 days in the presence of connective tissue growth factor (CTGF) (25ng / ml), TGFβ3 (10ng / ml), and ascorbic acid (4.4g / ml) to stimulate ECM deposition. Subsequently, they were digested with 0.25% trypsin and then attached to tissue hooks to form three-dimensional organoids. 1.14 Single-cell RNA sequencing The BD Rhapsody Single-Cell Analysis System was used to collect transcriptome profiles from single cells of wild-type mouse and human tendons. Single-cell RNA sequencing was performed by NovelBio Biopharmaceutical Technologies, Inc. Single-cell capture was achieved by randomly distributing a single-cell suspension into >200,000 microwells using limiting dilution. Oligonucleotide-barcoded microbeads were added to saturation, allowing the beads to pair with the cells in the microwells. Cells were lysed in the microwells, allowing mRNA molecules to hybridize with the barcoded capture oligonucleotides on the beads. The beads were collected in a single tube for reverse transcription and ExoIdi-digestion. After cDNA synthesis, each cDNA molecule was labeled at the 5' end (i.e., the 3' end of the mRNA transcript) with a unique molecular identifier (UMI) and a cellular barcode, identifying its cell of origin. Whole-transcriptome libraries were prepared using the BD Rhapsody single-cell whole transcriptome amplification (WTA) workflow, which includes random primer and extension (RPE), RPE amplification PCR, and WTA index PCR. The libraries were quantified using a high-sensitivity DNA chip (Agilent) and a Qubit high-sensitivity DNA detector (ThermoFisher Scientific) on a Bioanalyzer 2200. Sequencing was performed using an Illumina sequencer (Illumina, CA, USA) with a 150-bp paired-end run. 1.15 EdU proliferation assay: After injury, EdU (0.25 mg / mouse) was injected intraperitoneally once daily until sample collection. EdU staining was performed using the Shanghai Ruibo Cell light EdU Apollo 643 In Vitro Kit according to the manufacturer's instructions, and images were acquired using an Olympus FV3000 confocal microscope. 1.16. Kidney capsule transplantation experiment: First, 8-10 week old nude mice (Lingchang Company) were anesthetized, and then the hair on the left side of the abdomen was shaved, and the surgical site was disinfected. The kidney was exposed through a 1 cm incision, and a 2 mm pocket was created on the renal capsule. 5 μl of Matrigel (Corning, 356231) containing approximately 10,000 cells was implanted under the capsule, and then the kidney was placed into the body cavity. After 8 weeks, the animals were euthanized in carbon dioxide. Then, the kidneys were fixed with 4% PFA for 5 hours, and tendon formation was detected by frozen section. Example 2: Single-cell sequencing of mouse Prrx1-Cre+ periosteum The purpose of this example was to identify the true SSC population among Prrx1 lineage periosteal cells. Periosteal cells were obtained from 4-week-old Prrx1-Cre; Rosa26-Ai9 male mice, and Ai9-positive periosteal cells were sorted for single-cell RNA sequencing (scRNA-seq). Specifically, as described in Example 1, after agarose coating, collagenase digestion, and red blood cell lysis, Ai9-positive cells were sorted by flow cytometry, and then scRNA-seq analysis was performed using the 10X Genomics platform (Figure 1A). In the R-studio software, code filtering was used to screen and remove cells with CD45 expression levels higher than 0, and 2169 CD45-negative single cell data were obtained from the sequencing results for subsequent analysis. The Prrx1-Cre lineage cells obtained by sequencing were comprehensively analyzed, and 13 cell subpopulations were presented using the t-SNE dimensionality reduction display method (Figure 1B). These included periosteal stem / progenitor cells (cell subpopulations 0, 1, 2, 3, 7, and 10 expressing Col3a1 and Itgbl1, Figure 1C). In addition to periosteal stem / progenitor cells, osteoblasts (cell subpopulations 4 and 12, expressing Sp7 and Bglap), muscle cells (cell subpopulations 9 and 11, expressing Pax7 and Myob5), and endothelial cells (cell subpopulations 3, 6, and 8, expressing Emcn and Pecam1) were also identified (Figure 1D-F). Next, we analyzed the heterogeneity of Prrx1-Cre lineage cells and found that there were three cell states, and pseudo-time analysis of these three states showed a trajectory from state 2 (cell subpopulations 0, 1, 2, 3, 7, and 10) to state 1 and state 3 (Figure 1G and H). t-SNE plots showed the distribution of SSC markers (CD105 and CD200) and other classic skeletal precursor cell markers in Prrx1-Cre lineage cells (Figure 2A and B). Based on the consistency with SSC surface molecular markers, cell subpopulation 1 was defined as the periosteal skeletal stem cell population (periosteal skeletal stem cells, P-SSC) (Figure 2C). Periosteal cells were stained using anti-surface molecule antibodies, and the proportion of SSCs in the mouse periosteum was analyzed by flow cytometry. It was found that Itm2a could greatly enrich periosteal SSCs (SSCs accounted for 34.24% ± 1.3% in Itm2a-positive cells and 3.56 ± 0.44% in Itm2a-negative cells) (Figure 2D and E). Example 3: Membrane protein ITM2A is a good SSC sorting marker The purpose of this example is to identify the membrane protein characteristics of ITM2A and the effect of SSC sorting. 3.1. Using the pHAGE-GFP vector (Addgene, #106281) as the backbone, expression vectors encoding fusion proteins of mouse Itm2a (mItm2a, encoded by SEQ ID NO: 1) and human ITM2A (hITM2A, encoded by SEQ ID NO: 2) fused to EGFP (encoded by SEQ ID NO: 3) were constructed. The constructed vectors were transfected (see Suo et al., VGLL4 promotes osteoblast differentiation by antagonizing TEADs-inhibited Runx2 transcription, Sci Adv. 2020 Oct 23; 6(43): eaba4147) into 293 cells. Immunofluorescence analysis of the transfected cells revealed that the ITM2A protein could colocalize well with the cell membrane marker Dil (Figure 3A). 3.2, harvest periosteal cells from 4-week-old wild-type mice. Lin-Itm2a- and Lin-Itm2a+ cells were sorted and subjected to a clone forming capacity (CFU-F) experiment (see Han et al., Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation. J Clin Invest. 2019 May 1; 129(5): 1895-1909). Higher CFU-F capacity was observed in the Lin-Itm2a+ group (Figure 3B and C). Both Lin-Itm2a- and Lin-Itm2a+ cells showed in vitro clonal multipotency of chondrocytes, adipocytes, and osteoblasts (Figure 3D). In view of the finding in Example 2 that Itm2a can greatly enrich periosteum SSCs, the stem cell properties of Lin-6C3-CD90.2-CD105-CD200+Itm2a+ (hereinafter referred to as Itm2a-positive SSCs) and Lin-6C3-CD90.2-CD105-CD200+Itm2a- (hereinafter referred to as Itm2a-negative SSCs) were further compared. Specifically, the stem cell properties of Itm2a-positive SSCs were verified by transplanting cells into bone drill injury sites and under the renal capsule (Figure 4A). Actin-GFP mice (expressing GFP under the actin promoter) were used as the source of cells transplanted into bone drill and renal capsule transplant models, which can well determine the proliferation and differentiation of transplanted cells and distinguish them from the recipient mouse's own cells. GFP-labeled Itm2a-negative SSCs and Itm2a-positive SSCs were sorted, and then these cells were mixed with matrix gel. The matrix gel-encapsulated cells were injected into the cortical drill site of the femoral shaft (Figure 4A). μ-CT analysis (SkyScan1272, Bruker) of the femurs of mice 7 days after transplantation showed that the wound healing progressed better in mice injected with Itm2a-positive SSCs (Figures 4B and C). At the same time, in frozen sections of femurs 7 days after transplantation, immunofluorescence staining using an anti-osteopontin (OPN, an osteoblast marker) antibody showed a higher degree of colocalization of GFP and OPN in mice injected with Itm2a-positive SSCs, indicating that Itm2a-positive SSCs exhibited stronger osteogenic potential in vitro (Figure 4D). We also transplanted single-cell clones derived from GFP-positive cells beneath the renal capsule of 7- to 8-week-old recipient mice. As assessed by μ-CT and OPN immunofluorescence staining, Itm2a-positive SSCs exhibited significantly higher osteoid-forming ability than Itm2a-negative SSCs (Figures 4E to G). Example 4: Skeletal Stem Cell Lineage Evolution Ability of Itm2a-Positive Periosteal Skeletal Stem Cells The purpose of this example is to verify whether Itm2a-positive periosteal skeletal stem cells have the ability to evolve into the entire skeletal stem cell lineage. Flow cytometry analysis was performed on bone organoids formed under the renal capsule. The results showed that Itm2a-positive SSCs were able to reconstruct their entire skeletal stem cell lineage in vitro and maintain the ability to self-renew after transplantation, indicating their stemness characteristics (Figures 5A and B). In summary, Itm2a-positive SSCs are at the top of the periosteum skeletal stem cell lineage evolution tree and have the ability to self-renew and differentiate into downstream skeletal stem cell lineages. In comparison, Itm2a-negative SSCs do not have the ability to differentiate into Itm2a-positive cells and have a weaker ability to evolve into downstream cell groups. Example 5: ITM2A-positive stem cells in human periosteum samples The purpose of this example is to identify the ITM2A-positive stem cell population in human periosteum samples. Human periosteum samples were obtained clinically (Figure 6A). Through immunofluorescence staining of histological sections of periosteum samples, it was found that, like mice, there were ITM2A-positive periosteal cell populations in human periosteum samples (Figure 6B). By enzymatic digestion of periosteum samples, single-cell suspensions of human periosteum samples were obtained, and the suspensions were stained with antibodies for human skeletal stem cell markers (CD45-CD235-CD146-PDPN+CD73+CD164+) and ITM2A antibodies and analyzed by flow cytometry. The results showed that among the skeletal stem cells in human periosteum samples, approximately 60.3% of the cells were ITM2A-positive, that is, ITM2A was able to well enrich the skeletal stem cell population in human periosteum samples (Figure 6C). Example 6: Function of ITM2A cells in human periosteum The purpose of this example is to identify the function of ITM2A cells in human periosteum. ITM2a-positive cells isolated and sorted from human periosteum samples were cultured. In a trilineage differentiation assay (see Han et al., 2019, supra), ITM2a-positive cells demonstrated stronger osteoblast differentiation and weaker chondrogenic differentiation (Figure 7A). This supports observations in mice, demonstrating that Itm2a-positive cells play an important role in the fracture injury process, particularly in osteoblast ossification and repair of bone fragments. The colony-forming capacity (CFU-F) assay was performed on ITM2A-negative and ITM2A-positive cells, and a higher CFU-F was observed in the ITM2A-positive cell group (Figure 7B and C). Furthermore, ITM2A-positive cells also exhibited stronger bone organoid formation ability in the subrenal capsule transplantation experiment (Figure 7D and E). Example 7: Distribution and Function of Itm2a-Positive P-SSCs in Vivo The purpose of this example is to study the distribution and function of Itm2a-labeled P-SSCs in vivo. To this end, the CreERT-IRES-mCherry element (SEQ ID NOs: 4, 5, and 6) was inserted after the promoter of the Itm2a gene using CRISPR technology to construct Itm2a-CreERT-IRES-mCherry mice (hereinafter referred to as Itm2a-CreER mice) (Figure 8A). This mouse can indicate the real-time expression of the Itm2a gene through the expression of the mCherry red fluorescent protein, and can track Itm2a-positive cells and their progeny cells in vivo after specific induction time points through tamoxifen induction. The cell fate transition of Itm2a-positive cells in vivo can be tracked by co-localization of immunofluorescence staining with specific antibodies. To perform lineage tracing experiments using Itm2a-CreER mice, Itm2a-CreER mice were crossed with R26-LSL-ZsGreen mice (also known as R26-Ai6, i.e., Ai6 fluorescent reporter mice that express ZsGreen green fluorescent protein after Cre enzyme-mediated recombination) to obtain Itm2a-CreER;R26-Ai6 mice. Immunofluorescence analysis of frozen sections of 4-week-old Itm2a-CreER; R26-Ai6 mice 2 days after tamoxifen injection showed that Itm2a-CreER can specifically mark periosteal cells, but rarely mark bone marrow cells (Figure 8B). This is a great advantage compared to the previously identified stem cell markers of skeletal cells. At the same time, through co-localization analysis of osteoblasts and stem cell markers after antibody staining, Itm2a-CreER-labeled periosteal cells highly expressed CD200, but did not express the osteoblast marker OPN (Figure 8C and D). Next, 2 days after tamoxifen injection in 4-week-old Itm2a-CreER;R26-Ai6 mice, flow cytometry was used to examine the proportion of SSC lineage in periosteal cells. The data showed that Itm2a-CreER-positive cells were enriched for a higher proportion of skeletal stem cells (24.6% ± 1.1% vs. 3.88 ± 0.76%) (Figure 8E). Example 8: Effect of Itm2a-positive cells on fracture repair The purpose of this example is to further study the in vivo regenerative ability of Itm2a-positive SSCs and verify that Itm2a-positive SSCs can participate in the repair of fracture injuries. 8.1. Itm2a-positive SSCs can participate in fracture repair Fracture injury models were established in Itm2a-CreER; R26-Ai6 mice to determine the involvement of Itm2a-positive SSCs in the repair process. Specifically, 4-week-old Itm2a-CreER; R26-Ai6 mice were first induced with tamoxifen to fully label Itm2a lineage cells. One week after the last tamoxifen injection, fracture injury models were established, and samples were collected 3, 7, and 14 days after injury for section analysis (Figure 9A). 3 days after fracture injury, a significant increase in ZsGreen-positive cells in the periosteum was observed, and Itm2a-positive cells were clearly involved in the periosteal reaction (Figure 9B). Based on staining of chondrocyte, osteoblast, and bone marrow stromal cell markers and confocal microscopy imaging of callus samples at different time points after fracture, it was found that Itm2a lineage cells (labeled by green fluorescence) were able to form approximately 37.4% COL2A1-positive chondrocytes, approximately 56.6% OPN-positive osteoblasts, and approximately 12.6% LepR-positive bone marrow stromal cells within the fracture callus (Figures 9C to F). These data indicate that Itm2a lineage cells have the ability to remodel bone after fracture. 8.2. Bmp2 knockout in Itm2a lineage cells leads to impaired fracture repair Lin-Itm2a- and Lin-Itm2a+ cells in the periosteum of mice were sorted by flow cytometry and sequenced and analyzed (R-studio) by bulk-RNAseq (10X Genomics). The results showed that the upregulated genes in Lin-Itm2a+ cells were enriched with genes related to tissue damage repair and the BMP signaling pathway. The ossification and angiogenesis signaling pathways that are crucial for fracture healing were also enriched in Lin-Itm2a+ cells (Figure 10A). BMP2 is one of the key components of the BMP signaling cascade in fracture callus tissue. Bmp2 is essential for bone development and fracture healing. In the heat map analysis of genes in the BMP signaling pathway, we found that Bmp2 also had a consistent enrichment trend (Figure 10B). Previous studies have tested whether Bmp2 knockout in skeletal cells will lead to fracture damage repair disorders, but no studies have yet studied whether Bmp2 plays an important role in skeletal stem cells in the periosteum. To test this, 4-week-old Itm2a-CreER; Bmp2 fl / fl Mice (provided Itm2a-CreER mice and Bmp2 fl / fl Bmp2f l / flMice were induced with tamoxifen, and fracture models were established one week after the last injection. Samples were collected 7, 14, and 21 days after injury to observe callus formation during fracture recovery and evaluate the repair of fracture injuries (Figure 10C). X-ray examination (once a week for three weeks) showed that, as expected, Itm2a-CreER; Bmp2 fl / fl Mice showed impairment of hard callus formation during fracture healing (Figure 10D and E). In the results of μ-CT analysis and histological staining (safranin fast green staining) of sections on day 14 after fracture, good hard callus formation was observed in control mice, while Itm2a-CreER; Bmp2 fl / fl More tissue in the callus of mice showed μ-CT-negative cartilage-like tissue ( Figure 10F and G ), indicating that the loss of Bmp2 function in Itm2a lineage cells would lead to abnormalities in the entire fracture injury repair process. 8.3 Elimination of Itm2a-lineage cells leads to impaired fracture repair Itm2a-CreER mice were crossed with R26-DTA mice to obtain mice that were able to clear Itm2a-positive cells from the periosteum after tamoxifen induction (Figure 11B). Tamoxifen was induced in 4-week-old mice, and femoral fracture surgery was performed one week after the last injection (Figure 11A). As assessed by μ-CT and histological staining (safranin fast green staining), Itm2a-CreER; R26-DTA mice exhibited a nonunion phenotype, commonly known as a nonunion, with a decrease in Itm2a+ cells and an increase in the nonunion rate 28 days after fracture (28 dpf) (Figures 11C and D). Taken together, these data suggest that Itm2a-positive periosteal cells play an important role in the repair of fracture injuries, especially in hard callus formation and bone healing, and that this process is associated with the BMP signaling pathway. Example 9: Itm2a-positive skeletal stem cells combined with TNC can promote bone repair The purpose of this example is to study the effect of extracellular matrix proteins (such as TNC) on the osteogenic function of Itm2a-positive skeletal stem cells. To this end, we established a bone defect model in adult nude mice. The injury site was treated with Matrigel containing TNC (1.5 μg / ml) and / or Itm2a-positive skeletal stem cells (20,000 / 5 μl). Samples were then taken seven days after injury ( FIG12A ). Mice treated with Matrigel alone served as controls. The Matrigel treatment method was similar to that described in Example 1.5, except that the injection was performed at the bone injury site rather than the renal capsule. μ-CT showed that treatment with TNC protein or Itm2a-positive SSCs significantly promoted bone repair in mice compared to controls, while mice treated with both TNC protein and Itm2a-positive SSCs exhibited significantly better bone repair than mice treated with either TNC protein or Itm2a-positive SSCs alone (Figure 12B and C). These results indicate that both TNC protein and Itm2a-positive SSCs can promote bone repair, and that TNC can enhance the bone repair-promoting effect of Itm2a-positive SSCs. Example 10: Single-cell RNA sequencing analysis of tenosynovial cells The purpose of this example is to explore the main cell source for tendon repair. To this end, we performed tendon injury (partial transection) on adult wild-type mice (8 weeks old), collected tendons from uninjured, 7 days after injury, and 21 days after injury, digested them to obtain single-cell suspensions for single-cell RNA sequencing. After batch correction, cell quality control, and data dimensionality reduction and clustering (Seurat software), the obtained single cells were clustered using the uniform manifold approximation and projection (UMAP) method and mainly divided into 10 cell populations. We detected tenocytes expressing fibromodulin (Fmod) and tenamudin (Tnmd). We also found tendon sheath cells expressing Ace and PDGFRα, myogenic precursors expressing Pax7 and Myf5, endothelial cells expressing Emcn and Pecam1, proliferating cells expressing Mki67 and Top2a, pericytes expressing Acta2 and Tagln, T / B cells expressing CD19, CD2, and CD3e, fast skeletal muscle expressing Acta1 and Mylpf, neutrophils expressing Ptprc and S100a9, and monocytes / macrophages expressing CCR2 and CD74 ( Figure 13 , AB). Analysis of cellular composition before and after injury revealed an inflammatory response in the tendon after injury, with a significant increase in the proportion of macrophages and neutrophils (Figure 13, CD). Pseudo-time series analysis (monocle2 software) showed that tenosynovocytes were located anterior to tenocytes (Figure 13, E). RNA velocity analysis (scvelo software) revealed a similar tendon differentiation trajectory, with tenosynovocytes differentiating into tendon progenitors 7 days after injury and into normal tenocytes 21 days after injury (Figure 13, F). To identify membrane protein markers of tenosynovial cells, we analyzed the expression of membrane protein markers of tenosynovial cells by single-cell RNA sequencing, among which Itm2a and PDGFRα had the highest expression levels ( Figure 13 , G). We then constructed PDGFRα-CreER; R26-Ai9; ScxGFP transgenic mice and induced them with tamoxifen at 8 weeks of age so that cells expressing PDGFRα could be detected by tdTomato signaling. ScxGFP-positive cells represent tendon cells. We performed lineage tracing experiments on PDGFRα and found that PDGFRα-CreER marked tendon and tenosynovial cells (Figure 13, H). Further analysis revealed that the membrane protein Itm2a is expressed in the tenosynovial cell population during the uninjured stage, but not in the tenosynovial population. It begins to be expressed in tenocytes 7 days after injury (Figure 13, I). Immunofluorescence experiments further confirmed that Itm2a protein is specifically distributed in the tendon sheath (Figure 13, J). The above data suggest that Itm2a can be used as a specific marker for tenosynovial cells, and Itm2a-positive cells are a group of stem / progenitor cells. Example 11: Itm2a+ tendon sheath stem / progenitor cells participate in tendon repair The purpose of this example is to study the distribution and function of Itm2a+ tendon sheath stem / progenitor cells in vivo. To this end, we constructed Itm2a-CreERT-IRES-mCherry mice (Itm2a-CreER mice) ( Figure 14 , A). Laser confocal microscopy imaging showed that, as expected, Itm2a-mCherry was specifically expressed in the tendon sheath and partially localized around CD31+ blood vessels ( Figure 14 , B). To investigate its in vivo function using lineage tracing experiments, we crossed Itm2a-CreER mice with R26-LSL-Ai6 reporter mice (expressing ZsGreen after Cre-mediated recombination) to generate Itm2a-CreER;R26-LSL-Ai6 mice. To examine whether adult Itm2a lineage cells participate in tendon development and homeostasis, we traced Itm2a-CreER;R26-LSL-Ai6 mice at day 1 after birth and analyzed tendons collected 2 days, 2 months, and 6 months later. We found that Itm2a lineage cells did not give rise to tenocytes at any time point (Figure 14, C). To investigate whether Itm2a+ tenosynovial cells contribute to the generation of adult ScxGFP+ tenocytes, we generated Itm2a-DreER mice and derived Itm2a-DreER;ScxGFP;R26-RSR-tdTomato mice (Figure 14, D). We performed a repairable tendon "partial transection" model in adult Itm2a-DreER;ScxGFP;R26-RSR-tdTomato mice and collected samples 4, 7, 14, and 28 days after injury for confocal microscopy analysis (Olympus FV3000 confocal microscope). The results showed that under stable conditions, Itm2a-DreER+ cells did not participate in the formation of ScxGFP+ tenocytes. Interestingly, Itm2a+ cells began to proliferate 4 days after injury. Itm2a+ cells migrated to the injury site and formed ScxGFP+ tenocytes 7 days after injury. The number of Itm2a+ScxGFP+ tenocytes increased 14 and 28 days after injury (Figure 14, E). Second harmonic generation (SHG) can characterize the abundance of collagen. Itm2a-derived ScxGFP+ tenocytes showed SHG, indicating that they are involved in the formation of tendon collagen (Figure 14, F). These data suggest that Itm2a+ tenosynovial cells are not involved in tendon maintenance under steady-state conditions but participate in tendon repair under injury conditions. Example 12: Self-renewal and multipotency of Itm2a+ tendon sheath stem / progenitor cells The purpose of this example is to verify the properties of Itm2a+ tendon sheath stem / progenitor cells. To this end, we sorted Itm2a+ cells (approximately 1%) from adult Itm2a-DreER; ScxGFP; R26-RSR-tdTomato mice ( FIG. 15 , A). In vitro culture showed that Itm2a+ cells had colony-forming capacity (CFU-F) (Figure 15, BC). Furthermore, in vitro differentiation assays revealed that Itm2a+ cells possessed multipotential differentiation potential, including osteogenic, adipogenic, and chondrogenic differentiation potential (Figure 15, D). To test the tenogenic capacity of Itm2a+ cells, we utilized organoid technology and found that Itm2a+ cells could form tendon organoids in vitro (Figure 15, E). Furthermore, we transplanted Itm2a+ cells into the renal capsule of immunodeficient (nude) mice and found that they could generate ScxGFP+ tenocytes (Figure 15, F). Importantly, we sorted and transplanted Itm2a+ cells into injured mouse tendons and found that almost all of them differentiated into ScxGFP+ tenocytes and promoted repair (Figure 15, G). Taken together, these data indicate that Itm2a marks tendon sheath stem / progenitor cells and that Itm2a+ cells possess self-renewal capacity and are able to repair tendon injury. Example 13: Elimination of Itm2a+ tendon sheath stem / progenitor cells to inhibit tendon repair The purpose of this example is to verify the importance of Itm2a+ tendon sheath stem / progenitor cells for tendon injury repair. To investigate the effect of Itm2a+ cell depletion on tendon repair, we mated Itm2a-CreER mice with R26-DTA mice to generate Itm2a-CreER;R26-DTA mice, which can be depleted of Itm2a+ cells under tamoxifen induction (Figure 16, AC). The results showed that depletion of Itm2a+ cells inhibited tendon repair and collagen formation ( FIG. 16 , DE). Example 14: ITM2A+ tendon sheath stem / progenitor cells in human tendons The purpose of this example is to investigate whether human tendon contains Itm2a+ tendon stem cells. To this end, we obtained human Achilles tendon samples and digested them to obtain single-cell suspensions for single-cell RNA sequencing. After batch correction, cell quality control, and data dimensionality reduction and clustering, the obtained single cells were clustered based on the uniform manifold approximation and projection (UMAP) method and mainly divided into 9 cell populations. Specifically, we detected tenocytes expressing FMOD and COMP; we also found tendon sheath cells expressing CXCL14 and APOD, myogenic precursors expressing PAX7 and CADM2, endothelial cells expressing POSTN and PECAM1, pericytes expressing ACTA2 and MYH11, T / B cells expressing CXCR4 and IL7R, fast skeletal muscle expressing MYH1 and MYH2, mast cells expressing TPSB2 and CPA3, and monocytes / macrophages expressing CXCL8 and IL1B ( Figure 17 , AB). Consistent with the mouse single-cell sequencing results, ITM2A was also expressed exclusively in tenosynovial cells (Figure 17, C). Pseudo-time series analysis showed that human tenosynovial cells were located anterior to tenocytes (Figure 17, D). Immunofluorescence experiments confirmed the specific distribution of human ITM2A protein in the tendon sheath (Figure 17, E). To investigate the stem / progenitor cell properties of human ITM2A+ cells, Lin-ITM2A+ tenosynovial cells were isolated by flow cytometry and cultured in vitro, demonstrating that Lin-ITM2A+ cells possessed a stronger colony-forming capacity (CFU-F) than Lin-ITM2A- cells ( Figure 17 , FG). In vitro differentiation assays also demonstrated that Itm2a+ cells possessed multipotential differentiation potential, including osteogenic, adipogenic, and chondrogenic differentiation potential ( Figure 17 , H). To test the tenogenic ability of Lin-ITM2A+ cells, we performed analysis using organoid technology and used Lin-ITM2A- cells as a control. Briefly, sorted Lin-ITM2A+ cells were cultured to confluence on 6 cm dishes and then cultured for 6 days in a medium (α-MEM containing 10% FBS) supplemented with connective tissue growth factor (CTGF) (25 ng / ml), TGFβ3 (10 ng / ml), and ascorbic acid (4.4 g / ml) to stimulate extracellular matrix (ECM) deposition. Subsequently, they were detached with 0.25% trypsin and then attached to the scaffold to form three-dimensional organoids. The results showed that Lin-ITM2A+ cells had a stronger ability to form tendon organoids in vitro than Lin-ITM2A- cells ( Figure 5 , I). The above results prove that ITM2A can mark tendon sheath stem / progenitor cells in human tendons, and human ITM2A+ tendon sheath cells have the ability to self-renew and can repair tendon injuries. References 1.Wang,HN,YCHuang,and GXNi,Mechanotransduction of stem cells for tendon repair.World J Stem Cells,2020.12(9):p.952-965. 2.Millar,NL,GAMurrell,and IBMcInnes,Inflammatory mechanisms in tendinopathy-towards translation.Nat Rev Rheumatol,2017.13(2):p.110-122. 3.Chen, Y., et al., Targeted pathological collagen delivery of sustained-release rapamycin to prevent heterotopic ossification. Sci Adv, 2020.6(18):p.eaay9526. 4. Nourissat, G., F. Berenbaum, and D. Duprez, Tendon injury: from biology to tendon repair. Nat Rev Rheumatol, 2015.11(4):p.223-33. 5. Huang, ZZ, et al., Tendon Stem / Progenitor Cell Subpopulations and Their Implications in Tendon Biology. Frontiers in Cell and Developmental Biology, 2021.9. 6.Schneider,M.,et al.,Rescue plan for Achilles:Therapeutics steering the fate and functions of stem cells in tendon wound healing.Adv Drug Deliv Rev,2018.129:p.352-375. 7.Wei,B.and J.Lu,Characterization of Tendon-Derived Stem Cells and Rescue Tendon Injury.Stem Cell Rev Rep,2021. 8.Tan,Q.,et al.,Comparison of potentials of stem cells isolated from tendon and bone marrow for musculoskeletal tissue engineering.Tissue Eng Part A,2012.18(7-8):p.840-51. 9.Zhang,Y.,et al.,3D printing of chemical-empowered tendon stem / progenitor cells for functional tissue repair.Biomaterials,2021.271:p.120722. 10.Bi,Y.M.,et al.,Identification of tendon stem / progenitor cells and the role of the extracellular matrix in their niche.Nature Medicine,2007.13(10):p.1219-1227. 11.Agarwal,S.,et al.,Analysis of Bone-Cartilage-Stromal Progenitor Populations in Trauma Induced and Genetic Models of Heterotopic Ossification.Stem Cells,2016.34(6):p.1692-701. 12.Asai,S.,et al.,Tendon progenitor cells in injured tendons have strong chondrogenic potential:the CD105-negative subpopulation induces chondrogenic degeneration.Stem Cells,2014.32(12):p.3266-77. 13.Feng,H.,et al.,Tendon-derived cathepsin K-expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification.J Clin Invest,2020.130(12):p.6354-6365. 14.Lee,C.H.,et al.,Harnessing endogenous stem / progenitor cells for tendon regeneration.J Clin Invest,2015.125(7):p.2690-701. 15.Yin,Z.,et al.,Single-cell analysis reveals a nestin(+)tendon stem / progenitor cell population with strong tenogenic potentiality.Sci Adv,2016.2(11):p.e1600874. 16.Dyment,N.A.,et al.,Lineage tracing of resident tendon progenitor cells during growth and natural healing.PLoS One,2014.9(4):p.e96113. 17.Harvey,T.,S.Flamenco,and C.M.Fan,A Tppp3(+)Pdgfra(+)tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis.Nat Cell Biol,2019.21(12):p.1490-1503. 18.Howell,K.,et al.,Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing.Sci Rep,2017.7:p.45238. 19.Namkoong,S.,et al.,The integral membrane protein ITM2A,a transcriptional target of PKA-CREB,regulates autophagic flux via interaction with the vacuolar ATPase.Autophagy,2015.11(5):p.756-68. 20.Van den Plas,D.and J.Merregaert,In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway.Biol Cell,2004.96(6):p.463-70. 21.Lagha,M.,et al.,Itm2a Is a Pax3 Target Gene,Expressed at Sites of Skeletal Muscle Formation In Vivo.Plos One,2013.8(5). 22.Tuckermann,J.P.,et al.,Collagenase-3(MMP-13)and integral membrane protein 2a(Itm2a)are marker genes of chondrogenic / osteoblastic cells in bone formation:Sequential temporal,and spatial expression of Itm2a,alkaline phosphatase,MMP-13,and osteocalcin in the mouse.Journal of Bone and Mineral Research,2000.15(7):p.1257-1265. 23.Yujiao Han,et al.,Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation.J Clin Invest.2019;129(5):1895-1909. 24.Heng Feng,et al.,Tendon-derived cathepsin K-expressing progenitor cells activate Hedgehog signaling to drive heterotopic ossification.J Clin Invest.2020 Dec 1;130(12):6354–6365.

Claims

1. A pharmaceutical composition comprising: i) isolated skeletal stem cells, wherein the skeletal stem cells are Itm2a positive; and ii) a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the skeletal stem cells are human skeletal stem cells, and wherein the skeletal stem cells are CD45-CD31-TER119-CD235-CD146-PDPN+CD73+CD164+ cells.

3. The pharmaceutical composition of claim 1 or 2, wherein the skeletal stem cells have the ability to differentiate into osteoblasts.

4. The pharmaceutical composition of any one of claims 1-3, wherein the skeletal stem cells are self-renewing and clonogenic.

5. The pharmaceutical composition of any one of claims 1-4, wherein the skeletal stem cells are enriched from a bone tissue sample.

6. The pharmaceutical composition of claim 5, wherein the skeletal stem cells are expanded ex vivo.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the pharmaceutical composition is formulated for local administration, such as local administration at a bone fracture site.

8. A method for preparing an isolated skeletal stem cell population, wherein the skeletal stem cells are Itm2a positive, the method comprising: a) providing a cell pool from bone tissue; b) selecting Itm2a-positive cells from the cell pool derived from bone tissue; as well as c) optionally expanding the cells selected in step b) in culture.

9. The method of claim 8, wherein step b) comprises selecting with a binding molecule directed against Itm2a.

10. The method of claim 9, wherein the binding molecule is an anti-Itm2a antibody or an antigen-binding fragment thereof.

11. The method of any one of claims 8 to 10, wherein step b) comprises selection by flow cytometry.

12. The method of any one of claims 8-11, wherein the skeletal stem cells are human skeletal stem cells, and wherein the method further comprises selecting CD45-CD31-TER119-CD235-CD146-PDPN+CD73+CD164+ cells from the cell pool from bone tissue.

13. A method of treating a bone injury or defect in a subject in need thereof, the method comprising a) providing a cell pool from bone tissue; b) selecting Itm2a-positive cells from the cell pool derived from bone tissue; c) expanding the cells selected in step b) in culture; and d) administering the cells expanded in step c) to the subject in need thereof.

14. The method of claim 13, wherein the bone tissue is autologous bone tissue of the subject in need thereof.

15. The method of claim 13, wherein the bone tissue is allogeneic bone tissue.

16. The method of any one of claims 13 to 15, wherein step b) comprises selecting with a binding molecule directed against Itm2a.

17. The method of claim 16, wherein the binding molecule is an anti-Itm2a antibody.

18. The method of any one of claims 13 to 17, wherein step b) comprises selecting by flow cytometry.

19. The method of any one of claims 13-18, wherein the skeletal stem cells are human skeletal stem cells, and wherein the method further comprises selecting CD45-CD31-TER119-CD235-CD146-PDPN+CD73+CD164+ cells from the cell pool from bone tissue.

20. The method of any one of claims 13-19, wherein the expanded cells are administered locally at the site of the bone injury or defect.

21. The method of any one of claims 13-20, wherein the bone injury or defect is a bone fracture.

22. A method of treating a bone injury or defect in a subject in need thereof, comprising administering to the subject in need thereof the pharmaceutical composition of any one of claims 1-6.

23. The method of claim 22, wherein the pharmaceutical composition is administered locally at the site of the bone injury or defect.

24. The method of claim 22 or 23, wherein the bone injury or defect is a bone fracture.

25. The method of any one of claims 22-24, wherein the skeletal stem cells are autologous or allogeneic to the subject in need.

26. The pharmaceutical composition of any one of claims 1-7 for use in treating bone damage or defects in a subject in need thereof.

27. The pharmaceutical composition of claim 26, wherein the bone injury or defect is a bone fracture.

28. Use of the pharmaceutical composition of any one of claims 1-7 or the isolated skeletal stem cell population prepared by the method of any one of claims 8-12 in the preparation of a medicament for treating bone injury or defect in a subject in need thereof.

Images